The International Magazine on Banana and Plantain · 2018. 3. 28. · Evaluation of the agronomic characteristics of plantain hybrids Options for in vitro propagation of the banana

INFOMUSAINFOMUSAThe International Magazine on Banana and Plantain

INFOMUSA is published with the support of the Technical Centerfor Agricultural and RuralCooperation (CTA)

CTCTA

Vol. 11 N° 1June 2002IN THIS ISSUEIntegrated crop managementstrategies for plantain productionand control of black Sigatoka in DRCThe spread of black Sigatokathroughout Venezuela, 1997-2000Frequency of Paracercosporafijiensis and Pseudocercosporamusae in Dominico hartón plantain Effects of the natural fungicide F20on black Sigatoka disease onplantain and banana Seasonal fluctuations of R. similisand P. coffeae in certain cultivars of bananaHost plant response of Pisang JariBuaya and Mysore bananas to R. similisEffect of three arbuscularmycorrhizal fungi on root-knotnematode infection of MusaEndophytic fungal speciesassociated with root necrosis ofbanana in CubaEffects of mycorrhization onmicropropagated bananaArachis pintoi: a cover crop forbananas? Dynamics of boron in a soilcultivated with plantain in ColombiaEvaluation of the agronomiccharacteristics of plantain hybridsOptions for in vitro propagation ofthe banana hybrid FHIA-20Multiplication rate and regenerationpotential of somatic embryos from a cell suspension of banana (Musa AAA)Introduction, multiplication anddistribution of improved bananasand plantains in NicaraguaUsing RAPD technique foridentifying and classifying somebanana cultivars in VietnamConsumption and expenditurepatterns of banana and plantainconsumers in NigeriaThesisMusaNewsMusaForumINIBAP NewsBooks etc.Announcements

INFOMUSA Vol. 11, N° 1

CONTENTS

Integrated crop management strategies for plantain production and control of black leaf streak (black Sigatoka) disease in the Democratic Republic of Congo............................................................................................................ 3

The spread of black Sigatoka throughout Venezuela, 1997-2000 ...................... 6Frequency of Paracercospora fijiensis and Pseudocercospora musae

in Dominico hartón plantain............................................................................ 9Effects of the natural fungicide F20 on black Sigatoka disease (Mycosphaerella

fijiensis Morelet) on plantain (AAB) and banana (AAA) .............................. 14Seasonal fluctuations of Radopholus similis and Pratylenchus coffeae in certain

cultivars of banana ......................................................................................... 16Host plant response of Pisang Jari Buaya and Mysore bananas to Radopholus

similis ............................................................................................................... 19Effect of three arbuscular mycorrhizal fungi on root-knot nematode

(Meloidogyne spp.) infection of Musa .......................................................... 21A study of endophytic fungal species associated with root necrosis of banana in

banana and plantain plantations in Cuba..................................................... 23Effects of mycorrhization on the development of two cultivars of

micropropagated banana............................................................................... 25Arachis pintoi: a cover crop for bananas? Advantages and disadvantages as

regards nematology........................................................................................ 28Dynamics of boron in a soil cultivated with plantain (Musa AAB cv. Dominico

hartón) in the Quindío, Colombia ................................................................. 30Evaluation of the agronomic characteristics of plantain hybrids (Musa spp.).. 34Options for in vitro propagation of the banana hybrid cultivar FHIA-20......... 35Multiplication rate and regeneration potential of somatic embryos from a cell

suspension of banana (Musa AAA cv. ‘Grande naine’)................................. 38Introduction and multiplication of improved bananas and plantains in

Nicaragua and distribution to farmers .......................................................... 44Using RAPD technique for identifying and classifying some banana cultivars

in Vietnam ....................................................................................................... 48Consumption and expenditure patterns of banana and plantain consumers

in Nsukka Urban, Nigeria ............................................................................... 50Thesis ..................................................................................................................... 54MusaNews ............................................................................................................. 56INIBAP New ........................................................................................................... 60Books etc. .............................................................................................................. 65Announcements.................................................................................................... 66

The mission of the International Network for the Improvement of Banana and Plantainis to sustainably increase the productivity of banana and plantain grown on smallhold-ings for domestic consumption and for local and export markets.The Programme has four specific objectives:• To organize and coordinate a global research effort on banana and plantain, aimed at

the development, evaluation and dissemination of improved cultivars and at the con-servation and use of Musa diversity

• To promote and strengthen collaboration and partnerships in banana-relatedresearch activities at the national, regional and global levels

• To strengthen the ability of NARS to conduct research and development activities onbananas and plantains

• To coordinate, facilitate and support the production, collection and exchange ofinformation and documentation related to banana and plantain.

INIBAP is a programme of the International Plant Genetic Resources Institute(IPGRI), a Future Harvest center.

Vol. 11, N° 1Cover photo:In Tanzania, banana corms arefrequently distributed through schools.(D. Mowbray, Baobab Productions)

Publisher: International Network for the Impro-vement of Banana and PlantainManaging editor: Claudine PicqEditorial Committee: Emile Frison, Jean-Vincent Escalant,Suzanne Sharrock, Elinor Lipman,Charlotte LustyPrinted in FranceISSN 1023-0076Editorial Office: INFOMUSA, INIBAP, Parc ScientifiqueAgropolis II, 34397 Montpellier Cedex 5,France. Telephone + 33-(0)4 67 61 1302; Telefax: + 33-(0)4 67 61 03 34; E-mail: [email protected]//:www.inibap.orgSubscriptions are free for developingcountries readers. Article contributionsand letters to the editor are welcomed.Articles accepted for publication may beedited for length and clarity. INFOMUSAis not responsible for unsolicited mater-ial, however, every effort will be made torespond to queries. Please allow threemonths for replies. Unless accompaniedby a copyright notice, articles appearingin INFOMUSA may be quoted or repro-duced without charge, provided acknowl-edgement is given of the source.French-language and Spanish-languageeditions of INFOMUSA are also published.To avoid missing issues of INFOMUSA,notify the editorial office at least sixweeks in advance of a change of address.

Views expressed in articles are thoseof the authors and do not necessarilyreflect those of INIBAP.

INFOMUSAINFOMUSAThe International Magazine on Banana and Plantain

INFOMUSA is published with the support of the Technical Centerfor Agricultural and RuralCooperation (CTA)

CTCTA

Vol. 11 N° 1June 2002IN THIS ISSUEIntegrated crop managementstrategies for plantain productionand control of black Sigatoka in DRCThe spread of black Sigatokathroughout Venezuela, 1997-2000Frequency of Paracercosporafijiensis and Pseudocercosporamusae in Dominico hartón plantain Effects of the natural fungicide F20on black Sigatoka disease onplantain and banana Seasonal fluctuations of R. similisand P. coffeae in certain cultivars of bananaHost plant response of Pisang JariBuaya and Mysore bananas to R. similisEffect of three arbuscularmycorrhizal fungi on root-knotnematode infection of MusaEndophytic fungal speciesassociated with root necrosis ofbanana in CubaEffects of mycorrhization onmicropropagated bananaArachis pintoi: a cover crop forbananas? Dynamics of boron in a soilcultivated with plantain in ColombiaEvaluation of the agronomiccharacteristics of plantain hybridsOptions for in vitro propagation ofthe banana hybrid FHIA-20Multiplication rate and regenerationpotential of somatic embryos from a cell suspension of banana (Musa AAA)Introduction, multiplication anddistribution of improved bananasand plantains in NicaraguaUsing RAPD technique foridentifying and classifying somebanana cultivars in VietnamConsumption and expenditurepatterns of banana and plantainconsumers in NigeriaThesisMusaNewsMusaForumINIBAP NewsBooks etc.AnnouncementsPROMUSA News

P. Mobambo Kitume Ngongo

Plantain (Musa spp., AAB group) isan important staple food in manycountries of the humid tropics. It is

among the most important carbohydratesources in the diet of people in theseregions. Its low labour requirement andrelatively high-energy output make plan-tain a suitable staple for areas wherelabour shortage is usually the main con-straint to production. The crop is mainlygrown by small-scale farmers and it is anintegral component of most farming sys-tems in West and Central Africa, whereabout 50% of the world’s plantain is pro-duced (Wilson 1987, FAO 1990).

In spite of its importance to local peo-ple, plantain has long been ignored byagricultural researchers in the region,since it had no major disease problemsuntil the 1970s and was thereforeregarded as a disease-free crop in Africa(Wilson 1987). Twenty-five years ago,however, the crop was threatened byblack leaf streak (black Sigatoka), anair-borne leaf spot disease caused by thefungus Mycosphaerella fijiensisMorelet. The disease spread rapidly intoall plantain-producing regions of Africa.Black Sigatoka is the most destructiveleaf disease of plantain, as it is spreadinginexorably to all major lowland plantain-growing regions as the dominant leaf spot(Meredith and Lawrence 1970). Plantainyield loss of 76% due to black Sigatoka hasbeen reported during the second crop-ping cycle, while the whole complex ofdisease, pests and soil fertility declinetogether reduced yield by 93% (Mobamboet al. 1996a). As a perennial starchy crop,plantain requires a considerable time tomature, resulting in longer exposure todiseases, pests and in depletion of soilnutrients.

The soil-disease-pest complex can becontrolled by the combination of inor-ganic fertilizers, fungicides and insecti-cides/nematicides. In Africa however,chemical control strategies are socio-economically and environmentally

unsound in the framework of theresource-poor smallholders growingplantain. Chemicals are very expensiveand their applications may be hazardousto health in the village homesteadswhere the bulk of plantain is grown.Therefore, proper soil managementusing several crop residues mulches toimprove the organic matter and nutrientcontent of the soil could reduce the soil-disease-pest complex effects on plantainwith low inputs.

The objectives of the research reportedhere were to compare field performanceand yield of plantain under differentpractices of soil fertility management anddisease control.

Materials and methods

Location of the experimentInvestigations were carried out atKinshasa (4°22’S, 15°21’E, westernCongo), which is at 390 m above sea level(Anonymous 1985). The soil of the exper-imental site is a latosol derived fromdeposited sands, well drained, but poor innutrients and highly acidic. Annual rain-fall averages 1800 mm and average tem-perature is 24.5°C.

Plant materials and treatmentsMusa AAB cv. ‘Yumba’, locally wide-spread, was used in this experiment.Planting materials still constitute a con-straint for plantain production in ruralareas. As it is impossible to get many and uniform plantain suckers at once,the investigation started by vegetativemultiplication of planting materials(technique described by Auboiron 1997)in order to obtain 625 plants for theexperiment: 5 treatments x 5 replicationsx 25 plants per treatment.

Plantain corm stumps were split intosets of 50 g each and treated with woodashes. They were air-dried for 24 hoursbefore being planted in 15 cm-diameterplastic bags almost filled with forest top-soil. New sprouts emerged after 4 weeksfrom the date of planting and up to20 new plants were obtained from a corm.

Plants were grown in half-shade condi-tions and watered regularly. They weretransplanted in the field 3 months later,when they had 3-4 true leaves

Four different treatments to preventinfection by microorganisms, based oncultural practices, were compared: cropresidues mulches (wood sawdust or ricehusk), cover crop (Vigna unguiculata)and fertilizers (NPK). Non-treated plantswere used as control.

Field layout and cultural practicesThe experimental design was a random-ized complete block with five plot-treatments and five replications. The plotsize was 15 m x 10 m with 25 plants spacedby 3 x 2 m, resulting in a plant density of1667 per ha. Data were recorded only onthe nine central competitive plants.

Every 3 months, crop residues mulcheswere applied to the soil around the stemin mulched plots using one head-pan(10 kg). In fertilized plots, 300 kg N, 60 kgP2O and 550 kg K2O per ha per year weresplit into six applications during the rainyseason: urea at a rate of 65 g per plant perapplication, phosphorus at a rate of 20 gper plant per application, and muriate ofpotash at a rate of 89 g per plant perapplication.

For each treatment, soil samples weretaken at about 50% flowering stage usinga soil hand auger up to 20 cm depth,where plantain has the majority of itsroots (Swennen 1984, Purseglove 1988).These samples were air-dried in the labo-ratory, crushed, passed through 0.5 and2 mm sieves and analyzed.

Evaluation of host response to black Sigatoka, growth and yield parametersThe disease development was evaluatedevery week using the “symptom evolutiontime”, which is the number of daysbetween the appearance of symptoms ofstage 1 of the disease development(Fouré 1982) assimilated to stage b of thecigar (Brun 1963) and the appearance ofspots with dry centres (stage 6 of the dis-ease, Fouré 1982, 1987). The “youngest

INFOMUSA — Vol 11, N° 1 3

Integrated crop management strategies for plantain production and control of black leafstreak (black Sigatoka) disease in the DemocraticRepublic of Congo

Diseases Control of black Sigatoka

leaf spotted” which is the leaf with 10 ormore discrete necrotic lesions with drycentres (Meredith and Lawrence 1970,Fouré 1982, 1987) and the “life time ofthe leaf”, which is the number of daysbetween the cigar-stage b of the leaf andleaf death (100% leaf area necrotic),either due to senescence or blackSigatoka (Mobambo et al. 1994) were alsorecorded.

Disease severity was evaluated everytwo weeks, from two months after plant-ing until flowering. The percentage ofleaf area with symptoms was recordedusing the modified scale of Stover andDickson (1970) as described at theInternational Institute of TropicalAgriculture (Mobambo et al. 1993a).

Growth parameters evaluated includeheight of pseudostem, girth of pseudo-stem, number of leaves emerged andheight of the tallest sucker. They wererecorded on each plant from 2 monthsafter planting until flowering asdescribed by Swennen and De Langhe(1985).

Yield parameters evaluated were num-ber of hands per bunch, number of fruitsper bunch and bunch weight.

Data collected were analyzed using theANOVA procedures of Statistical AnalysisSystem (SAS 1988) for randomized com-plete block design. The Duncan MultipleRange (DMR) test at the 0.05 significancelevel was used to compare treatmentmeans for each parameter.

Results and discussion

Soil conditionsSoil analysis results presented in Table 1showed significant differences in theamounts of nutrients between cropresidues mulches (wood sawdust and ricehusk) and other management practices,such as cover crop (Vigna unguiculata)and mineral fertilizer (NPK). Meanwhile,statistical differences were foundbetween rice husk and wood sawdust,with rice husk as the best improving soilfertility level. According to the scales ofBlack (1965) and Brady (1984), in plotsmulched with crop residues the soil wasin general moderately acidic with veryhigh organic carbon, high total nitrogen,moderate calcium, moderate magnesiumand high potassium. In the non-mulchedplantain plots however, the soil wasextremely acidic with low organic carbon,moderately low total nitrogen, low cal-cium, very low magnesium and very lowpotassium.

These results indicate that theamounts of soil nutrients are higher in

the mulched plots than in the non-mulched plots. Crop residues mulchesconstitute better sources of nutrients andact therefore as a fertilizer. As pointedout by Lal and Kang (1982), organic mat-ter constitutes a key component of soilfertility, as a reservoir of nutrients, as amain source of cation exchange capacityand as major promoter of aggregate struc-tural stability.

Host response to black Sigatoka Significant differences were foundbetween plantain mulched with cropresidues (wood sawdust and rice husk)and the non-mulched plantain (control,cover crop and fertilizer) regarding symp-tom evolution time, youngest leaf spot-ted, percentage of leaf area with symp-toms and life time of leaf (Table 2). Theseverity of black Sigatoka on plantain wasmuch lower in the mulched plantain thanin the non-mulched plantain. Among cropresidues mulches, however, rice husk wasstatistically the best for slowing diseasedevelopment.

Black Sigatoka symptom developmentin the mulched plantain was slower thanin the non-mulched plantain. In the plan-tain mulched with crop residues, the dis-ease needed almost one month more todevelop the last symptom stage comparedto the control. For the fertilized andcover-cropped plantain, symptom evolu-tion time values were respectively 40 and36 days, i.e. 2-3 weeks less than for themulched plantain.

With respect to the youngest leaf spot-ted (YLS), results show the same trend asfor the symptom evolution time (Table 2).There were significant differencesbetween the plantain mulched with cropresidues and the non-mulched plantain.On mulched plantain, YLS was 11 for ricehusk and 9 for wood sawdust, whereas onboth the fertilizer-treated plantain andon the cover-cropped plantain the YLSwas 8. The non-treated plantain (control)had the lowest YLS value, 6.

These results indicate that when usingthe rice husk mulch the plant gains threehealthy leaves comparing with the fertil-izers or cover crop treatments, and fivehealthy leaves against the control.Therefore, with a leaf emergence time ofabout one per week for plantain in gen-eral, the soil fertility (Table 1) due to ricehusk mulch slowed the symptom evolu-tion by 3 and 5 weeks compared respec-tively to that of the fertilized and cover-cropped plots and the control.

Pronounced differences were alsoobtained between mulched and non-mulched plants regarding the percentageof leaf area with symptoms (Table 2).While in cover-cropped and fertilizedplots, plantain presented respectively10.3% and 6.9% of leaf area infected byblack Sigatoka, in plots mulched withcrop residues it lost only 3.8% to 4.2% ofits leaf area. The slower spread of the dis-ease in mulched plots is facilitated by anincreased functional leaf area comparedto that of non-mulched plots.

4 INFOMUSA — Vol 11, N° 1

Table 1. Selected soil chemical properties under different plantain managementpractices at Kinshasa, western Congo, 1998.

Treatment pH Organic Carbon Total Nitrogen Exchangeable cations (%) (%) (meq/100 g)

Ca Mg K

Control 4.2 a 1.15 a 0.11 a 1.22 a 0.21 a 0.15 a

Wood sawdust 6.2 c 3.51 c 0.25 b 6.51 c 1.87 c 0.87 c

Rice husk 6.8 d 3.79 d 0.28 b 7.52 d 2.10 c 0.98 d

Vigna unguiculata 5.2 b 2.15 b 0.22 b 4.07 b 0.78 b 0.46 b

N-P-K 5.6 b 2.23 b 0.26 b 5.63 c 1.06 b 0.96 cWithin columns, means followed by the same letter are not significantly different at the 0.05 probability level, according to theDuncan Multiple Range test.

Table 2. Host-plant response to black Sigatoka of plantain under differentmanagement practices at Kinshasa, western Congo, 1998.

Treatment Symptom Youngest % Leaf area Life time evolution time leaf spotted with symptom of leaf

(SET, days) (YLS) (% LAWS) (LTL, days)

Control 23.0 a 5.5 a 19.6 d 62.5 a

Wood sawdust 50.0 c 9.3 c 4.2 a 125.7 d

Rice husk 56.8 c 10.9 d 3.8 a 130.3 d

Vigna unguiculata 35.5 b 7.5 b 10.3 c 80.3 b

N-P-K 40.0 b 8.1 b 6.9 b 103.3 cWithin columns, means followed by the same letter are not significantly different at the 0.05 probability level, according to the Duncan Multiple Range test.

Regarding leaf life time, significantdifferences were also found betweenplants mulched with crop residues andthose that were not mulched (Table 2).Slower disease development on mulchedplantain prolonged the lifetime ofleaves. In plantain treated with ricehusk and wood sawdust, black Sigatokaneeded almost 9, 7 and 4 weeks longer todestroy the leaves as compared respec-tively to the control, the cover-croppedand the fertilized plantain. The controlplantain was the most affected by thedisease. As already reported, all plantaincultivars (Musa spp., AAB group) overthe world are susceptible to blackSigatoka (Fouré 1987, Mobambo et al.1996b).

The difference in the host response toblack Sigatoka between the plantainmulched with crop residues and non-mulched plantain is mainly attributed tothe difference in soil fertility. The higherthe soil fertility level, the lower the blackSigatoka severity. On better soils this isexpressed in a slower symptom develop-ment, older leaves bearing dry spots, lessleaf area with black Sigatoka symptomsand longer life time of leaves (Mobamboet al. 1994).

Growth and yield performancesResults presented in Table 3 show sig-nificant differences for all the parame-ters studied: plant height (PH), plantgirth (PG), number of emerged leaves(NEL), days to flowering (DF), days for fruit filling (DFF), days to harvest(DH) and height of the tallest sucker(HTS).

For all treatments (crop residuesmulches, fertilizer or cover crop) theplants had a similar height, whereasthey were shorter than the control.However, regarding plant girth and num-ber of emerged leaves, the plantainmulched with crop residues performedbetter than the non-mulched plantain.Bigger plant girth and lower number ofleaves were obtained on plants treatedwith rice husk and wood sawdust thanon the non-mulched plants. Plantsmulched with rice husk flowered signifi-cantly earlier and had a longer fruit-fill-ing period than those under other treat-ments. They flowered 5 months earlierthan the control and about 1 to 2 monthsearlier than the fertilized and cover-cropped plantains. The combined effectresulted in a shorter production cyclefor the plantain mulched with rice husk,whose bunches were harvested 104 and28 days earlier than in the control andfertilized plantain respectively. Plantain

mulched with rice husk was harvested16 days earlier than that mulched withwood sawdust. It also showed bettersuckering, the tallest sucker, i.e. thesucker that will continue as the nextproduction cycle, being significantlytaller than for other treatments. Thisshould normally result in a shorterratoon cycle for the plantain treatedwith rice husk as compared to othertreatments.

The yield components evaluated werethe number of hands per bunch, numberof fruits per bunch and bunch weight(Table 4).

There were significant differencesbetween the plantain mulched with cropresidues (wood sawdust and rice husk)and the non-mulched plantain (control,cover crop and fertilizer) regarding thenumber of hands per bunch and numberof fruits per bunch (Table 4). Themulched plantain had a higher number ofhands and fruits per bunch than the non-mulched.

Yield per hectare was calculated fromthe average bunch weight multiplied byplant density. Yield was significantly dif-ferent between the plantain mulchedwith rice husk and other treatments. Theyield of the best-performing plantaintreated with rice husk was 46%, 37% and26% higher than those of control, cover-cropped and fertilized plantain, respec-tively. The yield of plantain mulched with

rice husk was 14% higher than thatobtained with wood sawdust.

These results indicate that crop resi-dues mulches confer important advan-tages to plantain cultivation: higher yield,earlier maturity or shorter productioncycle and bigger girth allowing reductionof losses from wind-damage, anotherimportant constraint to plantain produc-tion (Mobambo et al. 1996a).

ConclusionThe research reported here compares dif-ferent management practices of plantainproduction. The effects of the cropresidues mulches (wood sawdust and ricehusk) were compared to those of fertil-izer application and cover crop for soilfertility, black Sigatoka severity, growthand yield parameters of plantain.

For all parameters evaluated, the plan-tain mulched with crop residues per-formed better than the non-mulchedplantain. Soil fertility is the critical fac-tor responsible for the differencebetween crop residues mulches, covercrop and fertilizer. Because of the highlevel of fertility due to the application ofcrop residues mulches, plantain was lessaffected by black Sigatoka and conse-quently better growing than when receiv-ing no mulch. Among crop residuesmulches, rice husk was statistically bet-ter than wood sawdust.


Table 3. Growth parameters of plantain under different management practices at Kinshasa, western Congo, 1998.

Treatment PH PG NEL DF DFF DH HTS (cm) (cm) at harvest

(cm)

Control 360.3 b 65.4 a 44 d 370 d 76 a 446 d 78.0 a

Wood sawdust 345.5 a 74.4 b 35 a 255 b 103 c 358 b 105.0 b

Rice husk 340.0 a 76.6 b 34 a 232 a 110 d 342 a 145.0 c

Vigna unguiculata 349.5 a 68.8 a 41 c 295 c 86 b 381 c 80.5 a

N-P-K 342.2 a 66.8 a 38 b 268 b 102 c 370 bc 86.8 aWithin columns, means followed by the same letter are not significantly different at the 0.05 probability level, according to the Duncan Multiple Range test.Legend: PH: Plant height; PG: Plant girth; NEL: Number of emerged leaves; DF: Days to flowering; DFF: Days for fruit filling; DH: Days to harvest; HTS: Height of the tallest sucker.

Table 4. Yield parameters of plantain under different management practices at Kinshasa, western Congo, 1998.

Treatment No. of hands No. of fruits Bunch weight Yield per bunch per bunch (kg) (t/ha)

Control 6.0 a 75 a 9.5 a 15.8 a

Wood sawdust 6.5 b 88 c 15.0 c 25.0 c

Rice husk 6.5 b 90 c 17.5 d 29.2 d

Vigna unguiculata 6.2 a 82 b 11.0 a 18.3 a

N-P-K 6.2 a 87 bc 13.0 b 21.7 bWithin columns, means followed by the same letter are not significantly different at the 0.05 probability level, according to the Duncan Multiple Range test.

Therefore, proper management oforganic matter is essential for the sus-tainable productivity of plantain, by mini-mizing the black Sigatoka severity withlow inputs. Since plantain is grownmainly by small-scale farmers in Africa,chemical fertilizers are not readily andeconomically available. Thus, the poten-tial of traditional organic fertilizers suchas compost, farmyard manure and cropresidues mulches need to be betterexploited. A study integrating organicresources and soil fauna may help tounderstand the mechanisms regulatingthe biological processes for the improve-ment of soil fertility in relation with thesustainability of plantain production, dis-ease and pest severity.

AcknowledgementsThis research was fully supported by theInternational Foundation for Science(IFS), Stockholm, Sweden. I am espe-cially grateful to Ms Ingrid Lindhe(Assistant, Crop Sciences Area) and MsJosiane Lindberg (Purchasing services)for their availability, assistance and effi-cient help during my research. ■

ReferencesAnonymous. 1985. Caractéristiques climatiques de

Kinshasa. Institut national de géographie. 85pp.Auboiron E. 1997. Propagation rapide de matériel de

plantation de bananiers et plantains. La multipli-cation sur souche décortiquée. Fiche technique.CRBP, Douala, Cameroun.

Black N.C. 1965. Physical and mineralogical proper-ties, including statistics of measurement and sam-pling. Pp. 515-545 in Methods of soil analysis. PartI: Physical methods (C.A. Black, ed.). AmericanSociety of Agronomy, Madison, Wisconsin.

Brady N.C. 1984. The nature and properties of soils.MacMillan Publishing Company, New York.750pp.

Brun J. 1963. La cercosporiose du bananier enGuinée. Etude de la phase ascosporée deMycosphaerella musicola Leach. Thesis. Facultédes Sciences, Université Paris-Orsay. 196pp.

FAO. 1990. Production Yearbook. Food andAgriculture Organization of the United Nations,Rome.

Fouré E. 1982. Les cercosporioses du bananier etleurs traitements. Comportement des variétés. 1:Incubation et évolution de la maladie. Fruits 37:749-766.

Fouré E. 1987. Varietal reactions of bananas andplantains to black leaf streak. Pp. 110-113 in

Banana and plantain breeding strategies (G.J.Persley and E.A. De Langhe, eds). Proceedings ofan International Workshop, 13-17 October 1986,Cairns, Australia. ACIAR Proceedings No. 21.

Lal R. & B.T. Kang. 1982. Management of organicmatter in soils of the tropics and subtropics.


Pp. 152-178 in Non-symbiotic nitrogen fixationand organic matter in the tropics. Symposiapapers 1. 12th International Congress of SoilScience, 8-16 Feb. 1982, New Delhi.

Meredith D.S. & J.S. Lawrence. 1970. Black leafstreak disease of bananas (Mycosphaerella

fijiensis): susceptibility of cultivars. TropicalAgriculture 27: 275-287.

Mobambo K.N., F. Gauhl, D. Vuylsteke, R. Ortiz,C. Pasberg-Gauhl & R. Swennen. 1993a. Yield lossin plantain from black Sigatoka leaf spot and fieldperformance of resistant hybrids. Field CropsResearch 35: 35-42.

Mobambo K.N., M. Naku & Z. Nganga. 1993b. Blackleaf streak disease in banana and plantain inZaïre. INFOMUSA 2: 14-15.

Mobambo K.N., K. Zuofa, F. Gauhl, M.O. Adeniji &C. Pasberg-Gauhl. 1994. Effect of soil fertility onhost response to black leaf streak of plantain(Musa spp., AAB group) under traditional farmingsystems in southeastern Nigeria. InternationalJournal of Pest Management 40: 75-80.

Mobambo K.N., F. Gauhl, R. Swennen & C. Pasberg-Gauhl. 1996a. Assessment of the cropping cycleeffects on black leaf streak severity and yielddecline of plantain and plantain hybrids.International Journal of Pest Management 42: 1-7.

Mobambo K.N., F. Gauhl, C. Pasberg-Gauhl &K. Zuofa. 1996b. Season and plant age affect eval-uation of plantain for response to black Sigatokadisease. Crop Protection 15: 609-614.

Purseglove J.W. 1988. Tropical crops:Monocotyledons. Longman Scientific & Technical,Singapore. 607pp.

SAS. 1988. Statistical Analysis Systems ProceduresGuide, Release 6.03. SAS Institute, Cary, NC.441pp.

Swennen R. 1984. A physiological study of the suck-ering behaviour in plantain (Musa cv. AAB). PhDThesis. Katholieke Universiteit te Leuven,Belgium. 180pp.

Swennen R. & E. De Langhe. 1985. Growth parame-ters of yield of plantain (Musa cv. AAB). Annals ofBotany 56: 197-204.

Wilson G.F. 1987. Status of bananas and plantains inWest Africa. Pp. 29-35 in Banana and plantainbreeding strategies (G.J. Persley and E.A. DeLanghe, eds). Proceedings of an InternationalWorkshop, 13-17 October 1986, Cairns, Australia.ACIAR Proceedings No. 21.

The author is a Professor in the Faculty of Agriculture,University of Kinshasa, BP 785 Kinshasa XI, DemocraticRepublic of Congo. Tel. +243 99 18257; E-mail: [email protected]

G. Martínez, J. Hernández, O. Tremont, R. Pargas

and E. Manzanilla

Since the first report of blackSigatoka (Mycosphaerella fijiensisMorelet) in Venezuela, great uncer-

tainty has existed as to the future ofbanana and plantain production there.The complex nature of the pathogen givesit considerable potential for adaptationto new climatic conditions, fungicidesand host genotypes (Ploetz 2000). This isclearly demonstrated by the loss in effi-cacy of certain products used for chemi-cal control such as the benzimidazolesand triazoles (Douglas and Ching 1992,

Estévez 1992, Stover 1993, Guzman et al.2000, Romero 2000).

This situation makes clear the extentof the problem posed by this disease andnecessitates the introduction of inte-grated control measures using resistantclones with a high yield potential (Roweand Rosales 1993). The existence of aclose relationship between certain cli-matic factors (relative humidity, temper-ature and rainfall) and the pathogeninfluences the incidence and severity ofthe disease (Fouré 1994, Gauhl 1994). Itis this which has enabled us to map thespread of the disease across the countryand also to take steps against its medium-term incidence in zones where its pres-ence has yet to be recorded, such as was

The spread of black Sigatokathroughout Venezuela, 1997-2000

Diseases Distribution of black Sigatoka

done in 1997 and 1998 (Martínez 1997,Martínez et al. 1998).

The work presented here aims todescribe the current situation of blackSigatoka in Venezuela, the course of itsspread, its relationships with various cli-matic factors which determine its aggres-siveness and measures taken for its con-trol. For this purpose, an expedition wasmade into different areas in the south-east of Venezuela, collecting samplesshowing typical symptoms of the diseasefor identification, questioning growersand analyzing weather data (relativehumidity, rainfall and temperature).

Current situation and spread of the diseaseBlack Sigatoka was detected for the firsttime in Venezuela in 1991 in the state ofZulia in the western region (Haddad et al.1992, Escobar and Ramirez 1995), andthen it spread into various zones andstates (Martínez 1997, Martínez et al.1998). Mention is made in this report ofits arrival in the state of Bolivar and,between 1999 and 2000, in the states ofDelta Amacuro and Amazonas, in theextreme east and south of the country,respectively. On the basis of their rainfalland relative humidity (Martínez 1997,

Martínez et al. 1998), these regions weredeclared to be at high risk of potentialinfection in the short term (Figure 1).

They are characterized by rainfall inexcess of 1500 mm/yr, a relative humidityabove 79% and a mean temperaturebetween 25 and 28ºC (Figures 2 and 3),which is significant in view of the rela-tionship established between climate andthe incidence and development of thedisease (Fouré 1994, Gauhl 1994,Mobambo 1995). They are very differentfrom the Maracay region where the meanrainfall is 922 mm/yr with a 6-month dryseason. This situation has enabled theestablishment of a model for comparingtwo totally different agro-ecologicalstates with which are correlated criticallevels of the severity reached by the dis-ease. This model serves as a reference forintroducing control measures on thebasis of climatic conditions and to pre-vent possible spread into zones possess-ing similar characteristics (Martínezet al. 2000).

Fouré (1994) mentions relationshipsexisting between climatic parametersand the spread of the disease which per-mit a better understanding of the dynam-ics of the epidemic in production zonesand of its potential for initiating future

infection. The liberation of ascospores israpid during rain because of the presenceof a film of residual water on the uppersurface of the leaves, whose lower surfaceexhibits more lesions. Dead leaves whichremain attached to the plant thereforerepresent an excellent source of inocu-lum (Gauhl 1994). As to the temperature,it is estimated that the ascospores ofMycosphaerella fijiensis germinatebetween 10 and 38ºC, with an optimum at27ºC, and noting that the relative growthrate of the germination tubes (hyphae)falls rapidly at temperatures below 20ºC(Pérez and Mauri, cited by Pérez 1996).Concerning the effect of wind, it has beenshown that the concentration of conidiain plantations is higher in the lowest airlayers than on the leaves whereas theconcentration of ascospores in the air isthe same: this confirms the importanceof the ascospores in the life cycle of thedisease (Stover 1984, Gauhl 1994).

It remains to emphasize the presenceof topographic accidents in well-definedgeographical areas which is apparentlycorrelated with the variation in climaticfactors mentioned above. These topo-graphic accidents therefore also affectthe development and severity of the dis-ease. The first report of the disease


AMAZONAS1991 - 1994

1994 - 1996

1996 - 1998

1998 - 2000

BRASIL

CARIBBEAN SEA

GUYANA

COLOMBIA

ZULIA

TACHIRA

MERIDA

FALCON

LARA

APURE

GUARICO ANZOATEGUI

MONAGAS

SUCRE

DELTA

AMACURO

BOLIVAR

MIRANDATRUJILLO

BARINAS

PORTUGUESACOJEDE

YARACUI CARABOBO

ARAGUA

DE

Figure 1. Spread of black Sigatoka in Venezuela from 1991 to 2000.

occurred in the Lake Maracaibo area,where the high relative humidity may bedue to the proximity of the lake and tothe topography of the landscape of theregion. These conditions are similar tothose around Lake Valencia (the point ofentry of the disease into the state ofAragua) and in the sectors close to theCaroni river, Hato Gil (Bolivar state). Inthe same way, the presence of the Andescordillera and the interior mountainchain, which constitute a natural barrierto the passage of the fungal spores toadjacent regions, ought to have pre-vented the spread of the disease intothese zones. However this has happened,and its appearance in these regions canonly be due to the transport of infectedmaterial.

Influence of the disease on plantation managementFarm surveys and visits to different areasof the country have shown us that thebiggest losses have occurred in fieldswhere there was no control of weeds,nematodes or insects. Hanging, dried-upleaves were not removed and no fertilizerwas used. There were also problems ofirrigation and drainage and an unsatis-factory spacing of plants in the field. Thegrowers are not in the habit of removingside shoots, nor of using chemicals tocontrol diseases. They lack technical sup-port and resources to buy agrochemicalsand equipment. Finally, there are no pro-

ducers’ organizations. With a low yieldwhich is all consumed by the family, thealternatives for the small producer are tosell his plantation, change the crop orsimply to abandon the farm completely(Martínez et al. 2000).

Medium-sized producers tend to adjustthe area of their plantation if productioncosts increase, allowing them to obtainyields which are dependent on theamount of investment. Large producerssucceed in living with the disease, as canbe seen to the south of Lake Maracaibo,where there are associations of produc-ers and firms which improve the qualityof the product in the plantations where itis destined for the international market.That which is rejected for export is soldon the national or local market wherethere is no quality control (Martínez etal. 2000).

Changes in crop managementobserved in the presence of black SigatokaThe presence of black Sigatoka in thecountry has resulted in radical changesin the way bananas are grown. The tradi-tional approach, which is to manage theplantations as perennial crops, tends tohave been gradually replaced by semi-perennial, and in some cases annual man-agement of the crop, with high plantingdensities possibly in association withother short-term crops, giving an increaseboth in yield and in the diversity of the

products obtained. This has been intro-duced thanks to research work carriedout by INIA, and also to the growingimportance of the organization amongthe producers.

In the course of all the field experi-ments the accent has been placed on effi-cient application of basic cultural prac-tices, such as removal of hanging deadleaves and the use of fertilizer, practiceswhich are not being applied at presenteven though it has been demonstratedthat they help to reduce the amount ofinoculum of the pathogen in the planta-tion and that they render the plant lessvulnerable to fungal attack (Gauhl 1994).

Quite clearly one should try to reducethe use of chemicals and aim for the bestpossible way of living with the pathogen.An alternative solution which may beadopted is the use of resistant cloneswhich can either be grown in commercialplantations in rows between the clonestraditionally grown in the country (so asto reduce the quantity of inoculum avail-able), or else as an entirely separate plan-tation, as is seen in the Ocumare regionof Costa, in Aragua state. There they havechosen to grow plantain FHIA-21, whosefruit has a softer texture than that of‘Hartón gigante’ and can be used for mak-ing “tostones”, or chips, of excellent qual-ity, much appreciated by the consumers,which has facilitated its introduction tothe market. Likewise it should be notedthat there are other possibilities for pro-duction such as the use of the hybridsFHIA-01, FHIA-02 and FHIA-03 whichyield well and a have a very good responseto the disease.

Conclusions1. The speed of spread of the pathogen

through the country has increasedrapidly: its passage from the westernzone to the central zone took five yearswhile the spread from the central zoneto the eastern zone and the southneeded only one. It seems clear that thisdevelopment has been encouraged byman. Causing an increase of 40-45% inthe costs of production, the disease hasparticularly affected small producersand cast doubt over the survival of theirholdings. The advance of the diseasethrough the national territory continuedinto the states of Bolivar, DeltaAmacuro and Amazonas between 1997and 2000.

2. The case of the state of Amazonas, onthe frontier with Brazil, is particular.The banana and the plantain, grown bythe indigenous communities, are majorelements of their diet. The ecosystemof the region is fragile and there is com-


80

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70

6588 89 90 91 92 93 94 95 96

90

85 San FelipeBarinasMaracayEl Vigía

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1000

1500

2000

2500

BarinasMaracayEl VigíaSan FelipeBolivar

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ipit

atio

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(mm

)

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Figure 2. Rainfall in Barinas, Maracay, El Vigia, San Felipe and Bolivar (Source: FAV.MARNR, DANAC).

Figure 3. Relative humidity in Barinas, Maracay, El Vigia and San Felipe (Source: FAV.MARNR, DANAC).

plex genetic and biological diversity.For this reason it is undesirable to usechemical control products and prefer-able to recommend introducing resis-tant clones which do not need theapplication of fungicides, even if thecultivars are not fully acceptable to thenative consumers.

3. Today it is evident that the presence ofblack Sigatoka in the country hasbrought about radical changes in theagronomic management of plantations.The effective use of basic cultural prac-tices, within the framework of inte-grated control, makes it possible to livewith the pathogen, as has been shownby numerous research studies carriedout by INIA.

AcknowledgementsWe thank particularly Mr Daniel Muñozfor his valuable collaboration in collect-ing information in the field, Mr MarcosSanoja, a specialist at the CorporaciónVenezolana de Guayana (CVG),Fundacite-Guayana for logistic support,the staff of the climatology section of theVenezuelan Air Force (FAV) at Maracay,the Ministry of the Environment andNatural Renewable Resources (MARNR)and the Polar Foundation, throughDANAC. ■

ReferencesDouglas M. & L. Ching. 1992. Monitoreo de sensibili-

dad de Mycosphaerella fijiensis al Benomil. Pp. 17-19 in Informe anual Corbana.

Escobar C. & M. Ramírez. 1995. Avance y establec-imiento de la Sigatoka Negra (Mycosphaerella

fijiensis) en el occidente de Venezuela.Universidad Nacional Experimental del Táchira y Ministerio de Agricultura y Cría-SASA. VIII Congreso Latinoamericano de Fitopatología.

XIV Congreso Venezolano de Fitopatología.Universidad de los Andes, Mérida, Venezuela.

Estévez M. 1992. Monitoreo sobre la resistencia de laSigatoka Negra a fungicidas sistémicos, pene-trantes, inhibidores de esteroles. Revista P.N.B.(Ecu): 32-33.

Fouré E. 1994. Leaf spot diseases of banana and plan-tain caused by Mycosphaerella fijiensis and M. musicola. Pp. 37-46 in The improvement andtesting of Musa: a global partnership. Proceedingsof the first Global conference of the InternationalMusa Testing Programme held at FHIA, Honduras(D. Jones, ed.).INIBAP, Montpellier, France.

Gauhl F. 1994. Epidemiology and ecology of blackSigatoka (Mycosphaerella fijiensis Morelet) onplantain and banana in Costa Rica, CentralAmerica. PhD thesis originally presented inGerman. INIBAP, Montpellier, France. 120pp.

Guzmán M., A. Jiménez, R. Vargas & R. Romero. 2000.Caracterización de cepas de M. fijiensis, causantede la Sigatoka negra, con menor sensibilidad afunguicidas triazoles. P. 64 in Reunión ACORBAT2000. Memorias.

Haddad O., M. Bosque, J. Osorio & L. Chávez. 1992.Aspectos fitosanitarios: Sigatoka negra medidasde prevención y control. FONAIAP Divulga 40: 44-45.

Martínez G. 1997. The present situation with regardto black Sigatoka in Venezuela. INFOMUSA

6(1): 16-17.Martínez G., R. Pargas, E. Manzanilla & D. Muñoz.

1998. Report on black Sigatoka status inVenezuela in 1997. INFOMUSA 7 (1): 31-32.

Martínez G., J. Hernández & A. Aponte. 2000.Distribución y epidemiología de la Sigatoka negraen Venezuela. Serie C.48. FONAIAP. FundaciteGuayana. 50pp.

Mobambo K. 1995. Factors influencing the develop-ment of black Sigatoka disease on plantain andplantain hybrids. INFOMUSA 4(1): 16-17.

Pérez L. 1996. Manual para el control integrado deSigatoka negra (Mycosphaerella fijiensis) ySigatoka amarilla (Mycosphaerella musicola

Leach ex Mulder) en banano y plátano. ProyectoTCP/CUB/4454. 27pp.

Ploetz R. 2000. La enfermedad más importante delbanano y el plátano: Una breve introducción a lahistoria, importancia y manejo de la Sigatokanegra. P. 117 in Reunión ACORBAT 2000.Memorias. Mesa redonda Sigatoka negra.

Romero R. 2000. Podemos evitar o disminuir el riesgode desarrollo de resistencia a los funguicidas enlas poblaciones de M. fijiensis? P. 118 in ReuniónACORBAT 2000. Memorias. Mesa redondaSigatoka negra.

Rowe P. & Rosales F. 1993. Diploid breeding at FHIAand the development of Goldfinger (FHIA-01).INFOMUSA 2(2): 9-11.

Stover R. 1984. Las manchas producidas por lasSigatokas en hojas de bananos y plátanos. Cursointernacional de reconocimiento, diagnostico ycontrol de Sigatoka negra del plátano y banano.14-18 May, Tulenapa, Colombia. 15pp.

Stover R. 1993. Cambios en la sensibilidad deMycosphaerella fijiensis al tilt. Informe UPEB16(97): 41-44.

The authors work in different centres of InstitutoNacional de Investigaciones Agrícola, INIA (formerFONAIAP), Venezuela. Gustavo Martínez, RafaelPargas and Edwuard Manzanilla at CENIAP-Maracay, apartado postal 4653, Maracay; E-mail: [email protected] and [email protected],Julitt Hernández at CIAE-Yaracuy and Omar Tremontat CIAE-Amazonas.


Frequency of Paracercospora fijiensisand Pseudocercospora musae in Dominico hartón plantain

Diseases Variability in leaf spot diseases

C. Lorena Cardona-Sanchez and J. Castaño-Zapata

Plantain (Musa sp.) is a subsistencecrop and in many areas is the sta-ple food for the population espe-

cially in rural areas, with an estimatednational per capita consumption of 68.5 kg/annum. Production is mostly with

minimal cultivation and as a result yellowSigatoka and black Sigatoka haveincreased in severity and dissemination(Merchán 1998), reducing production by 50% (Burt et al. 1997).

Nationally 384 957 ha are cultivated,75% with plantain Hartón and Dominicohartón, the latter cultivar increasinglycultivated in plantain plantationsthroughout the country, the high com-

mercial acceptance being due to theirflavour and other qualities and size; it ishighly susceptible to black and yellowSigatokas in the marginal coffee-growingzone (Merchán 1992).

Today, these diseases are found competing at altitudes greater than 1000 m above seal level. According tosome reports, black Sigatoka, caused by Mycosphaerella fijiensis Morelet, is

found attacking plantain Dominicohartón in the district of Victoria (Caldas)100 m above sea level, and is more aggres-sive than yellow Sigatoka, caused byMycosphaerella musicola Leach, a dis-ease which was displaced in less than sixmonths (Merchán 1992). A similar behav-iour was observed in the district ofPueblo Rico (Risaralda) 1560 m abovesea level (Merchán 1992). According tothe latest reports, black Sigatoka canaffect plantain from sea level up to analtitude of 1940 m (Belacázar et al. 1994).

The Sigatokas are difficult to differen-tiate from their external symptoms in thefield, and hence it is not possible to estab-lish clearly which of the two diseases ismore frequent when they occur together(Aguirre et al. 1998b). Microscopically,M. fijiensis and M. musicola are distin-guished mainly by the morphological dif-ferences of the anamorphs, in particularthe conidiophores and conidia character-istics, especially by the presence of scarspresent on conidiophores and conidia of Paracercospora fijiensis but absentfrom Pseudocercospora musae (Aguirreet al. 1998b).

Management of these diseases may bewith chemical products, a practice whichis not really applicable to the traditionalsystem of crop production. In order tosolve this problem more economicalalternatives have been sought, such ashost resistance to both diseases whichresults in a reduced sporulation of thecausal organisms.

The study was carried out with theobjective of determining the frequency ofP. fijiensis and P. musae spores in plan-tain Dominico hartón which is suscepti-ble to black and yellow Sigatoka diseases.

Materials and methodsThe investigation was carried out in theTolima department, 7 km from theDistrict of Fresno, on the road fromManizales (Caldas) to Mariquita (Tolima)in the village of La Ceiba, Campoalegreestate, located at an altitude of 1250 masland a temperature range of 18-25oC, a rel-ative humidity of 65-100% and a rainfallof 1800 mm/annum.

First, 1600 plants of the cloneDominico hartón, produced in vitro andmultiplied in the tissue culture labora-tory, Departamento de Fitotecnía de laFacultad de Ciencias Agropecuaria dela Universidad de Caldas, were trans-planted then acclimatized at theMontelindo farm of the University.

Evaluations were carried out weekly on53 clones selected at random. Recordswere taken starting 13 September 1998,the time of flower initiation, until harvest

on 13 March 1999. Impressions weretaken weekly from leaves attacked by theSigatokas with the aim of quantifyingspore populations of the anamorph state of M. fijiensis (Figure 1) and M. musicola (Figure 2).

Leaf impressions were made withsyringes of solidified agar in the form of adispenser, which was prepared from a 5 ml disposable syringe from which thefar end was removed forming a cylinder1.26 cm diameter. The dispenser wasfilled with crystal violet agar, prepared bymixing 1 g bacteriological agar with15 mlof a 1% solution of crystal violet in dis-tilled water and 100 ml water. The mix-ture was autoclaved at 121oC for 15 min.,after which 1 mg benomyl and two sensi-tivity discs of streptomycin (10 µg) wereadded (Aguirre et al. 1998b).

Conidial populations were quantifiedweekly by making impressions of eachmaterial evaluated, the conidia being

removed by pressing the agar surfaceagainst the lesion at leaf stage 4-5 of theyoungest leaf spotted. The cylinder ofcrystal violet agar and conidia was placedon a slide and transferred to a tray linedwith a paper towel moistened with sterilewater. The trays were covered with plas-tic bags and transferred to a hermeticpolystyrene container (icopor).

Both fungi were identified and theconidia/cm2 counted by means of a com-pound microscope (Olympus) with a 40 xobjective.

The variables analyzed were the num-bers of conidia/cm2 of P. fijiensis and P. musae, temperature (maxima, meansand minima), relative humidity andrainfall.

Each variable was subjected to analysisof variance, descriptive procedures formaximum, minimum and mean values,regression, Pearson’s correlation andChi-square test using the SAS (Statistical


Figure 2. Conidia of Pseudocercospora musae, anamorph of Mycosphaerella musicola.

Figure 1. Conidia of Paracercospora fijiensis, anamorph of Mycosphaerella fijiensis.

Analysis System) statistical programme(SAS Institute 1980). Conidial numberswere transformed with Lnx+1, which bestfits the behaviour of the data, where x isthe number of conidia/cm2.

Results and discussionAnalysis of variance of P. fijiensis and P. musae counts suggested highly signifi-cant differences for clones and dates ofevaluation. The interactions between the two factors were significant for P. fijiensis and highly significant for P. musae, indicating that a high or lowinoculum production depends on theplanting material and the effects of envi-ronmental conditions on the develop-ment of each material (Table 1).

The processes of infection and inocu-lum production were favoured by rainyperiods, and as rainfall increased so did the numbers of conidia shown by P. fijiensis and P. musae with two peri-ods of maximum conidial production at334 and 424 days after planting (dap).These coincided with the maximumrainfall recorded during the study, withan accumulated rainfall of 211.8 mmand 296.2 mm respectively (Figure 3A),which was in agreement with the stud-ies of Aguirre et al. (1998a). The authorsobserved that accumulated rainfall wasinversely related to the incubationperiod and development of black andyellow Sigatokas, and directly related tosporulation. This suggests that as weeklyaccumulated volume of rainfallincreases, the incubation period anddevelopment of both Sigatokas declinesand results in an increased diseaseseverity and hence greater inoculumproduction of the causal fungi. From 424 dap the relationship between coni-dial number and rainfall started todecline, being particularly evident at473 dap when, although there was anincreased rainfall (192.5 mm), conidialproduction was very low because foliagewas severely necrotic, and no healthytissue remained available for infection.

Temperature and relative humidityremained fairly constant with an aver-age of 21.5oC (Figure 3B) and 81% (figure 3C), conditions that are opti-mum for conidial production. In agree-ment with Mouliom Pefoura andMourichon (1990) and Tapia (1993),cited by Porras and Pérez (1997), tem-peratures higher than 20oC favour coni-dial development in P. fijiensis.According to Stover (1965), tempera-tures higher than 22oC favour conidialproduction in P. fijiensis, with a temper-ature of 26°C being optimum (Stover


Table 1. ANOVA components for the number of conidia of Paracercospora fijiensisand Pseudocercospora musae.

ANOVA P. fijiensisG.L. C.M. F Pr > F R2 C.V. (%)

Model 985 6.83 3.74 0.0237* 0.99** 35.8

Error 8 183

Clones 53 10.67 5.84 0.0061**

Dates of evaluation 18 56.59 30.97 0.0001**

Interaction of clone-date 859 5.53 3.03 0.0456*

Total 943

ANOVA P. musaeG.L. C.M. F Pr > F R2 C.V. (%)

Model 936 3.79 11.93 0.0004** 0.99** 23.31

Error 8 0.32

Clones 53 3.39 10.67 0.0007**

Dates of evaluation 18 40.50 127.67 0.0001**

Interaction of clone-date 860 3.04 9.59 0.0009**

Total 944* : Denotes significant differences, p = 5%

** : Denotes highly significant differences, p = 1%

Note: Data transformed as square root number of conidia.

Tem

per

atu

re (°C

)

334 361 389 424 452 473

23

22

22

21

21

20

20

B

Rel

ativ

e h

um

idit

y (%

)

334 361 389 424 452 473

69

72

75

78

81

84

Days after planting (dap)

C

Nu

mb

er o

f co

nid

ia

334 361 389 424 452 473

30000

24000

18000

12000

6000

0

350

300

250

200

150

100

50

0

Prec

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(mm

)

Precipitation

P. fijiensis

P. musae

A

Figure 3. Total conidia of P. fijiensis and P. musae in relation to time for 53 clones and their relationshipto climatic conditions. A. Rainfall, B. Temperature and C. Relative humidity.

1965). A relative humidity of about 100%favours production and viability ofspores, particularly when a water film ispresent on the leaf surface (Jacome andSchuh 1992).

Conidial populations of P. fijiensiswere always greater than those ofP. musae in a ratio of 2.3:1 (Table 2)hence confirming that black Sigatokatends to displace yellow Sigatokabecause of its greater aggressiveness;this is in agreement with the studies inthe same region by Aguirre et al.

(1998a) who demonstrated that blackSigatoka was more aggressive, occurringat times of the year when yellowSigatoka disappeared, tending to be dis-placed by black Sigatoka. In generalthere was a marked direct correlationbetween conidial numbers of P. fijiensisand P. musae and rainfall, which is alsoin agreement with the studies of Aguirreet al. (1998a) who observed that fluc-tuations in the numbers of conidiatrapped each week is highly correlatedwith rainfall.

At the time of flowering, which coin-cided with the start of the study, the stan-dard deviation remained high up to 452 dap, when buds had emerged frommost plants; the high standard deviationwas due mainly to the effects of rainfallon conidial production as mentioned forthe results where P. fijiensis had a higherfrequency than P. musae (Table 2).

From 319 to 347 dap the accumulatedrainfall was 242.5 mm, with a high fre-quency of conidia of both fungi. However,between 361and 404 dap, the accumu-lated rainfall of 378.6 mm was excessive,reducing the conidial populations of bothpathogens. It is noteworthy that at 438dap the highest correlation (r = 0.8575)occurred between conidial populationsand rainfall (Table 2).

Between 411 and 452 dap the majorityof leaf tissue started to become necrotic.During this period rainfall was 232 mmand conidial populations an average of 14 conidia/cm2/clone for P. fijiensis and4 conidia/cm2/clone for P. musae.

From 466 dap onwards, when the culti-vation cycle was complete, the standarddeviations were low because the majorityof clones were severely infected and no more leaf tissue was available forinfection, conidial populations of bothfungi were low, resulting in a low stan-dard deviation. At harvest, there were anaverage of 4 and 2 P. fijiensis and P. musae conidia/cm2/clone respectively.

The conidial populations of P. fijiensiswere always higher than those of P. musae,but populations of both fungi declined dueto a lack of healthy susceptible tissue(Figure 4).

Finally, the relationship observedbetween the two pathogens followed a regression between the numbers ofconidia/cm2 of P. fijiensis and P. musaein all clones evaluated. The correlationcoefficient was very low (r = 0.40656)(Figure 5), suggesting that the number ofconidia/cm2 of P. musae did not dependon the behaviour of P. fijiensis and viceversa, and that conidial production ineach of the clones depended on thesusceptibility of the material and envi-ronmental conditions, particularly rain-fall. In spite of the higher numbers of P. musae conidia, the numbers notalways increasing or decreasing, the coni-dia of P. musae followed the same pat-tern, but there was no direct or strongrelationship between inoculum produc-tion of the two pathogens.

In general, the clones evaluated pro-duced higher total numbers of conidia of P. fijiensis, confirming that blackSigatoka tends to displace yellowSigatoka (Table 3). ■


Table 3. Comparison of the mean number of conidia/cm2 of P. fijiensisand P. musae in clones evaluated in relation to rainfall.

Days after planting Mean number conidia Mean number conidia Rainfall (dap) of P. fijiensis of P. musae (mm)

319 45ª 37 81.60

326 42 17 34.40

334 26 12 95.80

340 32 13 9.70

347 23 13 21.00

361 13 6 44.00

375 31 9 51.80

389 11 3 109.10

404 22 8 173.70

411 15 5 42.20

418 16 4 25.30

424 14 5 55.00

438 14 2 51.90

452 10 2 57.60

466 5 2 103.20

473 4 1 89.30

493 3 0 220.80

Total 281 139 1266.00

Table 2. Mean conidia/cm2 with time for P. fijiensis and P. musae and their standarddeviations, and their agreement with rainfall (September 1998 – March 1999).

Days after Correlation Conidia Standard Conidia Standard Weekly planting (r) of deviation of deviation rainfall(dap) P. fijiensis P. musae (mm)

319 0.7782 ** 45 65.96 37 58.55 81.60

326 0.7466 ** 42 55.10 16 25.65 34.40

334 0.8464 ** 26 46.98 12 24.87 95.80

340 0.8115 ** 32 58.84 13 20.71 9.70

347 0.6622 ** 23 29.70 12 27.17 21.00

361 0.8557 ** 13 11.35 7 8.61 44.00

375 0.6407 ** 31 37.79 9 14.34 51.80

389 0.7373 ** 11 22.61 3 4.80 109.10

404 0.8552 ** 22 41.92 8 14.96 173.70

411 0.6903 ** 15 20.72 5 8.69 42.20

418 0.7697 ** 16 18.44 4 6.55 25.30

424 0.6966 ** 14 19.26 5 11.66 55.00

438 0.8575 ** 14 21.65 2 3.78 51.90

452 0.6361 ** 10 13.14 2 4.78 57.60

466 0.6028 ** 5 6.05 2 3.68 103.20

473 0.8074 ** 4 6.08 2 4.35 89.30

493 0.5474 ** 3 3.62 1 2.50 220.80

Mean 19 8 81.32** Correlations highly significant between the number of conidia of P. fijiensis and P. musae with time in relation to rainfall.

ReferencesAguirre M.C., J. Castaño-Zapata, J.A. Valencia,

L.E. Zuluaga & C. Arce. 1988a. Interacción deMycosphaerella fijiensis Morelet y M. musicola

Leach en siete genotipos de Musa spp. en un árealímite de expansión de la Sigatoka negra en lazona cafetera colombiana. Pp. 192-220 in

Memorias del Seminario Internacional sobreProducción de Plátano. Universidad del Quindío –Comité de Cafeteros del Quindío – SENA –INABAP – CORPOICA.

Aguirre M.C., J.Castaño-Zapata & L.E. Zuluaga.1998b. Método rápido de diagnóstico deMycosphaerella musicola Leach y M. fijiensis

Morelet, agentes causales de la Sigatoka amarillay Sigatoka negra. Agronomía 8(2): 26-30.

Belalcázar C.S., V.F. Salazar, M.J.A. Valencia, S.C.H. Silva, P.M.I. Arcila & R. Jaramillo. 1994.Reacción de variedades mejoradas de plátano al ataque de Sigatoka negra (Mycosphaerella

fijiensis Morelet). Pp. 192-214 in Mejoramiento

de la producción del cultivo del plátano. Segundo informe técnico 1984-1994. Región 9ICA-CORPOICA. Creced-Quindio, Armenia,Colombia.

Burt J.A., J. Rutter & H. González. 1997. Short dis-tance wind dispersal of the fungal pathogens caus-ing Sigatoka diseases in banana and plantain.Plant Pathology 46(4): 451-458.

Hernández G.J.C., J.A.C. Gómez & P.M.I. Arcila. 1994.Comportamiento agroeconómico de plántulas deplátano clon Dominico hartón Musa AABSimmonds, manejados bajo condiciones de almá-cigo. Pp. 41-54 in Mejoramiento de la produccióndel cultivo del plátano. Segundo informe técnico1984-1994. Región 9 ICA-CORPOICA. Creced-Quindio, Armenia, Colombia.

Jacome L.H. & W. Schuh. 1992. Effects of leaf wet-ness duration and temperature on development of black Sigatoka disease on banana infected by Mycosphaerella fijiensis var. difformis.Phytopathology 82(5): 515-520.

Merchán V.V.M. 1992. Informe de actividades conve-nio ICA-IRFA. Subdirección InvestigaciónEstratégica. CORPOICA Regional 9. Pp. 5-10.

Merchán V.V. M. 1998. Manejo de problemasfitosanitarios del cultivo del plátano en la zonacentral cafetera. Pp. 177-191 in Memorias delSeminario Internacional sobre Producción de Plátano. Universidad del Quindío – Comité de Cafeteros del Quindío – SENA – INIBAP – CORPOICA.

Porras A. & L. Pérez. 1997. The role of temperaturein the growth of the germ tubes of ascospores of Mycosphaerella spp., responsible for leaf spotdiseases of banana. INFOMUSA 6(2): 27-31.

SAS Institute Inc. 1980. The SAS applications guide.(K.A. Council, ed.). SAS Inst., North Carolina.195pp.

Stover R.H. 1965. Leaf spot of bananas caused by Mycosphaerella musicola: Effect of tempera-ture on germination, hyphal growth, and conidiaproduction. Tropical Agriculture (Trinidad) 42(4): 351-360.

Claudia Lorena Cardona-Sanchez is an undergrad-uate student, Programa de Agronomía, Facultad de Ciencias Agropecuarias, Universidad de Caldasand Jairo Castaño-Zapata is Associate Professor,Departamento de Fitotecnia, Faculdad de CienciasAgropecuarias, Universidad de Caldas, Apartado aéreo275, Manizales (Caldas), Colombia. Email: [email protected]

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Figure 4. Frequency of P. fijiensis and P. musae with time and the relationship with rainfall.

Figure 5. Relationship between the number of conidia of P. musae and P. fijiensis.


R. Sánchez Rodríguez, J.A. Pino Algora, C. Vallin Plous,

M.E. Pérez Rodríguez, Y. Iznaga Sosaand F. Malpartida Romero

The presence of black Sigatoka dis-ease (Mycosphaerella fijiensis) inCuba since 1990 has resulted in

increased costs of production in planta-tions of plantain and banana because ofthe increased frequency of aerial andground-based sprays to control the causalorganism. Thus, there is an urgent needto find alternatives with nationally pro-duced products in order to reduce thecosts of disease control.

The indiscriminate use of chemicalproducts has resulted in side effectsincluding, inter alia, the appearance offungicide resistance in the causal organ-ism, the formation of strains more viru-lent than indigenous strains and environ-mental contamination (Rodríguez andJiménez 1985, Fullerton and Olsen 1991,Mouliom Pefoura 1999).

The use of natural products obtainedfrom microorganisms presents consider-able advantages in comparison with com-mercial products, since their productionis much less damaging to the ecosystemand their in situ biodegradability resultsin compounds that are not toxic to theindigenous microflora. The search fornew and different products of natural ori-gin that do not contaminate the environ-ment, for the control of pests and dis-eases, is an important alternative forsustainable agriculture.

Product F20 comprises two antibiotics:the streptothricins B and F. These anti-biotics are produced mainly by micro-organisms of the genus Streptomyces.The structure has an aminosugar (glucosamine) joined to a ß-lysine pep-tide chain. Streptothricins F to A differ in the numbers of ß-lysine residues in the peptide chain, from 1-ß-lysine instreptothricin F to 6-ß-lysine in strep-tothricin A.

The physicochemical properties ofstreptothricins, their spectrum of antimi-crobial activity and toxicity are wellknown (Wienstein and Wagmans 1978).

There appear to be no publications onthe use of streptothricins for the controlof plant pathogens; specifically therehave been no reports up to the present onthe use of any antibiotic produced bymicroorganisms against diseases ofbanana and plantain.

In this work we demonstrate the possi-ble use of streptothricins for the controlof black Sigatoka disease in clones ofplantain (Musa AAB) cv. ‘CEMSA 3/4’ andof banana (Musa AAA) cv. ‘Parecido alRey’.

Material and methodsThe study was carried out at the Institutode Investigaciones en Viandas Tropicales(INIVIT). Product F20, whose main activeingredients are the streptothricins B andF, was obtained from the Centro deQuímica Farmacéutica (CQF) in collabo-ration with the Centro Nacional deBiotecnología de la Universidad NacionalAutónoma, Canto Blanco, Madrid, Spain,by fermentation of strains of Streptomyceslavendofoliae var. 383 (which producesstreptothricin B) and Streptomyces rocheivar. f20 (which produces streptothricin F),isolated from Cuban soils. The fermenta-tion broth was centrifuged, the super-natant submitted to chromatography onion exchange resin IRC-50, finally giving asolution saturated with sodium acetateafter elutriation with acetic acid.

The product was applied after the satu-rated sodium acetate solution containingthe antibiotics was dissolved in an aque-ous solution of 0.2 g/L commercial deter-gent as emulsifier and 60 ml/L mineraloil, to give a final concentration of 5-13 gstreptothricin/L, corresponding to a doseof 80-200 g streptothricin/ha. Applicationwas with a knapsack sprayer, with thespray lance modified to simulate an aer-ial spray of 12 L/h. The product wasapplied in weeks 8, 13, 17 and 22 to banana, and in weeks 5, 11 and 16 toplantain. Infected leaves were removedat 2 week-intervals for all treatments and mineral oil with 0.2 g/L commercialdetergent, as emulsifier, applied to the control treatment in order to ensuretheir maintenance for the whole of thebiological cycle.

Each clone separately, plantain‘CEMSA 3/4’ and banana ‘Parecido al Rey’,were arranged in an experimental designof randomized blocks with six plants perplot, and four replications.

The effect of F20 on black Sigatoka wascompared with the effect without phy-tosanitary treatment and with plotstreated chemically with propiconazole(Tilt 250 EC) at 400 ml/ha. F20 wasapplied at a similar dose to propiconazole.

The effect of the fungicides was deter-mined weekly by recording the develop-ment stage (DS) of the disease, theyoungest leaf with symptoms or streak-ing (YLSS), and the youngest leaf spot-ted (YLS) (Fouré 1982, Pérez 1996,Orjeda 1998).

Results and discussionSprays with F20 and propiconazole (Tilt)with mineral oil, resulted in a reductionin the disease index DS in comparisonwith untreated plots (UT) in clones‘Parecido al Rey’ and ‘CEMSA 3/4’.

Figure 1 shows the results of phyto-sanitary applications in both clones.Graphs A and B show the similar behaviour of F20 and Tilt with DS valuesnot significantly different (P>0.05), anda significant difference (P<0.01) of bothapplications in comparison with the control treatment.

The effects of disease control of theproducts were also evident in the graphsas a reduction in the numbers of oscilla-tions in DS values and by the amplitudeof their fluctuations in both clones. Forexample, when treated in week 5, DSvalues declined steadily from weeks 5and 10, from about DS = 3000 to aboutDS = 50 in clone ‘CEMSA 3/4’; the rate ofdisease development declined, whereasfor control plants, DS values variedbetween 1500 and 2500.

Analysis of the variable YLSS (Figure 2)showed no significant differences(P>0.05) between treatments with F20or Tilt. However, with clone ‘Parecido alRey’ and F20, minor disease symptoms,stage 1, were evident on leaf 9 (Pérez1996).

Figure 3 showed that YLS could reach avalue equal or greater than 9 before


Effects of the natural fungicide F20 on blackSigatoka disease (Mycosphaerella fijiensis Morelet)on plantain (AAB) and banana (AAA)

Diseases Control of black Sigatoka

flower initiation, thus confirming thatthere were no effects on weight or prema-ture fruit maturity in either clone; inCuba a strong negative correlationbetween the leaf area affected and the

YLS has been observed (Pérez et al. 1993,Pérez 1996).

All the data analyzed above suggest theuse of F20, produced from natural originsand mixed with mineral oil and commer-

cial detergent as an emulsion, for thecontrol of black Sigatoka disease inbanana and plantain crops. F20 is supe-rior to synthetic chemicals in terms of environmental effects. Attention is


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Figure 2. Youngest leaf with streak symptoms, (YLSS), weeks before flowering, after application of product to clones, A: ‘Parecido al Rey’ (AAA), and B: ‘CEMSA 3/4’ (AAB). UT: Control.

Figure 3. Youngest leaf spotted (YLS), weeks before flowering, after application of product in clones, A: Parecido al Rey’ (AAA), and B: ‘CEMSA 3/4’ (AAB).UT: Control.

drawn to the difference in behaviourbetween the two clones, with ‘CEMSA 3/4’having the higher infection. However, inorder to avoid the possibility of fungalresistance, it is important that this prod-uct should form part of an integratedcontrol programme in combination withother antifungal products (Pérez 1996,Romero 1997).

Conclusions• Product F20 showed no significant dif-

ferences in comparison with the com-mercial product Tilt in its effectivenessto control black Sigatoka.

• Maximum effectiveness of F20 wasobtained with an emulsion of mineraloil and commercial detergent, and theeffect was maintained for 3 or 4 weeksfrom the time of application.

• Application at doses of 80-200 g strep-tothricin/ha controlled black Sigatokain field plantations, irrespective of timeof the year. There are advantages incomparison with synthetic chemicalproducts because its biodegradabilityin situ results in compounds that arenot toxic to the indigenous microfloraand it has a lower toxicity. ■

ReferencesFouré E. 1982. Les cercosporioses du bananier et

leurs traitements : Etude de la sensibilité varié-tale des bananiers et des plantains à M. fjiensis

Morelet au Gabon. Fruits 37(12): 749-771.Fullerton R.A. & T. Olsen. 1991. Pathogenic vari-

ability in Mycosphaerella fijiensis Morelet. Pp. 105-114 in Banana diseases in Asia and thePacific. Proceedings of a Regional technicalmeeting on diseases affecting banana and plan-tain in Asia and Pacific. (R.V. Valmayor, B.E. Umali and C.P. Bejosano, eds). INIBAP-ASPNET Book Series No. 3.

Mouliom-Pefoura A. 1999. First observation of thebreakdown of high resistance in ‘Yangambí km 5’(Musa sp.) to the black leaf streak disease inCameroon. Plant Disease 83(1): 78.

Orjeda G. 1998. Evaluation of Musa germplasm forresistance to Sigatoka diseases and Fusarium

wilt. INIBAP Technical Guidelines No. 3. INIBAP,Montpellier, France.

Pérez L., A. Hernández & F. Mauri. 1993. Efficacy of a biological warning system for timing fungicide treatments for the control of blackSigatoka disease Mycosphaerella fijiensis

Morelet in banana plantations in Cuba. in Proceedings VI International Congress of Plant Pathology, 28/07-06/08/1993, Montreal,Canada.

Pérez L. 1996. Manual para manejo integrado de Sigatoka negra (Mycosphaerella fijiensis

Morelet) y Sigatoka amarilla (Mycospharella

musicola Leach ex Mulder) en banano y plátano.FAO, Representación de la FAO en Cuba, LaHabana. 54pp.

Romero R.A. 1997. Avances en epidemiología ymanejo de la Sigatoka negra del banano.Agronomía Costarricense 21(1): 77-81.

Rodríguez R. & L. Jiménez. 1985. El problema de la tolerancia de Mycosphaerella fijiensis al fungicida Benomil en plantaciones bananeras de Costa Rica. ASBANA. 16pp.

Wienstein M.J. & G.H. Wagman. 1978. Antibiotics.Isolation, separation and purification. Journal of Chromatography Library 15: 625-680.

R. Sánchez Rodríguez and J.A. Pino Algora work atthe Instituto de Investigaciones en Viandas Tropicales(INIVIT), Apdo. 6, Santo Domingo, Villa Clara, Cuba,CP 53000; C. Vallin Plous, M.E. Pérez Rodríguezand Y. Iznaga Sosa work at the Centro de QuímicaFarmacéutica (CQF), Calle 2000 y 21, Atabey 16042,Playa, Ciudad Habana, Cuba and F. MalpartidaRomero at the Centro Nacional de Biotecnología,Campus de la Universidad Nacional Autónoma, 28049Canto Blanco, Madrid, Spain. Author for correspon-dence: Robersy Sánchez, E-mail: [email protected]


Seasonal fluctuations of Radopholus similis andPratylenchus coffeae in certain cultivars of banana

Pests Nematode species in southern India

P. Sundararaju

The lesion-producing nematodessuch as Radopholus similis andPratylenchus coffeae are consid-

ered to be the economically importantnematode pests of banana and arewidely distributed in South India (Koshyet al. 1978, Rajendran et al. 1979). Theburrowing nematode, Radopholus sim-ilis enjoys wide geographical distribu-tion in the tropical and subtropicalbanana-growing regions of the world. InIndia, the first occurrence of the nema-tode was reported on banana fromPalghat District of Kerala (Nair et al.1966), causing up to 41% yield losses.Subsequently this nematode wasreported from banana in South India(Koshy et al. 1978), Gujarat (Sethi et al.1981), Maharashtra (Darekar et al.1981), Madhya Pradesh (Tiwari et al.2000), Goa (Koshy and Sosamma 1988),

Lakshadweep Islands (Sundararaju1990), Manipur (Anandi and Dhanchand1992), Orissa (Mohanty et al. 1992),Tripura (Mukherjee et al. 1994) andBihar, Uttar Pradesh and Nagaland(Khan 1999).

The root-lesion nematode, Pratylenchuscoffeae is reported to have spread to differ-ent banana-growing regions through theinfested corm. In India, the nematode isknown to occur on plantain (AAB) inSouth India, Gujarat, Orissa, Bihar andAssam (Sundararaju 1996). P. thornei, theother important species was found toinfest banana plants from Assam only(Choudhury and Phukan 1990).

Crop losses caused by nematodes tobananas are very high, with averageannual yield losses estimated at about20% worldwide (Sasser and Freckman1987). Soil temperature at a depth of 30 cm did not influence population size(Jimenez 1972). The populations werefound to fluctuate between samples,

trees, months and years. However, therewere definite periods for occurrence ofmaximum and minimum populationswithin a year. An extensive survey carriedout by Sundararaju (1996) from differentbanana-growing regions in the countryindicated the presence of 17 genera ofplant parasitic nematodes. Among them,the lesion nematodes, Radopholus similis and Pratylenchus coffeae are thepredominant species found to occur indifferent cultivars of banana in variousintensities. The burrowing nematode-infested field exhibited severe root rot-ting, resulting in serious economic losses.Yield was reduced up to 25-35% in theburrowing nematode-infested field com-pared to nematode-free plantations. Croplosses due to the root-lesion nematode,Pratylenchus coffeae in banana cv.Nendran were reported to be 25.4%(Sundararaju et al. 1999). Therefore,studies were initiated to determine theseasonal fluctuations of these nematode

populations in different cultivars ofbanana roots by periodic sampling ofnematode-infested banana plants at theNational Research Centre for Banana(NRCB) Farm. The main objective of thestudy was to find out the activity peaks, interms of highest and lowest populationsof these nematode parasites, in the rhi-zosphere so that while devising the man-agement schedules, the findings of thework could be considered.

Materials and methodsIn order to study the population fluctua-tion of lesion nematodes, the threebanana cultivars Kalyan bale (AB),Alukkal (ABB) and Kalibow (AAB), whichare highly susceptible to R. similis, and avariety Nendran (ABB), which is highlysusceptible to P. coffeae, were selected atNRCB Farm, Podhavur, Trichy, TamilNadu. The nematode-infested field wasselected for this study and the testedbanana cultivars were grown in the fieldand alluvial soil condition. Samples ofboth soil (250 cc) and root (10 g) werecollected from the base of the motherplants at monthly interval from the 5th

month up to harvesting stage during1997-98. Tender, main feeder roots ofwhite to creamy-white colour, with reddish-brown cortical lesions, were col-lected from the base of the plants. Carewas also taken to collect only the above-mentioned type of roots known to har-bour the maximum number of lesionnematodes. Root samples washed thor-oughly and cut into 2-2.5 cm pieces andlater sliced into 8 longitudinal pieceswere left in 15 cm-Petri dishes containing150 ml of tap water for 72 hrs at 10-14°Cin a refrigerator for nematode extraction(Koshy et al. 1975). Soil samples wereprocessed as per Cobb’s sieving methodfollowed by the modified Baermann’s fun-nel method for estimation of nematodepopulations. Soil temperature at 15 cmdepth was recorded from the fields dailyat 7 am. The mean temperature, soilmoisture and data on cumulative rainfallwere correlated with the nematode popu-lation density in the sample.

Results and discussionIt is seen from Figure 1 that a drasticincrease of R. similis population wasnoticed in all three cultivars during themonths of November to April; it laterdecreased to a negligible level from May to October which was in agreementwith Shafice and Mendez (1975). It isinteresting to observe that the maximumnematode population was recorded in April from all the cultivars: Kalayanbale (86/g root), Alukkal (78/g root) and

Kalibow (68/g root), and the minimumin July in cv. Kalyan bale (20/g root).Analysis of the soil samples also revealedthe same trend as in the case of rootsamples with maximum population

occuring during the month of Novemberto April with maximum rainfall and soilmoisture during the period. In the caseof P. coffeae on variety Nendran the maxi-mum population was recorded from


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Figure 3. Total monthly rainfall, mean temperature, soil moisture and relative humidity at NRCB Farm,Podhavur, during the experimental period.

Figure 2. Population fluctuation of Pratylenchus coffeae in roots of banana (cv. Nendran).

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October to December and the minimumpopulation from May to August (Figure2). Regarding the average population fora month, it is seen that a maximum of 92 per gram root was recorded inDecember, whereas it was only 23 pergram in June. The root-lesion nematodepopulation from soil samples alsoshowed the same trend as in the case ofroot samples with maximum populationduring the months October-Decemberand minimum population during themonths May-August. Peak rainfalloccurred during the Northeast monsoon(September to December) with an averagerainfall of 140 mm. The soil temperatureat 15 cm depth recorded from fields var-ied from 18-37.5°C. Analysis of the mois-ture content revealed that it was maxi-mal in those months where maximumnematode populations were recorded(Figure 3). Rainfall also influences thegrowth of roots. Thus, with the increasein the availability of root system, therewas an increase in the activity of R. similis during November to Apriland of P. coffeae during October toDecember.

Fluctuations in the populations ofPratylenchus spp. were correlated withrainfall (Cooke and Draycott 1971). Thebehaviour of P. coffeae in relation to soiltemperature and rainfall was similar to that of P. crenatus and P. penetransin corn (Miller et al. 1972). Lack of mois-ture coupled with high summer tempera-ture during April to August was found tobe unfavourable for the prevalence of P. coffeae in oil palm (Sundararaju andRatnakaran, in press). The present inves-tigation is in agreement with Kumar(1984) who reported that higher popula-tion of P. coffeae was recorded during themonth of October to December which isthe period of high rainfall and increasedroot activity in coffee plants.

Similar observations were reported inthe burrowing nematode R. similis oncitrus (DuCharme and Suit 1967), banana(Vilardebo 1976), coconut and arecanut(Koshy and Sosamma 1978).

Figure 1 indicates that the R. similispopulation fluctuates between themonths. A steady increase of R. similispopulation was recorded during themonths of November-January and grad-ual decrease was recorded in Februaryand March, whereas a drastic increase ofnematode population was recorded inApril in cv. Kalyan bale (Figure 1). A sim-ilar trend was noticed in cvs. Alukkal andKalibow (Figure 1).

In the case of P. coffeae a steadyincrease of nematode population wasrecorded from September to December

and gradually decreased from January to June (Figure 2).

This clearly shows that the populationbuild-up of R. similis and P. coffeae wouldgreatly vary depending upon the seasonand other ecological conditions such asrainfall, soil temperature, soil moistureand availability of susceptible rootswhich play their own roles in the popula-tion build-up.

AcknowledgementThe author is thankful to Dr H.P. Singh,former Director, NRCB, Trichy for provid-ing necessary facilities. Technical assis-tance given by Mr T. Sekar is dulyacknowledged. This research work wascarried out within the framework of theNRCB research programmes. ■

ReferencesAnandi Y. & D. Dhandachand. 1992. Nematodes of

banana plantation in Imphal district, Manipur.Curr. Nematol. 3: 153-58.

Choudhury B.N. & P.N. Phukan. 1990. Distributionand occurrence of certain plant parasitic nema-todes in different cultivars of banana. Curr.Nematol. 1: 153-156.

Cooke D.A. & Draycott. 1971. The effect of soil fumi-gation and nitrogen fertilization on nematodes in sugar-beet in sandy soils. Ann. Appl. Biol. 65:253-254.

DuCharme E.P. & R.F. Suit. 1967. Population fluctua-tion of burrowing nematodes in Florida citrusgroves. Proc. Fla. St. Hort. Soc. 80: 63-67.

Jiménez M.F. 1972. Seasonal fluctuations ofRadopholus similis in the banana growing area ofPococo, Costa Rica. Nematropica 2: 6.

Khan R.M. 1999. Distribution of Radopholus similis

in India, its spread in new regions and an analysisof the nematofauna of banana crop pathosystem.Nematol. Medit. 27: 239-245.

Koshy P.K. & V.K Sosamma. 1978. Studies on the population fluctuations of Radopholus similis

in coconut and arecanut roots. Indian Phytopath.31: 180-183.

Koshy P.K. & V.K Sosamma. 1988. Occurrence of theburrowing nematode Radopholus similis in thestate of Goa. Indian J. Nematol. 18: 130.

Koshy P.K., V.K Sosamma & C.P. Radhakrishnan Nair.1975. Preliminary studies on Radopholus similis

(Cobb, 1893) Thorne, 1949 infesting coconut andarecanut palms in South India. Indian J. Nematol.5: 26-35.

Koshy P.K., P. Sundararaju & V.K Sosamma. 1978.Occurrence and distribution of Radopholus sim-

ilis (Cobb, 1893) Thorne, 1949 in South India.Indian J. Nematol. 8: 49-58.

Kumar A.C. 1984. Investigations on Cannoncadodieback in coffee. J. Coffee Res. 14: 85-103.

Miller R.E., C.W. Bothroyd &W.F. Mai. 1972. Plantparasitic nematodes associated with corn roots inNew York. Phytopath. 52: 22 (Abstr.).

Mohanty K., N.K. Sahoo & S. Ray. 1992. Occurrence ofRadopholus similis (Cobb, 1893) Tgirbe 1949 on

banana and pepper in wide areas of Orissa, India.Afro Asian Nematol. Network. 1: 25-26.

Mukherjee B., R.C. Nath & M.K. Dasgupta. 1994. New record on the occurrence of burrowing nema-tode, Radopholus similis (Cobb, 1893) Thorne,1949 on banana in Tripura. Indian J. Ent. 28: 553-554.

Nair M.R.G.K., M.N. Das & M.R. Menon. 1966. On theoccurrence of the burrowing nematodeRadopholus similis (Cobb, 1893) Thorne, 1949 on banana in Kerala. Indian J.Ent. 28: 553-554.

Rajendran G., T.N. Naganathan & S. Vadivelu. 1979.Studies on banana nematodes. Indian J. Nematol.9: 54.

Sasser J. N & D.W. Freckman. 1987. A world perspec-tive on nematology: the role of the society. Pp. 7-14 in Vistas on Nematology (J.A. Veech and D.W.Dickson, eds). Society of Nematologist Inc.,Hyattsville, USA.

Sethi C.L., Siyandand & N. Srivastava. 1981.Occurrence of Radopholus similis (Cobb, 1893)Thorne, 1949 in Gujarat. Indian J. Nematol. 11: 116.

Shafice M. F. & J.M. Mendez. 1975. Seasonal fluctua-tions in Radopholus similis on three varieties of Musa species. Universidad de la Habana, Serie11, Sanidad vegetal No. 12. 12pp.

Sundararaju P. 1990. Occurrence of the burrowingnematode, Radopholus similis in the UnionTerritory of Lakshadweep Island. Indian J.Nematol. 18: 112.

Sundararaju P. 1996. Nematode pests of banana andtheir management. Pp. 17-19 in Souvenir,“Conference on challenges for banana productionand utilisation in 21st Century” held at Trichy on 24-25 September 1996.

Sundararaju P. & K. Ratnakaran. In press. Factorsinfluencing the prevalence of the root-lesionnematode Pratylenchus coffeae on oil palm(Elaeis guineensis Jacq.). Indian J. Nematol.

Sundararaju P., B. Padmanaban & S. Sathiamoorthy.1999. Control of root-lesion nematodePratylenchus coffeae in certain cultivars ofbanana. Pp. 46-47 in National Seminar onNematological Research in India held at C.S. AzadUniversity of Agri. & Tech. 17 December 1999(Abstract).

Tiwari S.P., I. Vadhera & G.S. Dave. 2000. Burrowingnematode Radopholus similis associated withbanana crop in Madhya Pradesh. Indian J.Nematol. 30(1)38: 41.

Vilardebo A. 1976. Population dynamics of Radopholus

similis in relation to climate factors and the physiology of the plant. Nematropica 6: 54-55.

The author works at the Crop Protection NationalResearch Centre for Banana, 44 Ramalinga NagarSouth Vayalur Road,Tiruchirapalli – 620 017, India.


Duong Thi Minh Nguyet, A. Elsen, Nguyen Thi Tuyet

and D. De Waele

Plant parasitic nematodes are amajor constraint of banana produc-tion worldwide (Gowen and

Quénéhervé 1990). Nematode infectioncan interfere with nutrient and wateruptake and transportation, resulting in slow growth, reduced fruit filling andsensitivity to wind lodging. Among the nematodes attacking banana,Radopholus similis (Cobb) Thorne isconsidered the most destructive species(Sarah et al. 1996).

The possibilities of controlling nema-todes in bananas are limited becausebananas are usually grown as a perma-nent crop by small-scale farmers andsources of resistance have proved hard tofind. Resistance to R. similis has beenreported in ‘Pisang Jari Buaya’ (MusaAA—Pisang Jari Buaya group) and‘Yangambi Km5’ (Musa AAA—Ibotagroup) (Pinochet 1988, Viaene et al. 1998,Fogain and Gowen 1998, Stoffelen 2000).The clone ‘SH-3142’ derived from a geno-type belonging to the Pisang Jari Buayagroup and ‘SH-1734’ was found to behighly resistant to R. similis (Pinochetand Rowe 1979, Pinochet 1988).Moreover, some ‘Pisang Jari Buaya’expressed favourable agronomic featuressimilar to those of commercial banana.

The Mysore banana (Musa AAB) is avery popular and delicious dessert.Information on resistance and/or toler-ance to R. similis of Mysore bananas isscarce. When testing 17 AAB Musa geno-types, Fogain (1996) reported that noneof the plants were immune, including‘Pisang Ceylan’, the only cultivar belong-ing to the Mysore group. The objective ofour study was to further investigate thehost plant response of Musa genotypesfrom the Pisang Jari Buaya and Mysoregroups to a R. similis population fromCosta Rica, to find additional sources ofresistance to the burrowing nematode.

Throughout the study, the terminologyof Bos and Parlevliet (1995) concerningresistance and susceptibility of hostplants to pathogens and the methodologyfor nematode resistance screening

in Musa as described by Speijer and De Waele (1997) were used.


Preparation of banana plantsThirteen diploid (AA) banana genotypesbelonging to the Pisang Jari Buaya group(Experiments 1 and 2, see Tables 1 andTable 2) and five triploid (AAB) bananagenotypes from the Mysore group(Experiment 3, see Table 3) wereincluded in the study. Two triploid (MusaAAA) bananas, ‘Grande Naine’ and‘Yangambi Km5’, were included as refer-ence genotypes because of their high sus-ceptibility and resistance to R. similis,respectively. The Musa genotypes used inthe experiments were provided by theINIBAP Transit Centre (ITC) at theCatholic University of Leuven. After pro-liferation, regeneration and rooting(Banerjee and De Langhe 1985), each in vitro propagated banana plantlet with3-4 leaves and 5-6 roots was transplantedin a 1-litre (12 cm-diameter) plastic potcontaining about 1000 cm3 autoclavedsubstrate of peat and quarts (2:1). Tokeep a high humidity, the pots wereplaced under a plastic cover, which wasslightly opened after 2 weeks andremoved after 4 weeks. The greenhouseconditions were maintained at 25-30°Cand 70-80% relative humidity with a 12-hour photoperiod. The pots were irri-gated as needed and fertilized with ahydroponics solution (Swennen et al.1986) every 3 weeks after nematode inoc-ulation. The plants were inoculated withnematodes either 4 weeks after plantingfor the Pisang Jari Buaya group, or 8 weeks after planting for the Mysoregroup, since the number of nematodeswas too low in the experiment withMysore genotypes.

Preparation of nematode inoculumThe R. similis population used in theexperiments was obtained from infectedbanana roots of ‘Valery’ (Musa AAA) atTalamanca in Costa Rica. The populationwas reared monoxenically on carrot discsand incubated at 28°C in the dark for sev-eral generations (Moody et al. 1973,Pinochet et al. 1995). The carrot discswere blended twice for 10 s (with 5 s

interval) and poured through 106 and 25 µm pore sieves. Carrot tissue collectedon the 106 µm pore sieve was discarded,while the nematodes were collected fromthe 25 µm pore sieve.

A suspension of 1000 living vermiformnematodes was poured in three holesmade in the substrate around the base ofeach plant. After inoculation, the holeswere covered.

Host plant response observationsEight weeks after inoculation, the plantswere harvested to observe the responseof the different banana genotypes to R. similis. The following data wererecorded:

Root necrosis percentage The procedure followed was thatdescribed by Speijer and De Waele(1997). Five 10 cm-pieces of functionalprimary roots were collected and slicedlongitudinally. The percentage of rootcortex showing necrosis was scored for ahalf of each root. The maximum rootnecrosis per root half is 20%, giving a maximum root necrosis of 100% for the five root-halves together.

Nematode population densitiesThe entire root system, including the 5 roots segments observed for necrosis,was weighed and cut into 2 cm-pieces.Fifteen grams of fresh roots were takenrandomly and macerated three times for10 s with 5 s intervals. The mixture waspoured through a series of 250-106-40 µmpore sieves and the sieves were rinsedwith tap water. Nematodes remaining onthe 40 µm pore sieve were collected in abeaker with distilled water. Nematodeswere counted in 6 ml aliquots of eachsample using a binocular microscope.

Experimental design and data analysisThree experiments were conducted,based on a completely randomizeddesign, with either eight replicates foreach genotype (Pisang Jari Buaya group,Experiment 1, Table 1; Mysore group,Experiment 3, Table 3) or nine repli-cates (Pisang Jari Buaya group,Experiment 2, Table 2). Prior to statis-tical analysis, the percentage of rootnecrosis was transformed to arcsin


Host plant response of Pisang Jari Buaya and Mysore bananas to Radopholus similis

Pests Nematode resistance

(x/100) and the nematode numberswere converted to log10 (x+1). All datawere subjected to analysis of variance(ANOVA) and means of the parameterswere compared using the Tukey HSDtest at P ≤ 0.05.

Results and discussionsThe results obtained from the Pisang JariBuaya group are presented in Tables 1

and 2. In Table 1, no significant differ-ences were observed in nematode num-bers per root system or per 1 g fresh rootsand root necrosis percentage betweenthe Pisang Jari Buaya genotypes and ‘Grande Naine’. Among the Pisang Jari Buaya genotypes, root necrosis per-centage was significantly higher in‘Morong Princessa’ compared with‘Pisang Tunjiuk’ and ‘Saing Todloh’. In

Table 2, reproduction of R. similis wasobserved in all the genotypes tested. Ingeneral, the nematode populations col-lected from roots of the Pisang Jari Buayagenotypes, including Pisang Jari Buayaaccession ITC0312, were not significantlydifferent from those recovered from‘Grande Naine’ but significantly highercompared to ‘Yangambi Km5’ and ‘SH-3142’. Only for ‘Pisang Sipulu’ was thenumber of nematodes per 1 g fresh rootsnot significantly different from ‘YangambiKm5’. The lowest nematode numberswere recorded on ‘SH-3142’ and‘Yangambi Km5’. The root necrosis per-centages of all Pisang Jari Buaya geno-types, ‘Yangambi Km5’ and ‘SH-3142’were significantly lower compared with‘Grande Naine’.

These results show that all Pisang JariBuaya genotypes tested are as suscepti-ble to R. similis as ‘Grande Naine’. Theyconfirm a previous report (Wehunt et al.1978) that ‘Pisang Jari Buaya’, ‘GabahGabah’, ‘Pisang Sipulu’ and ‘Pisang GigiBuaya’ are significantly less sensitive toroot damage (expressed as root necrosispercentage) compared with ‘GrandeNaine’. Surprisingly, ‘Pisang Jari Buaya’,which has previously been confirmedresistant to R. similis (Pinochet 1988,Viaene et al. 1998, Fogain and Gowen1998, Stoffelen 2000), did not appear tobe so in our study. Also, ‘Pisang Sipulu’,considered a promising banana genotypebecause less susceptible to R. similis(Wehunt et al. 1978, Binks and Gowen1996), did not show resistance either to R. similis in our study.

The results obtained from the Mysoregroup are presented in Table 3. Thenematode numbers per root system andper 1 g fresh roots of ‘Gorolo’ and ‘LadyFinger’ (South Johnstone) were signifi-cantly lower compared with ‘GrandeNaine’, while those recovered from theother Mysore genotypes were not signifi-cantly different compared with the ref-erence genotype. The root necrosis per-centage observed in ‘Thap Maeo’ and‘Gorolo’ was significantly lower com-pared with ‘Grande Naine’. In contrast,the root necrosis percentages of ‘Pisang Ceylan’, ‘Lady Finger’ (SouthJohnstone) and ‘Lady Finger’ (Nelson)did not differ significantly from those in‘Grande Naine’.

According to Price (1994) and Priceand McLaren (1995), AAB Musa geno-types are susceptible to R. similis whenexamined in field trials. Unfortunately,genotypes of the Mysore group were notincluded in their trials. Our study con-firms previous reports (Stanton 1994,Fogain et al. 1996) that ‘Lady Finger’


Table 1. Reproduction of Radopholus similis (Costa Rica population) on 8 diploid(Musa AA) banana genotypes belonging to the Pisang Jari Buaya group and on the reference genotype ‘Grande Naine’ measured 8 weeks after inoculation with 1000 vermiform nematodes per plant.

Musa genotype Genome ITC number Fresh root Root necrosis Nematodes Nematodes weight (%) per 1 g per root

(g) fresh roots system

Huwundu AA 0308 35.3 22.4 ab 1050 a 36188 a

Morong Datu AA 0309 41.0 14.5 ab 851 a 33526 a

Morong Princessa AA 0310 29.9 30.5 b 972 a 26022 a

Pisang Rotan AA 0313 41.1 16.6 ab 794 a 29033 a

Pisang Tunjiuk AA 0315 44.2 7.9 a 255 a 10770 a

Saing Todloh AA 0316 36.0 6.8 a 297 a 10038 a

Unnamed AA 0318 43.2 11.3 ab 442 a 17151 a

Umbarim AA 0317 32.6 17.1 ab 495 a 16030 a

Grande Naine AAA 1256 52.0 9.3 ab 528 a 23052 aITC= INIBAP Transit Centre.

Original data are presented, but data of nematode numbers were transformed to log10 (x+1) and data of root necrosispercentage were converted to arcsin (x/100) for statistical analysis. Means in the same column followed by the same letter are not significantly different (P ≤ 0.05) according to the Tukey HSD test.

Table 2. Reproduction of Radopholus similis (Costa Rica population) on 5 Pisang Jari Buaya genotypes, Yangambi Km5 and on the reference genotype‘Grande Naine’ measured 8 weeks after inoculation with 1000 vermiform nematodes per plant.



Gabah Gabah AA 0307 74.4 11 a 588 c 44571 b

Pisang Gigi Buaya AA 0310 64.0 8.4 a 741 c 45914 b

Pisang Jari Buaya AA 0312 54.9 11.9 a 1053 c 56541 b

SH-3142 AA 0425 52.7 8.9 a 108 a 5941 a

Pisang Sipulu AA 1308 60.6 8.9 a 579 bc 34355 b

Yangambi Km5 AAA 1123 57.2 7.8 a 120 ab 6384 a

Grande Naine AAA 1256 43.9 25 b 2041 c 87763 bITC= INIBAP Transit Centre.

See note in Table 1.

Table 3. Reproduction of Radopholus similis (Costa Rica population) on 5 triploid(Musa AAB) banana genotypes belonging to the Mysore group and on the reference genotype ‘Grande Naine’ measured 8 weeks after inoculation with 1000 vermiform nematodes per plant.



Thap Maeo AAB 1301 101.3 17.3 a 852 ab 82849 abc

Gorolo AAB 0723 45.6 22.8 ab 579 a 32562 a

Pisang Ceylan AAB 0650 58.3 29.6 abc 804 ab 46827 abc

Lady Finger(South Johnstone) AAB 0583 51.8 36.9 c 679 a 38616 ab

Lady Finger (Nelson) AAB 0582 87.8 33.1 bc 1128 ab 99009 bc

Grande Naine AAA 1256 83.9 42.5 c 1552 b 127439 cITC= INIBAP Transit Centre.

See note in Table 1.

(Nelson), ‘Lady Finger’ (South Johnstone)and ‘Pisang Ceylan’ are susceptible to R. similis.

AcknowledgementsThe authors would like to thank the INIBAP Transit Centre (ITC) at theCatholic University of Leuven for supply-ing the Musa genotypes and the equip-ment for completing this research. TheFlemish Interuniversity Council (VL.I.R.)is gratefully acknowledged for fundingscholarships for Ms Duong Thi MinhNguyet and Ms Nguyen Thi Tuyet to com-plete this study as part of their MSc thesis in the Postgraduate InternationalNematology Course. ■

ReferencesBanerjee N. & E. De Langhe. 1985. A tissue culture

technique for rapid clonal propagation and storageunder minimal growth conditions of Musa (bananaand plantain). Plant Cell Reports 4: 351-354.

Binks R.H. & S.R. Gowen. 1996. Field evaluation of nematode infestations in Musa germplasm at FHIA (La Lima, Honduras). INFOMUSA 5(2):15-17.

Bos L. & J.E. Parlevliet. 1995. Concept and terminol-ogy on plant/pest relationships: toward consensusin plant pathology and crop protection. AnnualReview of Phytopathology 33: 69-102.

Fogain R. 1996. Screenhouse evaluation of Musa forsusceptibility to Radopholus similis: evaluationof plantains AAB and diploid AA, AB and BB. Pp. 79-86 in Proceedings of a workshop on Newfrontiers in resistance breeding for nematode,Fusarium and Sigatoka, 2-5 Oct. 1995, KualaLumpur, Malaysia (E.A. Frison, J.P. Horry & D. De Waele, eds). INIBAP, Montpellier, France.

Fogain R. & S.R. Gowen. 1998. ‘Yangambi Km5’(Musa AAA, Ibota subgroup): a possible source of resistance to Radopholus similis andPratylenchus goodeyi. Fundamental and AppliedNematology 21: 75-80.

Fogain R., S.R. Gowen & F. Mekemda. 1996.Screening for susceptibility to Radopholus

similis: evaluation of plantain AAB and diploidAA, AB and BB. Tropical Agriculture, Trinidad73: 281-285.

Gowen S.R. & P. Quénéhervé. 1990. Nematode para-sites of bananas, plantain and abaca. Pp. 431-460in Plant parasitic nematodes in subtropical andtropical agriculture (M. Luc, R.A. Sikora & J. Bridge, eds.). CAB International, Wallingford,United Kingdom.

Moody E.H., B.F. Lownsberry & J.M. Ahmed. 1973.Culture of the root-lesion nematode Pratylenchus

vulnus on carrot disks. Journal of Nematology 5: 225-226.

Pinochet J. 1988. Nematodes problem in Musa spp.:pathotypes of R. similis and breeding for resis-tance. Pp. 66-70 in Proceeding of a workshop onNematodes and the borer weevil in bananas: pre-sent status of research and outlook, 7-11December 1987, Bujumbura, Burundi. INIBAP,Montpellier, France.

Pinochet J. & P.R. Rowe. 1979. Progress in breedingfor resistance to Radopholus similis on bananas.Nematropica 9: 76-78.

Pinochet J., C. Fernandez & J.L. Sarah. 1995.Influence of temperature on in vitro reproductionof Pratylenchus coffeae, P. goodeyi, andRadopholus similis. Fundamental and AppliedNematology 18: 391-392.

Price N.S. 1994. Field trial evaluation of nematodesusceptibility within Musa. Fundamental andApplied Nematology 17: 391-396.

Price N.S. & C.G. McLaren. 1995. Technique for fieldscreening of Musa germplasm. Pp. 87-105 in

Proceedings of a workshop on New frontiers inresistance breeding for nematode, Fusarium andSigatoka, 2-5 Oct. 1995, Kuala Lumpur, Malaysia(E.A. Frison, J.P. Horry & D. De Waele, eds). INIBAP, Montpellier, France.

Sarah J.L., J. Pinochet & J. Stanton. 1996. The bur-rowing nematode of bananas, Radopholus similis

Cobb, 1913. Musa Pest Fact Sheets (1). INIBAP,Montpellier, France. 2pp.

Stanton J.M. 1994. Status of nematode and weevilborer problems affecting banana in Australia. Pp.48-56 in Banana nematodes and weevil borers inAsia and the Pacific. Proceedings of a conference-workshop on Nematodes and weevil borers affect-ing bananas in Asia and the Pacific, 18-22 April1994, Serdang, Selangor, Malaysia (R.V. Valmayor,R.G. Davide, J.M. Stanton, N.L. Treverrow & V.N.Roa, eds). INIBAP/ASPNET, Los Baños,Philippines. ASPNET Book Series No. 5.

Speijer P.R. & D. De Waele. 1997. Screening of Musa

germplasm for resistance and tolerance to nema-todes. INIBAP Technical Guidelines (1). INIBAP,Montpellier, France. 42pp.

Stoffelen R. 2000. Early screening of Eumusa andAustralimusa bananas against root-lesion androot-knot nematodes. Doctoral thesis, UniversiteitK.U. Leuven, Belgium.

Swennen R., E. De Langhe, J. Janssen & D. Decoene.1986. Study of the root development of some Musa

cultivars in hydroponics. Fruits 41: 515-524.Viaene N., J. Duenas & D. De Waele. 1998. Screening

for resistance and tolerance to Radopholus

similis and Pratylenchus coffeae in banana andplantain [Abstract]. P. 125 in Programme andabstracts of the 24th International Symposium ofthe European Society of Nematologists, 5-8 Aug.1998, Dundee, Scotland (D. Brown, ed.).

Wehunt E.J., D.J. Hutchinson & D.I. Edwards. 1978.Reaction of banana cultivars to the burrowingnematode (Radopholus similis). Journal ofNematology 10: 368-370.

Duong Thi Minh Nguyet and Nguyen Thi Tuyetwork at the Vietnam Agricultural Science Institute(VASI), Van Dien, Thanh Tri, Hanoi, Vietnam. Tel: (84)4 861 43 25, Fax: (84) 4 861 71 67. Annemie Elsenand Dirk De Waele work at the Laboratory of TropicalCrop Improvement, Catholic University of Leuven,Kasteelpark Arenberg 13, 3001 Heverlee, Belgium.Tel: (32) 16 32 96 03, Fax: (32) 16 32 19 93.Corresponding author: e-mail:[email protected] or [email protected]


Effect of three arbuscular mycorrhizal fungi on root-knot nematode (Meloidogyne spp.)infection of Musa

Pests Nematode control

A. Elsen, S. Declerck and D. De Waele

Arbuscular mycorrhizal (AM) fungiare obligate symbionts of plantsthat biotrophically colonize the

root cortex and develop an extramatricalmycelium which helps the plant acquire

water and mineral nutrients from thesoil. AM fungi also may protect plantsagainst soil-borne plant pathogens,including nematodes. Several studieshave addressed the associations betweenAM fungi and root-knot nematodes,which are considered the most importantnematodes in the western hemisphere

on temperate agricultural crops. Many mycorrhizal associations are reported tohave a suppressive effect over sedentaryendoparasitic nematodes. In some cropsthis effect is significant enough to con-sider mycorrhizal infection as a more orless effective means of biological control(Pinochet et al. 1996).

In bananas, only a few studies were car-ried out on the effects of AM fungi onnematode development. Radopholussimilis populations in the roots as well asin the soil were suppressed in mycor-rhizal plants compared to non-mycor-rhizal plants (Umesh et al. 1988). Underin vitro conditions, using Ri T-DNA trans-formed Daucus carota roots, a R. similispopulation was suppressed with 50% inthe presence of AMF (Elsen et al. 2001).Pinochet et al. (1997) reported that mycorrhizal colonization did not effectnematode build-up in the roots, althoughplants infected with both Meloidogynejavanica and Glomus intraradices weremore galled.

In this experiment, three Glomusspecies (G. mosseae, G. macrocarpumand G. caledonium) were tested on theMusa cultivar Williams (ITC0570) fortheir effect on Meloidogyne javanica, aroot-knot nematode population isolatedfrom banana in Morocco. Tissue-culturederived plantlets were acclimatized in 1-litre pots filled with sterilized soil inthe greenhouse. During transplant theplantlets of the mycorrhizal treatmentwere mycorrhized with soil inoculum,consisting of ± 1850 spores and 0.25 gmycorrhized roots from Allium porrum.After one month, the plants were inocu-lated with a mixture of 5000 M. javanicajuveniles and eggs. The experiment wasplanned as a 4 x 2 randomly factorialdesign with 8 replicates per treatment:AM fungi (- AM, G. mosseae, G. macro-carpum and G. caledonium) x M. javan-ica (+ M. javanica, - M javanica). Threemonths after planting, the ‘Williams’plants were harvested and assessed formycorrhizal colonization and nematodedamage/development. A sub-sample ofthe roots was stained with 0.05% trypanblue in lactic acid (Koske and Gemma1989), in order to determine the mycor-rhizal colonization. The galls on the rootswere counted in a 5 g sub-sample afterstaining with phloxine B (Hadisoegandaand Sasser 1982).

Effect of the AM fungi on plantgrowthThe AM fungi had no effect on the plantgrowth since shoot weight, shoot diame-ter, plant height and root weight did notdiffer among the treatments (data notshown). In general, mycorrhization ofbanana plants resulted in a better plantgrowth compared to non-mycorrhizalplants (Declerck et al. 1994, 1995).Although, in some cases, it has beenobserved that the establishment of thesymbiosis resulted in a negative or neu-

tral effect on plant growth as long as themycorrhizal colonization was not welldeveloped (Jakobsen 1998). Thereforeat the time of harvest, the root coloniza-tion by the three Glomus strains testedwas relatively low. This may partlyexplain why in this experiment no effecton plant growth was observed. In addi-tion, it is important to note the differ-ences in colonization among the Glomusspecies in the plants without nematodes.Higher colonization was observed withG. mosseae as compared to G. caledo-nium and G. macrocarpum. Such dif-ferences were also reported in literature(Declerck et al. 1994, 1995). Glomusmosseae was shown the more infectiveon ‘Williams’ and other cultivars, as com-pared to G. macrocarpum (Declercket al. 1995).

Effect of AM fungi on nematodereproductionGlomus caledonium and G. macro-carpum significantly reduced galling inthe roots, while for G. mosseae thisreducing effect was not significant(Table 1). In literature, results are con-tradictory: according to Pinochet et al.(1997), Glomus intraradices did notreduce nematode build-up of M. javan-ica and resulted in more galled rootscompared to non-mycorrhized roots. Incontrast, G. mosseae suppressed rootgalling and nematode build up ofMeloidogyne incognita (Jaizme-Vegaet al. 1997).

Effect of M. javanica onmycorrhizal developmentMeloidogyne javanica significantlydecreased the intraradical developmentof G. mosseae. For G. macrocarpum andG. caledonium no such effect wasobserved: the presence or absence of theroot-knot nematode had no effect oninternal root colonization. In similarexperiments, root-knot nematodes had

no effect on the percentage root coloniza-tion in mycorrhizal plants (Pinochet et al.1997, Jaizme-Vega et al. 1997).

ConclusionThe results of this experiment suggest asuppressive effect of the three Glomusstrains studied over the rook-not nema-tode M. javanica. Mechanisms involvedin nematode suppression are still a matter of speculation. However somemajor factors are likely to be involved:enhanced nutrient status of the plant,biochemical changes in plant tissue(increase in chitinase, amino acids, per-oxidase and phytoalexins), anatomicalchanges (increased lignification), stressalleviation, microbial changes in the rhi-zosphere and induced changes to rootmorphology (increased branching,larger proportion of higher order roots)(Hooker et al. 1994). Further study is needed to confirm the suppressiveeffect of the AM fungi over the root-knotnematodes and to reveal the mecha-nisms involved. ■

ReferencesDeclerck S., B. Devos, B. Delvaux & C. Plenchette.

1994. Growth response of micropropagated plantsto VAM inoculation. Fruits 49: 103-109.

Declerck S., C. Plenchette & D.G. Strullu. 1995.Mycorrhizal dependency of banana (Musa

acuminata AAA group) cultivar. Plant and Soil176: 183-187.

Elsen A., S. Declerck & D. De Waele. 2001. Effects ofGlomus intraradices on the reproduction of theburrowing nematode (Radopholus similis) in dix-enic culture. Mycorrhiza 11: 49-51.

Hadisoeganda W.W. & J.N. Sasser. 1982. Resistanceof tomato, bean, southern pea and garden pea cul-tivars to root-knot nematodes based on host suit-ability. Plant Disease 66: 145-150.

Hooker J.E., M.C. Jaizme-Vega & D. Atkinson. 1994.Biocontrol of plant pathogens using arbuscularmycorrhizal fungi. Pp. 191-200 in Impact ofarbuscular mycorrhizas on sustainable agricul-


Table 1. Mycorrhization and effect of mycorrhization on reaction of “Williams”roots to infection with Meloidogyne javanica.

% mycorrhized root tissue Galls / 5 g roots

- AM – M. javanica / /

- AM + M. javanica / 41 ± 12 b

G. mosseae – M. javanica 29 ± 10 b /

G. mosseae + M. javanica 23 ± 12 a 29 ± 16 ab

G. macrocarpum – M. javanica 14 ± 3 a /

G. macrocarpum + M. javanica 15 ± 5 a 25 ± 17 a

G. caledonium – M. javanica 22 ± 5 a /

G. caledonium + M. javanica 16 ± 6 a 16 ± 8 aData are means of 8 replications. Means in the same columns followed by the same letter do not differ according Tukey’smultiple range test (P ≤ 0.05).

ture and natural ecosystems (S. Gianinazzi &H. Schüepp, eds). Birkhäuser Verlag, Basel,Switzerland.

Jaizme-Vega M.C., P. Tenoury, J. Pinochet &M. Jaumot. 1997. Interactions between the root-knot nematode Meloidogyne incognita andGlomus mosseae in banana. Plant and Soil 196:27-35.

Jakobsen L. 1998. Transport of phosphorus and car-bon in arbuscular mycorrhizas. Pp. 305-332 in

Mycorrhiza structure, function, molecular biologyand biotechnology (A. Varma & J.E. Hooker, eds).Springer-Verlag, New York, USA.

Koske R.E. & J.N. Gemma. 1989. A modified proce-dure for staining roots to detect VA mycorrhizas.Mycological Research 92: 486-488.

Pinochet J., C. Calvet, A. Camprubi & C. Fernandez.1996. Interaction between migratory endopara-sitic nematodes and arbuscular mycorrhizalfungi in perennial crops. Plant and Soil 185: 183-190.

Pinochet J., C. Fernandez, M. Jaizme & P. Tenoury.1997. Micropropagated banana infected withMeloidogyne javanica responds to Glomus

intraradices and phosphorus. Hortscience 32:101-103.

Umesh K.C., K. Krishnappa & D.J. Bagyaraj. 1988.Interaction of burrowing nematode, Radopholus

similis (Cobb, 1893) Thorne 199, and VA mycor-rhiza, Glomus fasciculatum (THAXT) Gerd. andTrappe in banana (Musa acuminata colla.).Indian Journal of Nematology 18: 6-11.

Annemie Elsen and Dirk De Waele work at theLaboratory of Tropical Crop Improvement, KULeuvenKasteelpark Arenberg 13, 3001 Leuven, Belgium;Stéphane Declerck works at the Mycothèque del’Université catholique de Louvain (MUCL). Unité demicrobiologie, Place Croix du Sud 3, 1348 Louvain-la-Neuve, Belgium.


A study of endophytic fungal species associatedwith root necrosis of banana in banana and plantain plantations in Cuba

Agronomy Pests and pathogens of roots

A. Battle-Viera and L. Pérez-Vicente

In Cuba a total of 108 700 ha are cul-tivated with Musaceae, 32 800 hawith cultivars of banana subgroup

Cavendish (AAA), 13 800 ha with plan-tain (AAB) and 62 000 ha with varietiesof type Burro/Bluggoe (ABB). Of the32 800 ha of banana plantations,13 800 ha are cultivated with localizedmicrojet irrigation systems, and theymust therefore remain in place for the next five years. However there isinterest in replanting these areas withtetraploid hybrids developed by theFondación Hondureña de InvestigaciónAgrícola (FHIA), and which are resis-tant to pests and diseases.

The nematode species most common-ly found in our plantations are Radopholus similis, Pratylenchus coffeae, Helycotylenchus multicinctus,Meloidogyne spp. and Rotylenchulusreniformis, the first three being the mostimportant in Cuba (Pérez et al. 1984).The pathogenicity of nematodes has usu-ally been established as the populationdensity found in the roots. However, evi-dence on the relationship of populationdensity of nematodes to damage in thecrop is contradictory. Over the last yearsplant lodging and root necrosis in Cubahave been recorded where low popula-tions of nematodes were very low.

Interactions at the root level betweenR. similis and species of fungi belonging

to the genera Cylindrocladium andAcremonium, which contribute to orincrease damage by the nematode, arewell documented (Booth and Stover 1981,Loridat 1989, Sarah 1990). Such associa-tions have been found in the majority ofsoils infested with nematodes in some ofthe Antilles islands. In Cuba there havebeen no studies to investigate and quan-tify such relationships at the level of theroot. However the relationship betweennematode populations, root damage andplant development is not strong.

The purpose of the present study wasto identify the species of fungi associ-ated with root necrosis of differentclones of banana and plantain in planta-tions in Cuba.

Material and methodsSamples were taken from banana plan-tations located in the provinces of Pinardel Río, La Habana, Matanzas, VillaClara, Ciego de Avila, Camagüey,Cienfuegos, Santiago de Cuba andGuantánamo.

Samples were taken from necroticroots of plants of Gran enano (AAA), GrosMichel (AAA), CEMSA 3/4 (AAB) andBurro CEMSA/ Bluggoe (ABB), some ofthem associated with plants that hadfallen over (lodged) apparently as aresult of nematode attack. From eachfield 10 plants were selected at random,holes 20 x 20 x 20 cm excavated 10 cmfrom the pseudostem and five affectedroots removed.

The roots were washed and necroticfragments, typical of R. similis attack,disinfected in 1% hypochlorite for twominutes and cultured on water-agar sup-plemented with 50 µg/ml streptomycin.Blocks of agar and fungal growth weretransferred to tubes of PDA, and incu-bated when the fungal species were readyfor identification. Fusarium specieswere identified according to the key ofBooth (1981). Cylindrocarpon specieswere identified according to the keys ofthe CMI edited by CAB.

The relative frequency of each speciespresent at each site was determined inrelation to the total numbers of isolatesobtained from the different sites.

Results and discussionA total of 59 isolates of endophytic fungiwere obtained from the tissue of rootsapparently necrotic as a result of R. similis. The species are described inTable 1.

Species of Cylindrocarpon musae andFusarium oxysporum Schlect. were iso-lated from almost all samples from all thesites. F. oxysporum was the most fre-quently isolated species (45.6% total iso-lates), followed by C. musae (19.2% totalisolates). F. equiseti (Corda) Sacc. wasalso isolated but with less frequency. Theresults were similar to those ofPocasangre (2000) who found thatFusarium species were predominant insoils from Cuba, Costa Rica, Guatemalaand Honduras.

Booth and Stover (1871) reported thepresence of C. musae associated withroot necrosis in banana in Costa Rica,however the fungus did not have the par-asitic capacity to cause lesions on healthyroots. Other species of Cylindrocarponare important pathogens of plants. Forexample C. destructans causes rootnecrosis and death in pine (Pinus sp.)(Chakravarty and Unestam 1987).

Recently there have been bioassays ofartificial inoculation of C. musae alone orof C. musae in co-inoculation with R. sim-ilis. The studies provided importantinformation on the effects of the patho-genicity of these species on root necrosisin the crop.

None of the sites yielded species ofCylindrocladium or Zythia as reportedby Loridat (1989), Mourichon (1993)and Risède (1994). These species havebeen associated with necrosis of bananain Martinique and Guadeloupe, and lat-terly in Cameroon (Abadie 1998, pers.comm.) and Côte d’Ivoire (Kobenan1991).

Conclusions1. F. oxysporum and C. musae were the

species most frequently associatedwith necrosis caused by nematodes inplantations of banana and plantain indifferent parts of Cuba. F. equiseti andRhizoctonia spp. were found less fre-quently.

2. F. oxysporum was the most frequentisolate accounting for 45.6% total iso-lates, followed by C. musae.


Figure 1. Roots of banana with necrosis causedby nematode attack..

Figure 2. Cylindrocarpon musae. Macroconidiaand chlamydospores.

Table 1. Species of endophytic fungi associated with roots of banana and plantainin plantations on different locations, Cuba.

Isolate Species Clone Location

1.1 F. equiseti (Corda) Sacc. Gran enano UBPC 14 La Cuba, Ciego de Avila

1.2 F. oxysporum Schlecht. Gran enano UBPC 14 La Cuba, Ciego de Avila

1.5 C. musae B. & St. Gran enano UBPC 14 La Cuba, Ciego de Avila



2.3 Unidentified dark colonies Gran enano UBPC 1 La Cuba, Ciego de Avila



3.2 F. equiseti (Corda) Sacc. Gran enano UBPC 7 La Cuba, Ciego de Avila



5.1 Unidentified dark colonies Gran enano Sola, Camagüey

5.2 C. musae B. & St. Gran enano Sola, Camagüey

5.5 Unidentified Monilia Gran enano Sola, Camagüey

6.1 F. equiseti (Corda) Sacc. Gran enano La Esperanza, Quemado de Güines, Villa Clara

6.3 C. musae B. & St. Gran enano La Esperanza, Quemado de Güines, Villa Clara

6.5 F. oxysporum Schlecht. Gran enano La Esperanza, Quemado de Güines, Villa Clara

7.1 F. semitectum Gran enano Margarita, Quemado de Güines, Villa Clara

7.2 C. musae B. & St. Gran enano Margarita, Quemado de Güines, Villa Clara

8.1 C. musae B. & St Parecido al Rey Lutgardita, Quemado de Güines, Villa Clara

8.2 Basidiocarp Parecido al Rey Lutgardita, Quemado de Güines, Villa Clara

9.1 Unidentified dark colonies Gran enano Güines, Quemado de Güines, Villa Clara

9.2 F. oxysporum Schlecht. Gran enano Güines, Quemado de Güines, Villa Clara

9.3 Unidentified dark colonies Gran enano Güines, Quemado de Güines, Villa Clara

9.4 Unidentified Monilia Gran enano Güines, Quemado de Güines, Villa Clara

10.1 F. oxysporum Schlecht. Gran enano Horquita, Cuban 11, Cienfuegos

10.2 F. oxysporum Schlecht. Gran enano Horquita, Cuban 11, Cienfuegos

11.1 F. oxysporum Schlecht. FHIA-03 Lenin, Matanzas

11.2 C. musae B. & St. FHIA-03 Lenin, Matanzas

11.3 F. oxysporum Schlecht. FHIA-03 Lenin, Matanzas

12.2 Unidentified dark colonies Parecido al Rey Lenin, Campo 48, Matanzas

13.1 Unidentified dark colonies Gran enano Lenin, Campo 52, Matanzas

13.2 F. oxysporum Schlecht. Gran enano Lenin, Campo 52, Matanzas

14.1 F. equiseti (Corda) Sacc. Gran enano Pinar del Río, Vitroplantas

15.2 F. oxysporum Schlecht. Robusta La Maya, Santiago de Cuba

15.3 F. oxysporum Schlecht. Robusta La Maya, Santiago de Cuba

15.4 C. musae B. & St. Robusta La Maya, Santiago de Cuba

16.1 F. oxysporum Schlecht. Gros Michel La Ciénaga, Baracoa, Guantánamo

16.2 F. oxysporum Schlecht. Gros Michel La Ciénaga, Baracoa, Guantánamo

17.1 F. oxysporum Schlecht. Gran enano Imías, Guantánamo

17.2 Unidentified dark colonies Gran enano Imías, Guantánamo

17.3 F. oxysporum Schlecht. Gran enano Imías, Guantánamo

18.1 F. oxysporum Schlecht. Gros Michel Vega del Jobo, Imías, Guantánamo

18.2 F. oxysporum Schlecht. Gros Michel Vega del Jobo, Imías, Guantánamo

18.3 F. oxysporum Schlecht. Gros Michel Vega del Jobo, Imias, Guantánamo

19 F. oxysporum Schlecht. Burro Palma Soriano, Santiago de Cuba

20 Fusarium oxysporum Schlecht. Burro Antero Regalado, Artemisa, La Habana

21 Rhizoctonia sp. Burro UBPC Emilio Hernández, Artemisa

22 F. oxysporum Schlecht. Burro CPA Niceto Pérez, Güira La Habana

24 Rhizoctonia sp. Burro Ojo de Agua, San Antonio, La Habana

25 F. oxysporum Schlecht. Pelipita CPA Niceto Pérez, Güira La Habana

26.1 Rhizoctonia sp. FHIA La Palma, Alquízar, La Habana

26.2 F. oxysporum Schlecht. FHIA La Palma, Alquízar, La Habana

27 C. musae B. & St. Burro Rpto. Hnos. Cruz, Pinar del Río

28 Unidentified dark fungus Burro San Juan y Martínez, Pinar del Río

29 C. musae B. & St. Cavendish enano Coifa, Boyeros, La Habana

30 Unidentified fungus Burro CEMSA CCS Pedro Lantigua, Bauta, La Habana

31 F. oxysporum Schlecht. Consejo de Estado, Plaza, La Habana

32 F. oxysporum Schlecht. Robusta Fca. Govín, Caimito, La Habana

Cylindrocladium and Zythia spp.,reported in other countries to be associ-ated with root necrosis, were neverfound.

ReferencesBooth C. 1981. The genus Fusarium. CAB, Farnham

Royal, England, 237pp. Booth C. & R.H. Stover. 1971. Cylindrocarpon musae

sp. nov., commonly associated with burrowingnematode (Radopholus similis) lesions onbanana. Trans. British Mycol. Soc. 63: 503-507.

Chakravarty P. & T. Unestam. 1987. Mycorrhizal fungiprevent disease in stressed pine seedlings.Journal of Phytopathology 118(4): 335-340.

Kobenan K. 1991. Parasites du système racinairedes bananiers en Côte d’Ivoire. Fruits 46(6): 633-641.

Loridat P. 1989. Etude de la microflore fongique etdes nématodes associés aux nécroses de l’appareilsouterrain du bananier en Martinique. Mise enévidence du pouvoir pathogène du genreCylindrocladium. Fruits 44(11): 587-598.

Mourichon X. 1993. Parasites fongiques du bananier.Fruits 48(1): 26-28.

Pérez J., O. García & E. Fernández. 1984.Distribución de los principales nematodos parási-tos del plátano en Cuba. Ciencia y Técnica en laAgricultura, Serie Protección de Plantas. 7(1): 27-58.

Pocasangre L. 2000. Biological enhancement ofbanana tissue culture plantlets with endophyticfungi for the control of the burrowing nematodeRadopholus similis and Panama disease(Fusarium oxysporum f. sp. cubense). PhDThesis, Bonn University. 95pp.

Risède J.M. 1994. Eléments de caractérisation deCylindrocladium sp. agent des nécroses raci-naires du bananier en Martinique. Fruits 49(3):167-178.

Sarah J.L. 1990. Les nématodes et le parasitisme desracines de bananiers. Fruits. (Numéro SpécialBananes): 60-67.

Alicia Battle-Viera and Luis Pérez-Vicente work atthe Instituto de Investigaciones de Sanidad Vegetal(INISAV), Gaveta 634, Zona Postal 13, Playa, Ciudadde la Habana 11300, Cuba.


Effects of mycorrhization on the development of two cultivars of micropropagated banana

Agronomy Effets of micorrhizae

M.C. Jaizme-Vega, M. Esquivel Delamo,

P. Tenoury Domínguez and A.S. Rodríguez Romero

The likelihood of using arbuscularmycorrhiza (AM) in crop produc-tion systems is increasingly more

realistic and studies have increased con-siderably in the last few years.

Banana (Musa AAA) in its early stagesof development is readily colonized bymycorrhiza and is moderately (40-50%)dependent on them (Jaizme-Vega et al.1998). Mycorrhization in vivo hasresulted in large increases in the growthand nutrition of this species (Lin andChang 1987, Rizzardi 1990, Declerk et al.1995, Jaizme-Vega and Azcón 1995)including in the presence of standardfertilization regimes in commercial nurs-eries (Tenoury 1996, Sosa Hernández1997), with favourable effects on plantbehaviour when confronted with varioussoil-borne pathogens such as Meloidogyneincognita (Jaizme-Vega et al. 1997),Pratylenchus goodeyi (Jaizme-Vega andPinochet 1997) and Fusarium oxyspo-rum f.sp. cubense (Jaizme-Vega et al.1998). These results demonstrate theadvantages of applying inoculum of fun-gal AMs during root production andacclimatization of micropropagated

banana plants, which gives rise to plantsthat are well developed and have anincreased tolerance to attack by soil-borne pathogens. However, at presentthere is no information on the effects ofsuch symbiotic fungi on the bananaplant during the later stages of develop-ment and with fertilizer regimes similarto those practised in commercial crops.

Therefore, the sequential effects ofearly mycorrhization on the growth ofmicropropagated banana plants werestudied from the earliest stages of devel-opment until nine months after trans-planting to the field in microplots.


Host plantMicropropagated material of the twomost widespread commercial cultivars ofbanana Musa acuminata Colla AAA, cvs.‘Grande naine’ and ‘Gruesa’ (a localselection of ‘Dwarf Cavendish’) was used.

Rooting stage

Inoculation with AM fungiMycorrhization was done during harden-ing off. Inoculum comprised a homoge-neous mixture of rhizosphere soil, sporesand rootlets of the host plant.

Each cultivar was inoculated with one oftwo AM fungi, each with 1500 g inoculum

per tray (capacity of tray 24 kg) with thefollowing isolates:• Glomus intraradices Schenck &

Smith, from stock collection, multi-plied on sorghum, and giving 68% colo-nization;

• Glomus manihotis Howeler, Sieverdingand Schenck, from stock collection,multiplied on tomato, and giving 70%colonization.At inoculation, plants were 10 cm

± 2 cm and had approximately threedeveloped leaves. Inoculation was in poly-ethylene (PE) trays (40 x 60 cm, H x L),each tray containing one cultivar/funguscombination with an additional two con-trol trays with non-inoculated plants, onetray per cultivar. Thus there was a total ofsix trays each with 35 plants.

The substrate comprised a steam-ster-ilized mixture of dark-coloured volcanicsoil and amended peat (TKS1®, Instant,Floragard, GmbH) in a proportion of5:2:1. This phase lasted six weeks in aglasshouse and under a tunnel of blackmesh for acclimatization. Irrigation waswith distilled water according to theneeds of the plants.

Nursery phaseAt the end of root production and beforetransplanting to individual containers,10 plants of each treatment/cultivar com-bination were selected and the effects

evaluated of inoculation with mycorrhizaon plant development, the mycorrhizaldependency under the fixed conditions offertilizer inputs, and the extent of colo-nization by the AM fungi.

Parameters relevant to the growth ofthe plant in general were evaluated ateach stage of the investigation as follows:fresh weight (g) of roots and aerial parts,dry weight (g) of aerial parts, length anddiameter (cm) of pseudostem, leaf num-bers and area (cm2). Leaf area was calcu-lated with an area meter Li-COR, inc.Lincoln, Nebraska, USA, model Li-3100.

The relative mycorrhizal dependency(RMD), defined by Gerdeman (1975) asthe degree of mycorrhization needed byplants to produce the maximum growthor yield depending on fertility of the soil,was calculated according to the formulaproposed by Plenchette et al. (1983) asthe numerical expression of this concept:

RMD = DW of plant with AM–DW of plant without AM

x 100DW of plants without AM

(DW : dry weight)

Infection by the mycorrhiza was con-firmed by observation with a light micro-scope. Root samples were bleached with10% KOH and then stained with 0.05% try-pan blue in lactic acid as described byPhillips and Hayman (1970) and modifiedby Koske and Gemma (1989). Percentageroot colonization was determined on 201-cm sections of stained root, mountedon slides and examined with a lightmicroscope as described by Brundettet al. (1985).

Once the determinations were com-plete 20 plants of each treatment weretransferred to 2 L PE bags containing asubstrate comprising equal volumes

(1:1:1) of steam sterilized soil (‘picón’)and enriched peat (TKS1®). This phasetook 14 weeks in glasshouse conditions attemperatures of 27-32°C, and a relativehumidity of 70-80%.

Fertilization was according to the fer-tilizer regime of a commercial banananursery. Plants were fertilized twiceweekly (100 cc/plant) on alternate days.One of the fertilizer applications was with(NO3)2Ca (3 g/L) and NO3H (0.4 cc/L),and the other application was with SO4K2(3 g/L) and PO4H3 (0.2 cc/L). The days onwhich fertilizer was not applied alter-nated with irrigation with running wateraccording to the needs of the crop. Plantsreceived a weekly foliar application ofmicronutrients consisting of 3% Wuxal®Super AA 8-8-6 (Argos Shering, Agrevo,S.A., Valencia, Spain).

Microplot phaseAfter growth for 3.5 months, plants weretransferred to larger containers, andburied in a plot within the boundaries ofthe ICIA estate situated 300 masl. Thesite was chosen on the basis of aspect, cli-matic conditions and as an area marginalfor this crop. Prior to this, and as with thefirst transplanting, 10 plants per cultivarand treatment were evaluated for theeffects of the AM fungi, that is the extentof root infection by mycorrhiza and myc-orrhizal dependency.

For this last phase of the trial, PE pots35 cm diameter and 50 L volume wereselected and filled with non-sterilizedmedium of the same materials and in thesame proportions as described for theprevious transplanting (1:1:1), andamended with 1.5 g/L of slow release fer-tilizer (Osmocote 17:10:10, Scotts,

O.M. Tarragona). Once in position in theirnew pots (10 per cultivar and treatment),the plants were placed amongst othersimilarly sized pots previously buried upto the upper edge of pot, in the trial plot.Plants were fertilized weekly (1 L/plant),via the localized irrigation system, withthe two combinations of fertilizer treat-ment described previously for bananaplants after the first transplanting. Foliarfertilizers were applied fortnightly. Thedays on which fertilizers were notapplied, plants were irrigated accordingto the needs of the plants.

Plants remained in position for ninemonths. The trial was then terminatedand the effects of symbiosis on develop-ment of the banana plants evaluated.

The following experimental variableswere studied: fresh weight of roots andaerial parts, numbers of suckers, num-bers of leaves, leaf area, N, P and K con-tent, and dependency for mycorrhiza.

On completion of the foliar analyses,the samples were transferred to a heaterfor 24 hours at 70°C after which nitrogen,phosphorous and potassium contentswere determined. For N determination,the sample was mineralized “via humidprocess”, P was determined colourimetri-cally and K by spectrophotometry ofatomic absorption.

Data were analyzed by means ofANOVA (Systat). Means were comparedby Fisher’s test of least significant differ-ences (LSD) using the statistical pack-age Systat version 5.0 (SPSS Inc.,Chicago, USA).

Results and discussionBy completion of the rooting stage, bothcultivars showed a positive response to


Figure 1. The different development phases of the banana plant in the trial.

Inoculum of AM fungi

RECORDING OF DATA RECORDING OF DATA RECORDING OF DATA

“In vitro”Rooting phase

(6 weeks)Nursery phase

(14 weeks)Microplots phase

(9 months)

Commercial fertilization

the two AM fungi used for inoculation(Tables 1a and 2a). In this phase, the rel-ative mycorrhizal dependency (RMD) ofboth cultivars to Glomus manihotis andGlomus intraradices were the highestthroughout the trial and were 35% and50% respectively. In this first phase thepercentage colonization by the two inoc-ulated AM fungi was similar for the twocultivars.

Following transplanting, the positiveeffect of the AM fungi on plant develop-ment was maintained for 3.5 monthsafter mycorrhization. For inoculatedplants of both cultivars, the majority ofexperimental variables were signifi-cantly different for both cultivars in com-parison with the controls (Tables 1b and2b). The development of RMD was simi-lar for both cultivars completing this

phase of the trial with averages of 40%for both AM fungi on ‘Grande naine’, and30% and 20% respectively for Glomusmanihotis and Glomus intraradices onGruesa (Tables 1b and 2b).

Root colonization of banana plants bymycorrhizae tended to differ dependingon the cultivar. Thus, roots of ‘Grandenaine’ inoculated with G. manihotis hadtwice the mycorrhiza infection in com-parison with the beginning of the study,similar results being maintained on rootscolonized by G. intraradices. Howeverwith plants of cv. ‘Gruesa’ no changes inroot colonization were observed in com-parison to the first transplanting. Duringthe trial, from 14 weeks onwards 15% rootinfection by contaminant AM fungi wasnoted in control plants of both cultivars(Tables 1b and 2b) but without signifi-

cant effects on plant development. Theseendophytes are able to disperse in irriga-tion water or by uncontrolled contamina-tion in the nursery containing the plants.

These data confirm those already pub-lished on the benefits of early mycor-rhization of plants in the first phases ofdevelopment of this crop (Declerck et al.1995, Tenoury 1996, Sosa-Hernández1997, Jaizme-Vega et al. 1997, 1998).

The results of the second phase of thetrial in which the effects of the AM fungion mycorrhiza-treated plants in thein vivo phase for three months and trans-planted to non-sterile medium, showedthat after nine months in microplot con-ditions and a standard fertilizer regime,banana plants inoculated withG. intraradices usually, particularly withcv. ‘Gruesa’, showed a beneficial effect of


Table 1. Effect of Glomus manihotis and G. intraradices on the development, colonization and mycorrhizal dependency of micropropagated banana cv. ‘Grande naine’ at a) 6 weeks after inoculation, b) 14 weeks after inoculation and c) 9 monthsafter transplanting to microplots.

Fresh weight (g) Dry weight (g) Pseudostem Leaf area Colonization RMD**

Root Aerial parts Aerial parts Diameter Length No. leaves (cm2) (%)

a) 6 weeks after inoculation (rooting phase)

Control 2.6 b* 8.6 b 0.5 b 0.9 b 10.4 b 5.2 b 143 b — —

G. manihotis 6.4 a 17.5 a 1.1 a 1.2 a 12.9 a 6.3 a 261 a 26 51

G. intraradices 5.5 a 17.8 a 1.0 a 1.2 a 12.1 a 6.0 a 269 a 37 46

b) 14 weeks after inoculation (nursery phase)

Control 13.1 b* 38.1 b 2.6 b 1.8 b 15.4 b 7.3 b 494 b 15 —



Fresh weight (g) No. leaves No. suckers Leaf area Colonization RMD Macronutrient content

Root Aerial parts (cm2) (%) (%) N P K

c) 9 months after transplanting to microplots

Control 5.3 b* 9.2 a 14.0 a 3.7 ab 50192 a 59 — 2.89 a 0.185 a 2.52 a

G. manihotis 6.8 ab 6.9 a 14.3 a 2.2 b 44256 a 71 5 2.99 a 0.180 a 2.80 a

G. intraradices 9.8 a 10.0 a 13.7 a 4.5 a 55774 a 74 8 2.71 a 0.183 a 2.41 a* Means of 10 replicates. Within each column, differences between numbers followed by the same letter are not statistically different by Fisher’s test (P ≤ 0.05).

** RMD: relative mycorrhizal dependency.

Table 2. Effect of Glomus manihotis and G. intraradices on the development, colonization and mycorrhizal dependency of micropropagated banana cv. ‘Gruesa’ at: a) 6 weeks after inoculation, b) 14 weeks after inoculation, and c) 9 months aftertransplanting to microplots.

Fresh weight (g) Dry weight (g) Pseudostem Leaf area Colonization RMD**

Root Aerial parts Aerial parts Diameter Length No. leaves (cm2) (%)

a) 6 weeks after inoculation (rooting phase)

Control 3.1 b* 8.2 b 0.50 b 0.97 b 8.7 b 5.5 a 155 b – –

G. manihotis 5.5 a 12.9 a 0.80 a 1.15 a 8.7 b 6.3 a 223 a 27 38


b) 14 weeks after inoculation (nursery phase)

Control 22.8 b* 40.5 b 2.8 b 2.0 b 14.7 b 7.7 a 514 b 14 –


G. intraradices 36.4 a 50.7 a 3.5 ab 2.4 a 15.9 ab 8.0 a 662 a 30 19

Fresh weight (g) No. leaves No. suckers Leaf area Colonization RMD Macronutrient content

Root Aerial parts (cm2) (%) (%) N P K

c) 9 months after transplanting to microplots

Control 7.1 a* 7.8 a 14.9 b 2.4 a 41845 b 59 – 2.84 a 0.176 a 2.65 a

G. manihotis 7.7 a 11.5 ab 19.1 ab 3.6 a 57733 ab 72 31 3.03 a 0.189 a 3.00 a

G. intraradices 9.5 a 13.6 a 23.2 a 4.0 a 61660 a 83 42 3.00 a 0.184 a 3.03 a* Means of 10 replicates. Within each column, differences between numbers followed by the same letter are not statistically different with Fisher’s test (P ≤ 0.05).

** RMD: relative mycorrhizal dependency.

symbiosis on plant development, withRMDs of approximately 40%. These valuesare considered to be relatively high forthe conditions of the trial (Tables 1c and2c); moreover there was an increase inthe other experimental variables.However, data on macronutrients (N, Pand K) although noticeably higher, didnot differ statistically (Tables 1c and 2c).This lack of response in nutrient contentof aerial parts can be interpreted as typi-cal for a mycorrhiza-treated plant receiv-ing soluble fertilizer.

Plants of cultivar ‘Grande naine’ showeda smaller response to the AM fungi afterthe microplot phase, plants inoculatedwith G. manihotis showing a develop-ment and nutritional state equal orslightly less than control plants.

At the end of this phase, root coloniza-tion by both Glomus species was rela-tively important in both cultivars (greaterthan 79%). Attention is drawn to the highlevel of colonization of roots of controlplants. This part of the trial used non-sterilized substrate which, together withother conditions in the trial, explainedthe data.

In conclusion, in general and particu-larly in the last phase of the trial, we canconfirm that, at the later stages of thecrop, this biotechnological resourceshowed promise for the improvement ofproduction.

AcknowledgementsThe authors thank Ana Rosa SocorroMonzón, head of the Laboratory for soilsand irrigation, ICIA, for the leaf analyses.

This study formed part of project INCO-DEV (International Cooperationwith Developing Countries) (1998-2002),Contract No. ERB IC 18 CT97-0208.

ReferencesBrundrett M.S., Y. Piche & R.L. Peterson. 1985. A

development study of the early stages in vesicular-arbuscular mycorrhizal formation. Canad. J. Bot.63: 184-194.

Declerck S., C. Plenchette & D.G. Strullu. 1995.Mycorrhizal dependency of banana (Musa acumi-

nata, AAA group) cultivar. Plant and Soil 176(1):183-187.

Gerdemann J.W. 1975. Vesicular-arbuscular mycor-rhizae. Pp. 575-591 in The development andFunction of Roots. (J.G. Torrey and D.T.Clarkson, eds). Academic Press, New York andLondon.

Jaizme-Vega M.C. & R. Azcón. 1995. Responses ofsome tropical and subtropical cultures to endomy-corrhizal fungi. Mycorrhiza 5: 213-217.

Jaizme-Vega M.C. & J. Pinochet. 1997. Growthresponse of banana to three mycorrhizal fungi inPratylenchus goodeyi infested soil. Nematropica27(1): 69-76.

Jaizme-Vega M.C., P. Tenoury, J. Pinochet & M.Jaumot. 1997. Interaction between the root-knotnematode Meloidogyne incognita and the mycor-rhizal association of Glomus mossae and GrandeNaine banana. Plant and Soil 196: 27-35.

Jaizme-Vega M.C., B. Sosa-Hernández & J. Hernández.1998. Efecto de Fusarium oxysporum f.sp.cubense (Foc) en platanera micorrizada bajo dosniveles de fertilización fosforada. ActaHorticulturae 490: 285-295.

Koske R.E. y J.M. Gemma. 1989. A modified proce-dure for staining roots to detect VA mycorrhizas.Mycol. Res. 92: 486 –505.

Lin Ch. & D.C.N. Chang. 1987. Effect of three Glomus

endomycorrhizal fungi on the growth of micro-propagated banana plantlets. Trans. Mycol. Soc.Rep. China 2(1): 37-45.

Phillips J.M. & D.S. Hayman. 1970. Improve proce-dures for cleaning roots and stain parasitic andvesicular-arbuscular mycorrhizal fungi for rapidassessment of infection. Trans. Br. Mycol. Soc. 55:158-161.

Plenchette C., J.A. Fortin & V. Furlan. 1983. Growthresponses of several plant species to mycorrhizain a soil of moderate P fertility. I. Mycorrhizaldependency under field conditions. Plant and Soil70: 191-209.

Rizzardi V. 1990. Effect of inoculation with vesicular-arbuscular mycorrhizal fungi on the growth ofmicropropagated Musa acuminata clone “GrandNain”. Revista de Agricultura Subtropicale eTropicale 84(3): 473-484.

Sosa Hernandez B. 1997. Estudio de la interacción delos hongos formadores de micorrizas arbusculares(MA) y el patógeno vascular Fusarium oxysporum

f. sp. cubense sobre platanera en fase de vivero.Proyecto Fin de Carrera. Universidad de La Laguna.Centro Superior de Ciencias Agrarias. 155pp.

Tenoury P. 1996. Estudio de la interacción del hongoformador de micorrizas arbusculares (MA)

Glomus mosseae y el nematodo agalladorMeloidogyne incognita en platanera. ProyectoFin de Carrera. Universidad de La Laguna. CentroSuperior de Ciencias Agrarias. 159pp.

The authors work in the Departamento de Proteccióndel Instituto Canario de Investigaciones Agrarias,Apartado 60, 38200 La Laguna, Tenerife, CanaryIslands, Spain.


Arachis pintoi: a cover crop for bananas?Advantages and disadvantages as regards nematology

Agronomy Use of cover crops

P. Quénéhervé, Y. Bertin and C. Chabrier

The legume Arachis pintoi L. (theperennial, wild or pinto peanut) hasbeen used for many years as a cover

crop in many tropical countries, notablyCentral America (Kerridge 1993). Itsresponse to nematodes is still barely documented. Possible resistance ofcv. Amarillo to Meloidogyne spp. inAustralia has been mentioned (Cook etal. 1990). In Mexico a noticeable reduc-

tion in Meloidogyne attacks on tomatoeswas observed in an intercropping experi-ment (Marban Mendoza et al. 1992). InCosta Rica, a field experiment showedthat Arachis pintoi is a good host forRadopholus similis (Cobb 1893, Thorne1949) with a concomitant mean infectionrate of about 30 individuals per g of root(Araya 1996). Also in Costa Rica, trialscarried out with bananas and plantainshave shown a beneficial effect of wildpeanut used as a cover crop in reducingthe density of Radopholus similis onneighbouring banana plants (Vargas

1998). Lastly, in 1999, Jonathan et al.showed from an artificial inoculationexperiment that the legume Arachis pintoi is not a host for certain species ofMeloidogyne Goeldi 1892 (M. incognita,M. arenaria, M. javanica) nor forRotylenchus reniformis Lindford &Oliveira 1940.

Before experimenting with, and per-haps recommending the use of Arachispintoi as a possible cover crop forbananas, we wanted to check its behav-iour towards nematodes of banana inMartinique. A controlled inoculation trial

using the main species present(Radopholus similis, Pratylenchus coffeae, Hoplolaimus seinhorsti, Meloi-dogyne incognita) together withMeloidogyne mayaguensis, a specieswhich is very pathogenic in Martiniquealthough not yet observed on bananas,was therefore carried out in a controlledenvironment chamber at the IRDNematology laboratory before attemptingany field experiments.

Materials and methodsSeeds of Arachis pintoi cv. Amarillofrom Costa Rica were inoculated by coat-ing at the moment of sowing with theirsymbiotic bacterium Rhizobium sp.These seeds were then grown in 237 cm3

PVC culture tubes filled with sterile soil(steam sterilization for 1 h at 100ºC).The substrate was a volcanic andosol ofpH 6.2, with 7.3% organic matter and acation exchange capacity of 10.3 meq per100g soil. The experiment was carriedout in a controlled environment cham-ber with eight replicates, using a 14-hphotoperiod and a temperature in thelight of 27±1ºC, and in the dark of22±1ºC, daily watering and weekly appli-cation of a Hoagland nutrient solution.Four weeks after sowing and develop-ment of the peanut, the five nematodespecies, meanwhile grown in the labora-tory (Radopholus similis, Pratylenchuscoffeae, Hoplolaimus seinhorsti,Meloidogyne incognita and Meloidogynemayaguensis) were individually inocu-lated at a rate of 400 individuals perplant. The infestation of the root systemwas checked 45 days later after extrac-tion of the nematodes from the roots byspraying (Seinhorst 1950). The nema-tode densities were then expressed asnumbers of nematodes per root systemand per g of dry root (after oven-dryingat 60ºC for 24h).

Results and discussionThe results of this experiment (Table 1)show that at 45 days only three species ofnematode are supported: R. similis,H. seinhorsti and P. coffeae. The inocula-tion of the different species of nematodehas had no effect on the growth either ofshoots or roots of the peanut, whichtherefore appears, over this short periodof time, to be tolerant of attack by thesenematodes.

Arachis pintoi was unable to maintainor permit the multiplication of the twospecies of Meloidogyne, M. incognitaand M. mayaguensis. This result con-firms and completes for M. mayaguensis

earlier results on the inability of thispeanut to act as host to the main speciesof root-knot nematodes, with the excep-tion of M. hapla (Jonathan et al. 1999).

Arachis pintoi does however act ashost to three other species, and accord-ing to the criteria applied to bananaweeds (Quénéhervé et al. 2002), one mightsay that it is a bad host for R. similisbut a very good host for H. seinhorsti andP. coffeae. The hosting capacity ofArachis pintoi to R. similis alreadyobserved (Araya 1996) is thus confirmed,but also (which is new) its great suscep-tibility to P. coffeae and H. seinhorsti,two species of nematodes whose patho-genicity to bananas was demonstratedfor one (P. coffeae) and seems very likelyfor the other.

These results may be compared withothers from the field under conditions ofnatural infestation. In fact, root produc-

tion in Arachis pintoi is extremely slow,with a shoot/root ratio of about 7.5 in ourexperiment, and it would be interestingto quantify the real “reservoir” capacityof this plant for nematodes in the field aswas done by Araya in 1996. However onecan already regard it as a “non-host” forMeloidogyne spp., and especiallyM. mayaguensis, and reconsider thiscrop as a “cleaning fallow crop” (for reha-bilitation) which provides protectionagainst nematodes before a susceptibleannual or perennial crop.

For many years agronomists havesought plants useful as fallows of short,medium or long term, or as cover cropswhich can, inter alia, reduce parasiticpressure (for example by nematodes) andalso reduce the effects of weeds, improvesoil fertility and limit erosion (Terisienand Melin 1989). In the Caribbean zone,two species have been used for theiractivity against nematodes, with bothadvantages and disadvantages: the foragegrass Digitaria decumbens and the for-age legume Mucuna pruriens cv. utilis,of African origin.

Each of these plants has its valueaccording to the cropping system beingconsidered. Digitaria decumbens fits intolong-term rotations combining livestockand field vegetable growing, as practisedon the vertisols in the south of Martinique.Mucuna pruriens, widely grown in thesoutheastern United States and in Africa,can also find a place in Martinique as ashort-term intercrop or in certain inten-sive vegetable systems to control nema-todes, particularly Meloidogyne spp.(Quénéhervé et al. 1998).

This third plant, Arachis pintoi,recently introduced by CIRAD-FLHOR intoMartinique, seems to offer certain advan-tages, but also possesses disadvantages:• advantages: commercially available

seed, propagation by seed, or vegeta-tive; “non-host” plant to several nema-tode species including Meloidogynespp.; suitable as a cover crop; suppliesnitrogen (about 60 kg/ha/year).


Table 1. Results of nematode counts and Arachis pintoi weighings 45 days afterinoculation.

No./g roots Roots (mg) Shoot (mg) Host quality1

Control - 260 ± 10 1470 ± 140 -

Hoplolaimus seinhorsti 382 ± 132 240 ± 40 1880 ± 110 ***

Pratylenchus coffeae 2918 ± 447 240 ± 30 1630 ± 350 ***

Radopholus similis 112 ± 95 250 ± 50 1820 ± 50 *

Meloidogyne mayaguensis 0 240 ± 60 1600 ± 300 NH

Meloidogyne incognita 0 270 ± 30 2010 ± 180 NH

ANOVA NS NS1 Very good host = ***; Good host = **; Poor host = *; Non-host = NH

Figure 1. Root system of Arachis pintoi.

• disadvantages: plants are host to seri-ous migratory endoparasites, includingR. similis and P. coffeae; slow to estab-lish; requires inoculation with a spe-cific associated bacterium.The introduction and use of Arachis

pintoi as a cover crop for bananas couldtherefore take place under certain con-ditions:• in the absence of the nematodes

R. similis and P. coffeae, which wouldlimit its use immediately after bananaor another crop infested by P. coffeae,such as yam or dasheen;

• after crop rotation but in the presenceof Meloidogyne spp. so as to reduce theinfestation potential of these root-knotnematodes before replanting with tissue-cultured banana plants.This plant could also find a place in

Martinique and elsewhere in the WestIndies in various other ecosystems, whichremain to be experimented with:• in orchards such as citrus and espe-

cially guavas which suffer seriousattacks from M. mayaguensis in theWest Indies (Quénéhervé et al. 2001);

• as a fallow crop or intercropped covercrop for vegetables. ■

ReferencesAraya M. 1996. Capacidad hospedante de Arachis

pintoi a Radopholus similis. CORBANA 21: 19-24.Cook B.G., R.G. Williams & G.P.M. Wilson. 1990.

Arachis pintoi Krap et Greg. nom. nud (pintopeanut) cv. Amarillo. Tropical Grasslands 24: 124-125.

Jonathan E.J., K.R. Barker & T.B. Sutton. 1999.Host status of wild peanut Arachis pentoi forroot-knot and reniform nematodes. INFO MUSA

8: 9-10.Kerridge P.C. 1993. Biology and agronomy of forage

Arachis. International Center for TropicalAgriculture (CIAT), Colombia.

Marban-Mendoza N., M.B. Dicklow & B. Zuckerman.1992. Control of Meloidogyne incognita on tomatoby two leguminous plants. Fundam. Appl.Nematol. 15: 97-100.

Quénéhervé P., P. Topart & B. Martiny. 1998.Mucuna pruriens and other rotational crops for control of Meloidogyne incognita andRotylenchulus reniformis in vegetables in polytunnels in Martinique. Nematropica 28: 19-30.

Quénéhervé P., Y. Bertin & A. Kermarrec. 2002.Meloidogyne mayaguensis: a root knot nematodecausing severe decline of guava trees in theCaribbean (Abstr.). African Plant Protection(in press).

Quénéhervé P., C. Chabrier, A. Auwerkerken,P. Topart, B. Martiny & S. Marie-Luce. 2002.Status of weeds as reservoirs of nematodes inbanana fields in Martinique. Nematropica (sub-

mitted).Seinhorst J.W. 1950. De betekenis van de toestand

van de grond voor het optreden van aanstastingdoor het stengelaattje (Ditylenchus dipsaci)

(Kühn) Filipjev). Tijdschr. Plziekt. 5: 291-349.Ternisien E. & Ph. Melin. 1989. Etude des rotations

culturales en bananeraie. Première partie : bilandes cultures de rotation. Fruits 44: 373-383.

Vargas A. 1998. Banana (Musa AAA) and plantain(Musa AAB) cultivation in the presence andabsence of a green cover crop (Arachis pintoi

CIAT-18748). CORBANA 22: 23-39.

Patrick Quénéhervé works at the Institut pour larecherche et le développement (IRD, formerORSTOM), BP 8006, 97259 Fort-de-France Cedex,Martinique, FWI; Yves Bertin and ChristianChabrier work at CIRAD-FLHOR, BP 153, 94202 Fort-de-France Cedex, Martinique, FWI.


Dynamics of boron in a soil cultivated with plantain(Musa AAB cv. ‘Dominico hartón’) in the Quindío,Colombia

Agronomy Micronutrient studies

M.M. Bolaños Benavides and A. Garcia Alzate

Boron (B) is the only non-metallicelement of the six essentialmicronutrients; it has a constant

valence of +3, and has the smallest ionicradius. It is found mainly in sedimentaryrocks. Of the igneous rocks it is mostabundant in granites, in the form ofborosilicates, with tourmaline (3-4%boron) the most common of the minerals.It is found in soil in four states: a) form-ing part of the crystalline structure ofminerals; b) adsorbed or bound by soilcolloids; c) as an anion in the soil solu-tion and d) associated with soil organicmatter (Bonilla et al. 1994).

The total content of boron in soilsvaries from 2 to 200 ppm most of which is

not taken up by plants. Compared withother micronutrients, boron has severalspecial features, thus in soil solution it isalways found in combination with oxygen,behaving as an anion (borate) in all reac-tions. The borate anion is highly mobileand hence is easily lost by leaching. Theavailable boron in soil can be consideredas belonging to a cycle where a smallamount originates from tourmaline and alarge part from soil organic matter.

Organic material is decomposed bymicroorganisms which liberate availableboron to the soil solution, from whence itis taken up by plants; part can be washedout by percolating water, and a small partis fixed or bound by clays (Berger andPratt, cited by Bonilla et al. 1994).

The multiple functions involving boronin plant metabolism include the follow-ing: it affects, inter alia, the processes of

flowering and fruiting, germination ofpollen grains, cell division, cell wall syn-thesis and the metabolism of nitrogen,carbohydrates and pectic substances.These substances are reported toincrease in plants that are deficient inboron (Rajaratnam and Lowry 1974).

Another function of boron is theabsorption of water by protoplasm andthe absorption of mineral salts. Themain function of boron is reported tofacilitate the transport of highly polarsugar molecules across the cell wall.Boron is a constituent of cell membranesand is immobile in the plant; thereforeany boron deficiency is immediatelyreflected in a change in the metabolismof carbohydrates (which accumulate inleaves). This condition could be thecause of almost all the remaining func-tions attributed to boron (Gómez and

Leguizamón 1975). In spite of the impor-tant advances in mineral nutrition, therole of boron in plant metabolism stillraises many questions.

At present, in the coffee productionarea of central Colombia, many plantaincrops show symptoms associated withboron deficiency. According to León et al.(1985) ten examples of boron deficiencywere reported in the country. In the pre-sent study we tried to obtain a soundfoundation in order to deal more clearlywith the problems above. The objectivewas to determine the importance ofboron in the cultivation of plantain(Musa AAB cv. Dominico hartón) in theQuindío, and to study the dynamics over aperiod of ten years, in a soil fertilizedwith the major elements.

Materials and methodsThe study plot was located at theExperimental station El Agrado, Districtof Montenegro, Department of Quindío,Colombia. The station was 1320 m abovesea level, with an average rainfall of2000 mm/annum, a mean annual tem-perature of 22oC and a relative humidityof 76%.

According to the classification ofHoldridge, the ecosystem corresponds topremontane humid woodland. The soilsare derived from volcanic ash (andisols)and have an average natural fertility, amedium to heavy texture, a low moistureretention capacity, and are leached andsusceptible to erosion.

For the study the soils were taken foranalysis from 2 May 1990 until 2 March2000. Soil samples, replicated five times,were taken every two years. Rainfall datawas analyzed for the same period.

Samples were analyzed for pH, organicmatter content, exchangeable calcium,phosphorus (P), magnesium (Mg), potas-sium (K) and boron (B). The analyticalmethods are described in Table 1. Thedata were analyzed to determine the cor-relation between: boron-weight of bunch(for each production cycle), boron-potas-sium, boron-calcium, boron-percentage

soil organic matter, and boron-pH. Therelationships between Ca/Mg, Mg/K, Ca/Kand (Ca+Mg)/K were also analyzed.

Results and discussionThe data obtained from soil analyses,averaged over the five replicates, changedover the years as shown in Table 2.

Boron contentAs can be seen from the chemical analy-ses during the 10 years of the study,boron content declined considerablyfrom levels sufficient for cultivation ofplantain, according to Buriticá (1985)from 0.4 ppm to 0.01 ppm boron, a valuewhich gave rise to deficiencies. However,one must consider the edaphic cycle ofboron which determines its concentra-tion in soil solution and hence the avail-ability for uptake by plants (Mengel1980).

Relationship between pH and boroncontentAs can be seen from Table 3, boronshowed a direct and close correlationwith pH, hence this is in a range optimumfor absorption of boron; fixation of thismicroelement to hydroxides of Fe and Al,as is with clays, increases with pH being amaximum between pH 8 and 9 and a min-imum at about pH 5 (Lora 1994).According to Domínguez (1988) theincrease in pH reduces the availability ofboron but this does not become evidentuntil more than pH 6 which did not occurafter the start of the experiment.

According to Marschner (1986) theavailability of boron to plants decreaseswith increased soil pH, as happens in cal-careous soils or in soils with high claycontent, presumably as a result of the for-mation and absorption of B(OH)4.

In agreement with the soil chemicalanalyses, the pH value (5.1-6.08) fluctu-ated in the range sufficient for themicronutrient to be available. Thisexplains why symptoms of boron defi-ciency only became evident in the lastyears. The explanation for the correlation

between boron content and pH, are basedon the following:• Profoundly influences many biological

processes in soil,• Affects the availability of micronu-

trients,• Alters the absorption of an element

and its effect on microbial activity,• Results in changes in the ability of

roots to absorb or transport ions oncethey have been taken up,

• Causes variations in the stability of soluble and insoluble organic com-plexes,

• Changes the solubility of antagonisticions and changes conditions in the rhi-zosphere.

Relations between nutrientsThe results also show a close inverse cor-relation between K and boron (Table 3)explicable because the K content over theyears had reached levels greater than0.3 meq/100 g soil, which, according toGómez and Leguizamón (1975) caninduce boron deficiency.

The potassium-boron interaction doesnot appear to follow a particular pattern.Revé and Shive (1944) cited byDomínguez (1988), demonstrated that ina boron-rich medium, absorption of boronincreased as the soil became enrichedwith K but, in contrast, when boron levelsin the medium are low, boron deficiencybecomes worse as K increases. The direc-tion of the interaction between K andboron appears to depend on the amountof boron in soil solution. The trend in thisstudy showed that increasing applica-


Table 3. Correlations between soil boron,pH, K, Ca, P and weight of bunches(WB) in kg.

Correlations

pH – B 0.82

K – B -1

Ca – B 0.8

P – B -0.86

WB – B 1

Table 1. Methods of chemical analysisof soils.

Factor Analytical methods

PH Potentiometer, ratio 1:2.5

EA (exchangeable acidity) KCl 1N

MO Walkley–Black

P (ppm) Bray II

Exchangeable bases Ammonium acetate (1N) and neutral (pH 7)

Table 2. Changes in chemical properties of soil under study (1990–2000).

Changes in soil fertility

Year pH MO K Ca Mg P B (%) (meq/100 g) (meq/100 g) (meq/100 g) (ppm) (ppm)

1990 6.08 3.79 0.95 5.2 0.93 22 0.40

1993 5.18 3.66 0.69 4.4 1.03 71.6 0.12

1995 5.72 3.72 1.22 2.8 0.64 34.6 0.19

1997 5.78 4.80 1.30 3.8 0.94 61.0 0.06

2000 5.10 4.80 1.79 6.0 0.60 29.0 0.01

tions of K resulted in a small reduction inboron availability.

When boron interacts with other ele-ments, it is necessary to consider the pos-sibility of nutritional imbalances in soil,given that this implies an antagonismthat affects the plant directly, as one orother elements would not then be avail-able. Such is the situation with potassiumthat is absorbed in smaller amounts whenboron content is very low.

As regards calcium, the levels increasewhen boron is deficient. In the soil understudy, where we did not find a high avail-ability of Ca, this could favour boronuptake. However, the interaction cal-cium-boron has been studied with ratherhigh concentrations, and with the rela-tionship of Ca/boron in the plant. Revéand Shive (1944) cited by Domínguez(1988), indicated that high concentra-tions of calcium exacerbate symptoms ofboron deficiency in tomato. The toxicityof boron, in a medium with too much ofthe element, can on the other hand havea reduced increase in the amount of cal-cium in the medium. It is possible thatwith all the preceding, boron deficienciesin the cultivation of plantain studied, onlybecame apparent in the last years (1999-2000) even when the low levels of this ele-ment were present from 1993.

Table 3 shows an inverse correlation ofP to boron. According to studies byRobertson and Loughman (1974), it wasevident that there was a clear reductionin the uptake of phosphorus in boron-deficient plants. This idea is based on therole of boron as a stimulant of the utilisa-tion of glucose1-phosphate. This suggeststhat when boron is low, the P found in soilwill be taken up very slowly, leading to asteady accumulation of P in soil.

According to the relationships obtainedbetween the different cations (Table 4)and later comparisons with the criticallevels, K was never seen to be deficient,

which is explained by the large quantitiesof potassium containing fertilizers thatwere applied over the years, as well as bythe recycling that occurs with this ele-ment in residues from the harvest of theplantain. According to Belalcázar (1991),cultivation of plantain removes a largepercentage of elements such as potas-sium (76.02%) and calcium (13.62%), fol-lowed by nitrogen, magnesium and phos-phorus. Those with the higher percentagethat are removed are nitrogen (25.55%)followed by magnesium (20.09%) andphosphorus (19.80%), whilst those thatare reincorporated or recycled in higheramounts are calcium (94.47%) and potas-sium (89.77%).

With reference to magnesium, amongstthe factors that gave rise to the deficiencyin this macronutrient, were unsuitablerelationships with the other bases in thesoil, mainly potassium (Table 4).

The relationship Mg/K appeared to beunbalanced, explaining a deficiency ofMg. Therefore, the high levels of K func-tion in a manner antagonistic to Mg,implying a low absorption for this ele-ment. The losses of magnesium in soil aregreater when added with potassium fertil-izers. Many authors consider that a soil tobe low in magnesium when it has lessthan 1.0 meq/100 g is present, whilst oth-ers classify soils as poor in magnesiumwhen there is less than 1.5 or even2.0 meq /100 g (Suárez and Carrillo 1984).

Intensive and continual fertilisationwith K, as employed in this zone, possibly

contributes to Mg deficiency, resulting inan imbalance in the relationship Mg/Kand as a result an inhibition of Mguptake. It should be noted that in thezone where this study took place, it iscommon to come across crops with symp-toms of magnesium deficiency. In accor-dance with Fried and Dean (1952) nutri-tional deficiencies resulting from animbalance can be corrected by a pro-gramme of balanced fertilisation.

Soil organic matter and boron in soilIn accordance with the results of thisstudy there was no correlation betweenorganic matter and boron, however, itshould be noted that various authors(Gómez and Leguizamón 1975) claim thatin mineral soils rich in organic matter,boron deficiency is rarely seen becausesoil organic matter is a major source ofboron. Similarly, Berger and Truog (1945)cited by Domínguez (1988), obtained apositive relationship between assimilableboron (water soluble boron) and theorganic matter content of soil. Morerecently, Olsen and Berger (1946) citedby Domínguez (1988), demonstrated thatmineralization of organic material leadsto a release of assimilable boron.

On the other hand, boron absorbed onorganic and inorganic soil colloids consti-tutes a reserve that maintains the con-centration of boron in solution; this helpsto replenish the demand by crops andreduces losses by washing. Furthermore,soils with higher organic matter content


3500

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Figure 1. Production of plantain in relation to boron content in soil. Figure 2. Boron content of soil vs. rainfall.

Table 4. Relationships between cations in the experimental soil (1990-2000).

1990 1993 1995 1999 2000Relationship

Mg/K 1.58 1.93 0.87 0.73 1.00

Ca/Mg 5.72 4.14 5.00 4.88 5.00

Ca/K 8.97 7.96 5.37 3.58 6.66

(Ca+Mg)/K 10.50 9.88 6.26 4.33 7.66

have higher concentrations of boron,since an important fraction of soil boroncomes from soil organic matter.

Production of plantain and soil boronThere was a close and direct correlationbetween yield and boron (Table 3). Thisis explicable as a result of the chemicaldegradation of the soil, as shown in Table 2. The gradual loss of the micronu-trient boron, drastically affects the fillingof the fruit (Figure 1) to the point wherethe young fruits become deformed,mature prematurely and size is reduced,hence yield declines and they are diffi-cult to market. Clearly productive capac-ity of plantain is harmed. The reductionin yield can also be associated with theregulation of uptake and translocation ofboron by the plants, which is more lim-ited in comparison with other minerals.

At the same time, the low quantity andquality of production could be caused byan early boron deficiency, which checksgrowth of the apices and restricts cellelongation (Lovatt et al. 1981, Robertsonand Loughman, 1974b) and cell division(Cohen and Lepper 1977).

According to Leguizamón (1975) inmany situations the affected developingbunches do not produce a yield and inthis situation, bunches are small anddeformed.

From the above it can be concludedthat boron is a nutrient fundamental togood production as well as fruit qualityand in quantity.

Rainfall and boronBoron content tends to decline as a resultof the high rainfall which occurs in thearea of the study as shown in Figure 2;this, linked to the sandy loam type tex-ture, and the definite mobility of theboron anion, gave rise to an increasedrate in leaching of boron. Thus, the nutri-ent should be applied in a more finelydivided form.

This result was in agreement withMarschner (1986) who suggested thatunder conditions of high rainfall boron iswashed out as B(OH)3.

Boron and the physiology of the plantA general aspect of boron deficiency isthe poor development of meristematictissues, as found at the tips of young rootsand in the buds. With boron deficiency,irregularities in development are the firstsymptoms (Domínguez 1988). This checkto the growth of the root tips possibly con-tributes to one of the main problems ofthe cultivation of plantain, which harmsthe plant. Primavessi (2000) confirmed

that the addition of boron aids rootgrowth, and that if this continues in thecoat with organic matter and roots do notwant to penetrate the soil, there may notbe sufficient boron.

Boron deficiency in plants is not easilyidentified except by leaf or soil analysis.This is important in the cultivation ofplantain because the micronutrient playsa key role in the transport of sugars, as aresult of the transformation of boron-sugar complexes (Marschner 1986) andtherefore affects the filling of the fruits;in such situations, a deficiency directlyand adversely affects the quality andquantity of the plantain harvest.

Conclusions• Boron is crucial for optimal yields of

plantain, as well as for the quality andquantity of fruit. This was confirmed bythe correlation between bunch weightand boron.

• The availability of boron in soil solutionis closely linked to rainfall and the loosesandy loam texture of the soil and thedefinite mobility of the borate anion.

• In the experimental soil, from 1990 to2000, as soil potassium contentincreased so boron content declined,and as a result yields of the experimen-tal crop of plantain declined. This couldbe associated with the repeated appli-cation of potassium fertilizer to the soil.

RecommendationFurther investigations are needed toimprove accuracy of the recommendationfor fertilization with boron in systems ofplantain cultivation, in relation to boroncontent of leaves and based on differentcritical levels of extraction of the borateanion.

AcknowledgementsThe authors would like to thank theComité de Cafeteros del Quindío, for theeconomic support for the soil analyses,which were essential to the presentstudy; Dr Fabio Aranzazu H., research scientist at CORPOICA, Regional 9; and Huberto Morales Osorno and LuzDary Celis García, research assistants, CORPOICA, Regional 9. ■

ReferencesBelalcázar C.S., C.A. Salazar M., G. Cayón S., J.E.

Lozada Z., L.E. Castillo & J.A. Valencia M. 1991.Manejo de plantaciones. Pp. 147-214 in El cultivodel plátano en el trópico. Manual de AsistenciaTécnica No. 50. Instituto Colombiano Agro-pecuario, Colombia.

Bonilla C.R., A. García, L.E. Castillo & Ch.F. Salazar.1994. Boro y zinc: dos elementos limitantes enColombia. ICA Programa de Suelos, Colombia.

Buriticá C.P. 1985. Situación del plátano enColombia y el sistema de producción de tecnologíapara su cultivo. Rev. ICA – Informa (Colombia) 13:24-31.

Cohen M.S. & Lepper R. Jr. 1977. Effect of boron oncell elongation and division in squash roots. PlantPhysiol. 59: 884-887.

Domínguez A. 1988. El boro. Pp. 155-181 in Losmicroelementos en agricultura (A. Loué, ed.).Mundi-Prensa, Madrid, Spain.

Fried M. & L. Dean. 1952. A concept concerning themeasurement of available soil nutrients. SoilScience 73: 263-271.

Gómez A. & J. Leguizamón. 1975. Importancia delboro en las plantas. Cenicafé (Colombia). Avancetécnico No.43.

Leguizamón J. 1975. Deficiencia de boro en cultivosde plátano en el Valle del Cauca. Cenicafé(Colombia). Avance técnico No.39.

León L.A., A.S. López & P.L.G. Vlek. 1985.Micronutrient problems in tropical Latin America.Pp. 95-129 in Micronutrients in tropical foods(P.L.G. Vleck, ed.). Martinus Nijhoff/Dr. W. JunkPubl., The Netherlands.

Lora S., R. 1994. Factores que afectan la disponibili-dad de nutrientes para las plantas. Pp. 29-55 inFertilidad de suelos: diagnóstico y control(F. Silva M., ed.). Soc. Col. Ciencia del Suelo(SCCS).

Lovatt C.J., L.S. Albert & G.C. Tremblay. 1981.Synthesis, salvage and catabolism of uridinenucleotides in boron-deficient squash roots. PlantPhysiol. 68: 1389-1394.

Marschner H. 1986. Mineral nutrition of higherplants. San Diego CA Academic Press. 674pp.

Mengel U.S. 1980. Borax plant food. Borate Meeting,Lafayette, Ind.

Primavessi A. 2000. Manejo ecológico del suelo.Presentación al Simposio “Biología de SuelosTropicales”, 14-18 agosto 2000, Universidad deCaldas, Manizales, Colombia.

Rajaratnam J.A. & J.B. Lowry. 1974. The role of boronin the oil-palm (Elaeis guineensis). Ann. Bot.(London) (N.S.) 38: 193-200.

Robertson G.A. & B.C. Loughman. 1974a. Reversibleeffects of boron on the absorption and incorpora-tion of phosphate in Vicia faba L. New Phytol. 73:291-298.

Robertson G.A. & B.C. Loughman. 1974b. Responseto boron deficiency: a comparison with responsesproduced by chemical methods of retarding rootelongation. New Phytol. 73: 821-832.

Suárez S & I.F. Carrillo. 1984. Comportamiento detres fertilizantes potásicos en un dystrandept

típico. Cenicafé (Colombia) 35(2): 31-39.

Martha M. Bolaños Benavides is a research biologistwith CORPOICA, Avenida Bolíva, Sector Regivit 28Norte. A.A. 1807, Armenia, Colombia. Email: [email protected] and Alexander GarcíaAlzarte is an undergraduate, Facultad de CienciasAgropecuarias, Programa de Agronomía, Universidadde Caldas, Manizales, Caldas, Colombia. Email:[email protected]


P. Orellana Pérez, I. BermúdezCaraballoso, L. García Rodríguez

and N. Veitía Rodríguez

Plantains are an important foodsource for the populations of LatinAmerica and certain African coun-

tries. Plantains of the ‘Horn’ type, tradi-tionally the most commonly grown, areseriously affected by black leaf streak(Mycosphaerella fijiensis Morelet),which has greatly reduced the supply ofthis product both in local markets and forexport. This is the main disease whichthreatens the production of this source ofboth food and income (Jacome 1998).The fact that plantains are usually grownon smallholdings, sometimes in moun-tainous areas and very often interplantedwith other crops, makes chemical controlof the disease difficult. The consequence,not only on volume of production but alsoon the quality of the product, is that cur-rent levels of production fail to meet thegrowing demand of certain local andexport markets.

In the 1990s, the first plantain hybridsresistant to black leaf streak and destinedfor commercial use, developed by theFundación Hondureña de InvestigaciónAgrícola (FHIA), offered the possibility of introducing new clones into commer-cial production and of restoring adequateproduction levels at lower cost.

However, because of their genetic con-stitution, which includes a contributionfrom clones of the tall ‘French’ type, thenew hybrids must be characterized mor-phologically and studied agronomicallybefore being exploited commercially.

The work presented here shows theresults of evaluation of various agronomiccharacters of the FHIA hybrids in thecentral region of Cuba.

Materials and methodsThe studies were made on tissue-culturedplants produced by micropropagationusing the method proposed by Orellana(1994), planted out on the multi-cropfarm “La Cuba”, situated in the provinceof Ciego de Avila.

Fifty plants of each hybrid wereplanted 3 metres apart, in two furrowsalso situated 3 m apart. On 20 plants ofeach hybrid, starting at the beginning of

flowering, the following characters wererecorded:• Plant height• Number of functional leaves (with more

than 75% of green leaf area) at the startof flowering (NFLF)

• Number of leaves showing lesions typicalof black leaf streak (Stage 5 on the Sto-ver scale modified by Gauhl (1984) atthe start of flowering (NLSF)

• Number of functional leaves (with morethan 75% of green leaf area) at harvest(NFLH)

• Number of leaves with black leaf streaklesions at harvest (NLSH)

• Number of hands per bunch• Length and diameter of the central fin-

ger of the first and penultimate hands• Number of days between harvest and ri-

pening (second hand at ripening degree1, using the scale in ‘Descriptors for ba-nana’ (IPGRI-INIBAP/CIRAD 1996))

• Growing period in days from planting toflowering and until harvest.Using the results of counts of the num-

ber of functional leaves showing typicalblack leaf streak lesions, two formulaewere devised to serve as indicators of thereduction in functional leaf area: theFunctional Leaf Reduction Index (FLRI)and Relative Infection Index (RII),reflecting the damage caused by the dis-ease. The latter index depends on thenumber of functional leaves showing typi-cal lesions at the start of flowering andwhen the bunch is harvested.

Formulae:• FLRI = NFLF/NFLH• RII = FLRI X NLSH/NFLH

= NFLF X NLSH/(NFLH)2

As there is only one production cycle inCuban plantains, the counts all apply tothe mother plant.

Results and discussionThe results show that with the exceptionof FHIA-19, which has the lightestbunches, the hybrids did not differ intheir bunch weight. For all the hybrids,the majority of the weight of the bunch isconcentrated in the first four hands(59.71% of the total weight). FHIA-19 had the highest proportion in thesehands (71%), which is confirmed by theobservation of the length and breadth ofthe fingers of the first hand (Table 1).The concentration of the majority of

the weight in the first hands is a charac-teristic of the plantain. It justifies theremoval of the terminal hands of hybridsthat have developed more than eighthands per bunch in order to encouragegreater development of the fingers interms of length and diameter. This latteraspect is very important if the hybridsare to claim to rival the ‘Horn’ type plantains. No differences were observedbetween the hybrids as regards otherbunch characteristics. Arcila et al.(2000) recommended leaving five handsand removing the rest 20 days after thestart of flowering.

It is important to emphasize that thehybrids with the longest interval betweenharvest and ripening under natural conditions (11 days) are FHIA-20 andFHIA-22; for FHIA-21 it is only 8 days.This shows that the first two have advan-tages for local sale and for export overshort distances.

As for response to black leaf streak, theFLRI shows that FHIA-04, which has only1.3 functional leaves at harvest (FLRI =9.31) is the hybrid whose leaf area wasmost reduced during the filling process ofthe fingers, resulting in insufficient fill-ing of these fingers. The other hybridshad lower values of this index and similarfor them all (Table 2). For these hybrids,the number of functional leaves at har-vest was not less than four, allowing fill-ing of the fingers.

The results indicate that hybrid FHIA-04 is also the most affected by blackleaf streak, with an RII of 9.31 due to thefact that all its functional leaves bore typ-ical lesions of the disease, which devel-oped rapidly after flowering. At the timeof harvest, FHIA-20 and FHIA-22, withmore than two functional leaves unaf-fected by the pathogen, had the lowestvalues of RII: 1.38 and 1.40 respectively.FHIA-05, FHIA-19 and FHIA-21, althoughhaving higher values, responded well tothe disease itself even though all theirfunctional leaves at the time of harvestbore typical lesions. The results confirmthat the time needed for the developmentof the disease on FHIA-04 was very muchless than on the other hybrids, as alreadyreported by Jones (1994).

The results suggest the possibility ofusing FLRI as an expression of the reduc-tion of leaf area during the process of fill-


Evaluation of the agronomic characteristics of plantain hybrids (Musa spp.)

Evaluation Performance of plantain hybrids

ing of the fingers, and RII as an expres-sion of the time needed for the develop-ment of the disease as a function of theleaf area affected, given by the number offunctional leaves and that of leaves whichare necrotic at harvest time, a relation-ship which has always been difficult toquantify numerically.

According to Ortiz and Vuylsteke(1994), cited by Craenen (1998), itrequires at least eight functional leavesduring the whole growth period and asimilar number of healthy leaves beforeflowering to guarantee a good yield.

From this point of view, FHIA-20 hasthe shortest growth period from plantingto harvest, at 481 days, whereas for theother hybrids this period varies from 493to 518 days.

Conclusions• The hybrids FHIA-20 and FHIA-22 should

have good yield potential owing to thelonger period of time between harvestand fruit ripening. FHIA-20 also has theshortest growth period.

• The hybrids FHIA-05, FHIA-19 and FH-21also have good yield potential. However,the RII shows us that by the time of har-vest all their functional leaves are affec-ted by black leaf streak, which in ex-treme infection conditions can haverepercussions on yield.

• The indicators FLRI (functional leaf re-duction index) and RII (relative infec-tion index) proposed in this study, seemto be suitable for comparing the reduc-tion in active leaf area and the time nee-ded for black leaf streak to develop indifferent plantain clones during the per-iod between flowering and harvest. ■

ReferencesArcila M.I., J.A. Valencia & S. Belalcázar. 2000. Efecto

del desmane sobre la calidad y la produccin thisstudy, seem to be suitable for compari in

Postcosecha y agroindustria del plátano en el ejecafetero de Colombia. (D.G. Cayón & G. Giraldo,eds). CORPOICA. Universidad del Quindío, ASIPLAT,Comité departamental de Cafeteros del Quindío,Colciencias. Fudesco, Armenia, Colombia.


Options for in vitro propagationof the banana hybrid cultivarFHIA-20

Tissue culture Mass propagation

Table 1. Bunch characteristics and yields of the hybrids studied.

Hybrid Bunch Weight of % of total Number of Finger length Finger diameter Number of weight first four bunch hands per (mm) (mm) days from

(kg) hands (kg) weight bunch harvest to First Penult. First Penult. ripening

FHIA-04 20.3 a 12.8 63 8.5 21.4 14.8 39.3 32.4 8

FHIA-05 21.5 a 13.5 63 8.6 20.8 15.5 39.0 33.5 8

FHIA-19 16.8 b 12.0 71 8.0 22.0 13.8 40.2 30.0 9

FHIA-20 20.6 a 12.1 59 9.7 19.0 14.0 39.8 32.0 11

FHIA-21 21.3 a 13.1 62 8.7 21.1 14.8 38.7 31.5 7

FHIA-22 22.2 a 14.0 61 8.6 20.0 14.0 41.0 31.0 11(a, b): means of values followed by different letters are significantly different according to Duncan’s multiple range test (P ≤ 0.05).

First: Central finger of the first hand; Penult.: Central finger of the penultimate hand.

Table 2. Response of hybrids to black leaf streak attack.

Hybrid At flowering At harvestNFLF NLSF NFLH NLSH FLRI RII

FHIA-04 12.1 3.13 1.3 1.3 9.31 9.31

FHIA-05 10.2 3.80 4.0 4.0 2.55 2.55

FHIA-19 9.0 4.0 4.0 4.0 2.25 2.25

FHIA-20 12.7 4.0 5.6 3.4 2.27 1.38

FHIA-21 11.5 4.8 4.5 4.5 2.56 2.56

FHIA-22 12.5 4.6 5.5 3.4 2.27 1.40NFLF: Number of Functioning Leaves at Flowering; NLSF: Number of Leaves with Streak at Flowering; NFLH: Number ofFunctioning Leaves at Harvest; NLSH: Number of Leaves with Streak at Harvest; FLRI: Functional Leaf Reduction Index; RII: Relative Infection Index.

Craenen K. 1998. Black Sigatoka disease of bananaand plantain: a reference manual. IITA, Ibadan,Nigeria. 60pp.

Gauhl F. 1994. Epidemiology and ecology of blackSigatoka (Mycosphaerella fijiensis Morelet) onplantain and banana in Costa Rica. INIBAP,Montpellier, France. 120pp.

IPGRI-INIBAP/CIRAD. 1996. Descriptors for banana(Musa spp.). International Plant GeneticResources Institute, Rome, Italy/InternationalNetwork for the Improvement of Banana andPlantain, Montpellier, France/Centre de coopéra-tion internationale en recherche agronomiquepour le développement, Montpellier, France. 55pp.

Jacome L. 1998. Sigatoka negra, la situación enAmérica Latina y el Caribe. Pp. 18-23 in MemoriasPrimer Simposio Internacional sobre Sigatoka

Negra (M.M. Robles et al., compil.). 8-10 de julio1998, Manzanillo, México. SAGAR, INIFAP, INI-BAP, Universidad de Colima, IICA.

Jones D.R. 1994. International Musa TestingProgramme Phase I. Pp. 12-20 in The improve-ment and testing of Musa: a global partnership(D.R. Jones, ed.). Proceedings of the first globalconference of the International Musa TestingProgramme held at FHIA, Honduras 27-30 April1994. INIBAP, Montpellier, France.

Orellana P.P. 1994. Tecnología para la micropropa-gación in vitro de clones de Musa spp. Thesis forthe obtention of a PhD in Agronomic Siciences,Instituto de Biotecnología de Las Plantas.Universidad Central de Las Villas, Cuba. 120pp.

The authors work at the Instituto de Biotecnologíade Plantas, Santa Clara, Villa Clara, Cuba.

L. García Águila, B. Pérez Mederos, Z. Sarría Hernández, and J. Clavero García

Currently, several Musa cultivarsare propagated by direct organo-genesis from axillary buds by in

vitro culture (Vasil 1994). This tech-

nique is the basis of mass propagation of bananas and plantains with the aim,for many countries at the present time,of large-scale commercial distributionof completely disease-free plants (Afzaet al. 1996).

Genotype is known to have an influenceon the efficacy of in vitro propagationand so, when new varieties or hybrid

clones are introduced into productionprogrammes, it is necessary to modify themicropropagation techniques used.Banerjee et al. (1986) (cited by Afza et al.1996) found considerable differencesbetween clones in shoot formation. Thisseems to be correlated with the presenceof one or two B genomes.

In vitro propagation of the hybridFHIA-20 (AAAB) has proved difficult.Conversion of apices into plants hasbeen observed during the initiationphase, as well as buds growing out in theform of rosettes and presenting whitebulbous structures during the multipli-cation phase, resulting in a reduction ofthe multiplication coefficient. In view ofthese problems and the need to multiplythe hybrid FHIA-20 efficiently in vitro,the development of alternative protocolsfor the manipulation of apices during theinitiation phase, and of axillary budsduring the multiplication phase, appearnecessary.

Materials and methodsFor the study, young plants with a meanheight of 25.6 cm, grown in the green-house, were selected (Figure 1). Theprocess of introduction into the labora-tory, including manipulation of theplants, disinfection of the corms, culturemedia for initiation and multiplication,together with the culture conditions,were as described by Orellana (1994).

The plants were cultivated in chambersunder natural light conditions at a tem-perature of 27 ± 2ºC. In every case, thebase of the apex or bud was placed down-wards on the culture medium.

Influence of the size of the apex and the physical state of the culturemedium during the initiation phaseThis study was made in order to establishthe conditions for manipulation andgrowth of the apex during the initiationphase. For this purpose, the followingtreatments were studied (Figure 2):1. 0.5 cm2 apex cultured in liquid medium

(control)2. 0.5 cm2 apex cultured in a semi-solid

medium3. 1.0 cm2 apex cut in halves and cultured

in liquid medium4. 1.0 cm2 apex cut in halves and cultured

in semi-solid mediumAt the end of 20 days’ culture, the fol-

lowing variables were recorded:• Percentage regeneration of apices• Percentage infection of apices• Percentage mortality of apices• Number of buds per apex

There were 20 replicates and the sta-tistical method used to compare percent-

ages was the comparison of proportions -ANOVA. The analysis of the variable“number of buds per apex” was a simpleanalysis of variance and the comparisonof means was made using Tukey’s test atP < 0.05%.

14.5 x 2.0 cm test tubes were used con-taining 10 ml of culture medium. For liq-uid culture media, a paper filter supportwas used, forming a bridge on which theapices were placed. In the case of semi-solid media, 2 mg.L-1 of the gelling agentGellan gum (Spectrum) was added.

The plants obtained during the initia-tion phase were transferred, after separa-tion and decapitation, onto multiplica-tion culture media. It was observed thatthe growth of the buds continuedthroughout this phase as small rosettesand presented white bulbous structures.This behaviour of the buds of FHIA-20during the multiplication phase resultedin a reduction in the multiplication coef-ficients (buds obtained/buds initiated).

Effects of doses of 6-benzylaminopurine and type of manipulation on the growth of buds during the multiplication phaseWith the aim of resolving problemsencountered during the growth of buds inthe multiplication phase, the effect of adose of 2 mg.L-1 of 6-benzylaminopurine(BAP) was studied, the rate of 4 mg.L-1,proposed by Orellana (1994) serving ascontrol. Each dose was combined withtwo manipulation protocols.

Protocol 1. The buds were separated,decapitated 0.5 cm from the top and cutin two.

Protocol 2. Buds which had notreached 1 cm length were left in groupsof two or were not separated from themother plant, and there was no decapita-tion. Buds of more than 1 cm were sepa-rated, decapitated at this height and cutinto two when the pseudostem was com-posed of more than three leaves.

Four treatments were thus obtained:1. Multiplication medium with 4 mg.L-1

BAP combined with protocol 1 (control).2. Multiplication medium with 4 mg.L-1

BAP combined with protocol 2.3. Multiplication medium with 2 mg.L-1

BAP combined with protocol 1.4. Multiplication medium with 4 mg.L-1

BAP combined with protocol 2.The variables recorded were the num-

ber of buds per initial explant and thepercentage of buds growing as rosettes.The counts were made after three subcul-tures carried out every 21 days (culturein growth chambers with natural lightand a temperature of 27 ± 2ºC).

Five explants were inoculated in 250 mlflasks containing 30 ml of semi-solid cul-ture medium (2 mg.L-1 of Gellan gum(Spectrum). There were 10 replicates.The data were analyzed by multifactorialvariance and the means compared withTukey’s test. Results in percentages wereanalyzed as in the previous experiment.


Influence of the size of the apex and the physical state of the culturemedium during the initiation phaseThe use during the initiation phase ofapices of 1 cm2, cut in halves and placedon a semi-solid culture medium, gave 85%regeneration after 20 days of in vitro cul-ture. On each section of the apex axillarybuds were noted, guaranteeing a largernumber of future explants at the multipli-cation phase, significantly different fromthat obtained with the other treatments(Table 1).

The technique of decapitating the api-cal dome proved necessary to induce theformation of new buds from the axillarybuds, normally inhibited by apical domi-nance (Ma and Shi (1972) and Swami etal. (1983) (cited by Afza et al. (1996)).Pérez et al. (1998) emphasized theimportance of increasing the multiplica-tion coefficient during in vitro propaga-tion of plantains, because each increaseof this indicator by one unit correspondsto a cost reduction of about 10%.

In this study, mortality occurred onlyin apices cut and grown in liquidmedium, possibly because the cuttingprocedure produced pieces which weretoo small to be grown in a liquid medium(Table 1). According to Orellana (1998),there are differences in tissue growthdepending on the physical state of theculture medium: it proceeds differentlyaccording to whether the medium is solidor liquid.

The incidence of infection during thisphase did not differ significantly with thetreatment. However, various authorsmention the influence of the size of theinitial explant on the incidence of infec-tion and have noticed that the smallerthe explant, and the closer it is located tothe apical meristem, the more the popu-lations of micro-organisms fall (Garciaand Noa 1998, Leifert et al. 1994).

Effect of doses of 6-BAP and type of manipulation on the growthof buds in the multiplication phaseBy reducing the dose of cytokinin in themultiplication medium, the differentia-tion of buds into plantlets could be stimu-lated whilst little by little the growth of


rosettes disappeared (inasmuch as thethree subcultures were made with the2 mg.L-1 dose of BAP). The manipulationsor cuts made during this phase, combinedwith the reduction in the dose ofcytokinin encouraged the biologicalresponse of the plants and caused theappearance of a larger number of budsper initial explant when using protocol 2(Table 2). These buds, once transferredonto rooting media, had no difficulty incontinuing their growth and reached theheight, girth, and number of leavesneeded to allow their transfer to theacclimatization phase.

On the other hand, for the treatmentsreceiving the 4 mg.L-1 dose of BAP, wecontinued to note the appearance ofrosette growth for both protocols,although the highest percentage of thistype of growth occurred with the normalprotocol (i.e. separation, decapitation at0.5 cm and cutting buds into two). Itseems that this treatment accentuatesthe presence of this particular growth inthe clone FHIA-20: in fact, the presenceof rosettes tended to diminish when protocol 2 was used (Table 2).

The development of cultures in vitrorequires an adequate ratio between auxins and cytokinins in the culturemedium. One must also consider theendogenous concentrations of these hor-mones in the different types of explantsor species (Jiménez 1998). Certain speciesare cultivated without the addition of anyexternal regulator, probably because thereis enough endogenous hormone present.

ConclusionsThe results obtained from this work makeit possible to propagate the hybrid FHIA-20 in vitro with a distinct improve-ment in the efficiency of the process ofpropagation by organogenesis by virtue ofan increase in the number of buds.During the initiation phase it is necessaryto culture apices of 1 cm2, cut into two,on a semi-solid medium. In this way,

85% of them regenerate plants by the endof 20 days’ culture. During the multiplica-tion phase, the cytokinin dose should bereduced to 2 mg.L-1 in the culture mediumand the explants separated into well-defined buds which are not less than 1 cmtall (those which are should be kept aspairs or should remain on the motherplant). Buds of 1.5-3.0 cm having morethan three leaves can be decapitated at aheight of 1.0 cm and cut in half. This wayrosette growth is reduced by 2% and onaverage 4.7 buds per explant are obtainedin the multiplication phase. ■

ReferencesAfza R., M. Van Duren, R. Morpurgo & F.J. Novak.

1996. Banana tissue culture and its prospectiveuse in the developing countries. Pp. 58-70 in PlantTissue Culture (A.S. Islam, ed.). Oxford & IBHPublishing Co. Pvt. Ltd., New Delhi/Calcutta.

García L. & J.C. Noa. 1998. Obtención de plantaslibres de patógenos. Pp. 135-148 in Propagación ymejora genética de plantas por biotecnología(J.N. Pérez Ponce, ed.). Instituto de Biotecnologíade las Plantas. Universidad Central de las Villas,Santa Clara, Cuba.

Jimenéz E. 1998. Generalidades del cultivo in vitro.Pp. 13-22 in Propagación y mejora genética de


Figure 1. Young plants of the clone FHIA-20used for introduction into the laboratory.

Figure 2. Treatments studied during the initiation phase in vitro.

Table 1. Behaviour of apices during the initiation phase after 20 days’ in vitroculture.

Treatments Regeneration Number Infection Mortalityof apices (%) of buds per apex (%) (%)

1 (control) 40.0 b 0.25 c 15 a 0.0 b

2 40.0 b 1.10 b 20 a 0.0 b

3 35.0 b 0.00 c 15 a 40.0 a

4 85.0 a 2.42 a 15 a 0.0 b*identical letters in the same column indicate that the results do not differ significantly at P < 0.05%.

Treatments

1. 0.5 cm2 apex cultured in liquid medium (control).

2. 0.5 cm2 apex cultured in semi-solid medium.

3. 1.0 cm2 apex cut in two and cultured in liquid medium.

4. 1.0 cm2 apex cut in two and cultured in semi-solid medium.

Table 2. Growth behaviour of buds during the multiplication phase.

Treatments Number of buds per explant Buds with rosetted growth (%)

1 (control) 1.24 b 44.0 a

2 2.10 b 24.0 b

3 2.20 b 6.00 c

4 4.70 a 2.00 c*identical letters in the same column indicate that the results do not differ significantly at P < 0.05%.

Treatments

1. Multiplication medium with 4.0 mg.L-1 of BAP + protocol 1 (control).

2. Multiplication medium with 4.0 mg.L-1 of BAP + protocol 2.



plantas por biotecnología (J.N. Pérez Ponce, ed.).Capitulo 8. Instituto de Biotecnología de lasPlantas. Universidad Central de las Villas, SantaClara, Cuba.

Orellana P. 1994. Tecnología para la propagaciónin vitro de clones de Musa spp. Tesis para optarpor el grado de Doctor en Ciencias Agrícolas.Instituto de Biotecnología de las Plantas,Universidad Central de las Villas. 104pp.

Orellana P. 1998. Propagación vía organogénesis. Pp.151-176 in Propagación y mejora genética de plan-tas por biotecnología (J.N. Pérez Ponce, ed.).

Instituto de Biotecnología de las Plantas.Universidad Central de las Villas, Santa Clara,Cuba.

Pérez Ponce J.N., E. Jiménez & D. Agramonte. 1998.Aumento de la eficiencia en la micropropa-gación. Pp. 179-190 in Propagación y mejoragenética de plantas por biotecnología (J.N. PérezPonce, ed.). Instituto de Biotecnología de lasPlantas. Universidad Central de las Villas, SantaClara, Cuba.

Leifert C., C. Morris & W.M. Waites. 1994. Ecology ofmicrobial saprophytes and pathogens in tissue

culture and field-grown plants: reasons for conta-mination problems in vitro. Critical Reviews inPlant Sciences 13(2): 1 39-183.

Vasil I. 1994. Automation in plant propagation. PlantCell, Tissue and Organ Culture 39(2): 105-109.

The authors work at the Instituto de Biotecnologíade Plantas, Universidad Central “Marta Abreu” de LasVillas, Carretera a Camajuaní Km. 51/2, CP 54830,Santa Clara, Villa Clara, Cuba. E-mail: [email protected]


Multiplication rate and regeneration potential of somatic embryos from a cell suspension of banana(Musa AAA cv. “Grande naine”)

Tissue culture Cell suspensions

S.L. Lerma, P. Acuña, A.S. Riveros and J.A. Sandoval

The production of bananas and plan-tains is widely distributed in tropi-cal and sub-tropical regions. The

area cultivated, estimated to be around10 million hectares, gives an annual yieldin the order of 88 million tonnes. Thiscrop, whose fruit forms part of the diet ofmore than 400 million people, ranksfourth in the world in the category of sta-ple food products, after rice, wheat andmilk (FAO 1999).

In view of the interest in banana grow-ing, considerable research effort hasbeen devoted to the improvement andcontrol of its mass propagation by meansof biotechnological techniques such assomatic embryogenesis, for which threeprotocols have been described using vegetative tissue such as fragments of thecorm and leaf bases (Novak et al. 1989,Ganapathi et al. 1999), cultures of prolif-erating meristems (Dhed’a et al. 1991,Dhed’a 1992, Schoofs 1997, Schoofs et al.1998), and immature male or femaleflowers (Escalant et al. 1994, Grapinet al. 1996).

The use of cell suspensions in somaticembryogenesis and the discovery of thefactors involved in the metabolic synchro-nization of cell suspensions and its tim-ing constitute two fundamental aspectseither of the procedures for applyingmethods of temporary immersion formass micropropagation of economically

important plant material (Escalant et al.1994, Gómez-Kosky et al. 2000), or oftheir use in genetic improvement pro-grammes by the induction of mutations,the study of selections in vitro (by meansof fungal toxins or plant extracts) andgenetic transformation by particle bom-bardment. Despite all the research car-ried out internationally in various labora-tories, it is still difficult to maintain cellsuspensions effectively. The setting up ofbanana cell cultures free from bacterialcontamination, changes due to oxidationor possible fungal attacks requires a lotof time, and their maintenance thereforeproves to be difficult (Schoofs et al.1999). The aim of the work presentedhere was to determine, by using sourcesof carbon and growth regulators, the opti-mum experimental conditions for the set-up and multiplication of a cell suspensionon the one hand, and for the regenerationof the somatic embryos on the other.


Maintenance of cell suspensions and homogenization of culturesThe plant material used to initiate thecell suspensions consisted of immaturemale flowers of Musa AAA cv “Grandenaine” which had been placed on M1induction medium [Murashige & Skoog(1962) salts - MS, 1 mg/L biotin, ANA andAIA, 4 mg/L 2,4-D, 6 g/L agarose, 30 g/Lsaccharose, pH 5.71] proposed by Grapinet al. (1998) for forming calluses. The fri-able embryogenic tissue obtained was

transferred into M2 cell suspensionmedium [MS salts, 100 mg/L glutamineand malt extract, 1 mg/L 2,4-D, 45 g/L sac-charose, pH 5.3] until its establishment.This technique for somatic embryogene-sis was originally developed by Escalantet al. (1994) and it is currently applied in the biotechnology laboratory of CORBANA on this same clone (Acuña andSandoval 2000).

From this initial suspension, new cul-tures were started during the phase ofmaintenance in M2 medium onto amedium made up of 35 ml of fresh M2medium and 13 ml of the previous M2medium (in which the suspension hadbeen maintained during the precedingcycle), a mixture into which were intro-duced 2 ml of cells made up to a total volume of 50 ml per erlenmeyer flask.These suspensions were subjected tofour treatments: T0 = 45 g saccharose; T1 = 45 g saccharose + 100 mg/Lmyoinositol; T2 = 30 g saccharose + 100 mg/L myoinositol; T3 = 15 g sac-charose + 100 mg/L myoinositol. Therewere 10 replicates (Figure 1).

Four subcultures were made whichwere each incubated for 14 days as pro-posed by Escalant et al. (1994). Thenumber of cells and the percentage via-bility of the suspensions were recordedon the 1st, 7th, and 14th days of the cul-ture using a haemocytometer. Therewere three replicates and 5 counts foreach, making a total of 15 readings pertreatment. Also, every 15 days, theincrease in cell volume was measured by

the sedimentation method (SCV) pro-posed by Schoof (1997) and the packedcell volume (PCV) as used by Reinertand Yeoman (1982). Four extra repli-cates were also made for monitoring thepH (2 in the inoculated medium and 2 inthe non-inoculated medium), the mea-surements being made at the beginningand end of each subculture.

In order to evaluate the effect of thegrowth regulators on the quality of thecell suspension in the M2 medium, thetreatment was selected which showed thehighest multiplication rate and cell via-bility during the first four subcultures ofthe maintenance phase. For this study,we added to the selected M2 medium: A1= 0.5 mg/L 2,4-D, A2 = 1 mg/L 2,4-D andA3 = 2 mg/L 2,4-D. The material was han-dled the same way as the treatmentsinvolving different saccharose concentra-tions. For evaluation, the same parame-ters were used as in the maintenancephase of the cell suspensions (mentionedabove). The cell morphology was alsonoted, as clusters or solid masses, andphotographs were taken using both opti-cal and electron microscopes.

Regeneration of somatic embryosThe viability of the process was evaluatedby observing the embryos obtained on theculture medium of Schenk andHildebrandt (1972), called modified M3[10 mg/L biotin, 100 mg/L of glutamineand malt extract, 230 mg/L proline,1 mg/L ANA, zeatin and 2-IP, 10 g/L lac-tose, 45 g/L saccharose and a pH of 5.3].The M3 medium was put into Petri dishesand sterile filter papers were placed onthe surface on which were inoculatedaliquots of 1 ml of the cells of treatmentscorresponding to the different growthregulator concentrations. The type ofmaterial regenerated was evaluated bymaking three evaluations per Petri dishfrom the zones where the distribution ofthe suspension was most homogeneous.All the cultures were maintained underconditions of controlled temperature(27ºC), relative humidity (80%) and pho-toperiod (12 hours).

Statistical analysisThe results obtained for the phase ofmaintenance of cell suspensions andhomogenization of cultures for variationin pH, cell volume, number of cells andpercentage viability, were analyzed by alinear modelling scheme and subjectedto analysis of variance using the SAS pro-gramme (1990). Results showing hetero-geneity of variance were adjusted tohomogeneity by using a square root trans-formation.


Effect of different saccharose or saccharose + myoinositolconcentrations on the maintenance of cell suspensions and the homogenization of the culturesThe results for the increase in numbersof cells, presented in Figure 2, indicatethat the dose of 30 g of saccharose pro-vides enough carbon for the suspension,as its behaviour differed little from thatof the suspension maintained with 45 g ofsaccharose. In general, the addition ofmyoinositol (T1-T2) did not alter thebehaviour of the cells and a tendency wasnoted to stabilization in subculture 4(relation of T1 and T2 with T10).

There were no significant differencesbetween the percentages of viability ofthe treatments with and withoutmyoinositol (T1 and T0 respectively). Nodifferences were noted between theobservations at different times (7 or14 days) nor any interactions betweenthe subcultures and time of observation(P = 0.1574).

On the other hand, as for the behaviourof this same percentage viability fortreatments with myoinositol associatedwith different saccharose concentrations(T1 and T2), it was found that the differ-ences for the four subcultures depended

on the treatment (P = 0.0040). This dif-ference in behaviour of distinct cell linesof a single clone may be an intrinsic char-acteristic of the material (Schoofs et al.1999), which would indicate the need toredouble efforts towards improving thesemethods.

Treatment T3 (15 g saccharose +myoinositol) was eliminated because itshowed progressive diminution of 5.18,4.20 and 2.06 ml in subcultures 1,2 and 3respectively. This low success rate of cellproliferation may be attributable to thelow availability of sugars in the mediumcompared with the demand of the cells inphase G1 of the cell cycle or to theosmotic shock due to the medium.Whichever it is, it is well known that sac-charose as a source of carbon is a stabi-lizer of culture media (Takeuchi andKomamine 1982, Vardi et al. 1982, Smithet al. 1984).

The differences in cell volume foundbetween treatments T0, T1 and T2 (P = 0.02602) were much reduced in sub-cultures 1 and 2 (Figure 3a and b) butwere accentuated in subcultures 3 and 4(Figure 3c and d) for which the differ-ence between treatments T0 and T1 canbe attributed to the action of myoinositol.Subcultures 2, 3 and 4 of T1 (withmyoinositol) produced a mean cell vol-ume 0.67 ml greater (P = 0.0188) than


Initial suspension M2

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S 30 gr. +100M mg/L

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Figure 1. General scheme of the protocol followed to study a cell suspension of banana (cultivar“Grande naine”). a) Experiment 1: M2 cell suspension medium; S = saccharose; M = myoinositol; T = treatment; No. = replications. b) Experiment 2: Different concentrations of 2,4-D. c) Evaluation of embryo formation.

that reached in the T0 subculture homo-logues. These results agree with thosefrom other research on banana and othercrops: for example Cronauer andKrikorian (1983) and Aftab et al. (1999)confirm the stimulatory action ofmyoinositol on mitosis and morphogene-sis of plant cells.

The difference between the four sub-cultures was on average 0.23 ml in favourof treatment 1. The treatment whichresponded best was that containing 45 gsaccharose and 100 mg/L myoinositol. Itwas also noted that the cell volume ofsubculture 1 was 5.95 ml and that of sub-culture 4 was 7.59 ml, representing amean increase of 0.65 ml for each. Thereis a positive correlation (Figure 3e)between the number of subcultures andthe cell volume, since this latterincreases with the number of subcul-tures, finally to stabilize at the fourthsubculture.

After mixing, the 35 ml of freshmedium and the 13 ml of old medium hada pH of 4.74. During the 14 days of cul-ture, the non-inoculated media remainedbetween pH 4.1 and 4.2 and the inocu-lated media between 4.4 and 4.6 (unpub-lished results). In the cell suspensions,

the pH varied with time, treatment andtheir interaction (P = 0.0001): similarbehaviour was observed by Skirvin et al.(1986). These same authors suggestedthat acidification of the medium couldbe due to ionic exchange between thecell and the culture medium, leading toan optimum pH for the normal function-ing of the cell wall.

Response of the suspension to differentconcentrations of 2,4-DAnalysis of the results obtained for thevariables “number of cells” and “percent-age viability” on media containing differ-ent concentrations of 2,4-D did not showany marked differences in their behav-iour. Treatments A1, A2 and A3 of the foursubcultures had mean numbers of cells of7.9, 6.0 and 7.0 and percentage viabilitiesof 59, 62 and 59 respectively.

When one studies the cell volumeobtained with varying concentrations of2,4-D (Figure 4) it is seen that treatmentA1 (1 mg/L of 2,4-D) is that which main-tains the best cell suspension with amean volume of 7.6 ml and a maximum of8.8 ml in subculture 3. The 2 mg/L dose of2,4-D was the best for standardizing thecell volume of several subcultures, which

is a useful parameter for carrying outstudies of the cell cycle or cell metabo-lism and other phenomena connectedwith synchronized cell populations.

The final results from subculture 4measured by the PCV method show thatall the treatments have progressivelyincreased the cell volume without largefluctuations during the 14 days of incuba-tion and that this volume doubled on thesixth day, when the cells begin a phase ofactive division (Figure 5). These resultsagree with those obtained by Bieberach(1995) for various Musa clones.

As to the use of 2,4-D and appropriatedoses, the results given here supplementthe information about the action of thisgrowth regulator on the embryogenicprocess and the doses required by differ-ent plant species. Lazzeri et al. (1987)emphasize the importance of auxins inthe regulation of the somatic embryogen-esis of soyabean and show that there isbetter somatic embryo production when2,4-D is used alone than in combinationwith acetic a-naphthalene.

The morphology of cells in suspensionwas observed by optical microscopy atmagnifications of 20x and 40x. Thepreparations showed cell clusters and


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Figure 2. Number of cells obtained with three treatments of multiplication media for banana cell suspensions (Musa AAA cv. “Grande naine”). Treatment mean for four subcultures n = 3. T0 = 45 saccharose; T1 = 45 g saccharose + myoinositol; T2 = 30g saccharose + myoinositol.Bars represent standard errors.

isolated cells (Figures 6a and 6b), whichconforms with descriptions by Grapin(1996) who reported that in suspensionsof ‘French Sombre’ clusters were seenwhich could reach 70 to 80% of the vol-ume of the suspension very similar towhat we observed in the course of thiswork. The clusters are formed by pre-embryogenic cells (Figure 6c) possess-ing partitions or cellular plates typical ofthe last stage of mitosis and of cellswhich are empty or in the course of dif-ferentiation.

The isolated cells are round, with adense cytoplasm and a well-definednucleus: one may regard them as proto-


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Figure 3. Cell volumes in the multiplication media of cell suspensions of banana (Musa AAA cv. “Grande naine”); a: means of treatments in subculture 1 (n = 10); b: means of treatments in subculture 2 (n = 9);c: means of treatments in subculture 3 (n = 9); d: means of treatments in subculture (n = 6); e: means of treatments in subcultures 0, 1, 2 (n = 23); T0 = 45 g saccharose; T1 = 45 g saccharose + myoinositol; T2 = 30 g saccharose + myoinositol; T3 = 15 g saccharose + myoinositol.Bars represent the standard errors.

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Figure 4. Cell volumes in the multiplication media of banana (Musa AAA cv, “Grande naine”). Mean of treatments A1, A2 and A3 containing 2,4-D in subcultures 1, 2, 3 and 4.Bars represent the standard errors.


plasts, initial cells with a primary cellwall that is characteristic of undifferenti-ated cells and with an active cell cycle.These observations are shared both byBieberach (1995) who noted the pres-ence of cells with identical morphologi-cal characteristics in cell suspensions ofthe cultivars “Dominico”, “Grande naine”

and “Gros Michel” and also by Sannasgala(1989) who described pre-embryos madeup of protein bodies and starch.

The characters described above are afactor indicating the embryogenic condi-tion of the cell suspension (Williams andMaheswaren 1986). Certain isolated cellswith elongated cytoplasm containing

lacunae are non-viable cells in a suspen-sion because they have already formedtheir secondary cell wall.

Under the scanning electron micro-scope, round cells were seen of 50-80µmdiameter, with rough walls with irregularornamentations surrounded by a polysac-charide mucus (Figures 7a and 7b).

Regeneration of somatic embryosMixtures of cell samples treated withgrowth regulators were inoculated andmaintained for 55 days on the semi-solidM3 medium to develop embryos. By theend of 22 days their growth began to beapparent, without traces of oxidation.The presence was detected of small clus-ters of 1 cm2 containing globular embryosin the shape of a heart or torpedo. Theembryos were sorted with a view to laterregeneration of the plants (Figure 8a). Atotal of 200 “torpedo-type” embryos weretransferred into eight Petri dishes at therate of 25 embryos per dish. At the end of20 days there was 63% germination, andafter 41 days the plants possessed normalmorphological characteristics. Until thenthe percentage germination for somaticembryos of the genus Musa oscillatedbetween 45% and 80% according to thegenotype and culture medium (Bieberach1995, Escalant et al. 1995, Côte et al.1996, Schoof 1997, Grapin et al. 1998).

Figure 6. Photomicrograph of cells and clusters,a: viable isolated cells, b: pre-embryogenic cells,c: clusters. N = nucleus, T = cell plates, C = clusters, CD = differentiating cells, CI = individual cells, CP = pre-embryogenic cell.

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Figure 5. Increase in cell volume (PCV) of a suspension of banana (Musa AAA cv, “Grande naine”) with different growth regulator concentrations.a, b, c: means of treatments (n = 10) in subculture 4.

Figure 8b shows the potential regene-ration of somatic embryos from cell sus-pensions. Escalant et al. (1994) haveobtained the highest germination per-centages, achieved by using temporaryimmersion systems with other bananacultivars.

ConclusionsThese experiments have enabled us tostandardize a protocol for obtainingembryos of Musa AAA cv. “Grande naine”from cell suspensions and by usinggrowth regulators. The initial cell suspen-sion was maintained with 45 g saccharose+ 100 mg/L myoinositol. An initial pH of4.74 and four subcultures of 14 days eachguaranteed a sufficient cell volume andembryos with a good percentage viability.The optimal dose of growth regulator forthe efficacy of the process is 1 mg/L of2,4-D applied as an exogenous hormone.The morphological observations revealthat the protocol has allowed the devel-opment of viable cells which are easilytransformed into embryos. The germina-tion of embryos has validated the entiremethod and the doses used.

AcknowledgementsThe authors are pleased to thank theBiotechnology Unit of the NationalBanana Corporation (CORBANA) ofCosta Rica for permitting the experimen-tal part of the work, and the University ofCosta Rica, where the photomicrographsshown in this article were taken.

Note: Extract from the Biology thesisof Sandra Liliana Lerma defendedbefore the Faculty of Sciences,University of Tolima, April 2001. Ibagué,Tolima, Colombia. ■

ReferencesAcuña P. & J. Sandoval. 2000. Embriogénesis

somática en banano (cv. “Gran enano”) a partir deflores masculinas. Pp. 20-22 in Informe Anual.Dirección de Investigaciones, CORBANA, SanJosé‚ Costa Rica.

Aftab F. & J. Iqbal. 1999. Plant regeneration fromprotoplasts derived from cell suspension ofadventive somatic embryos in sugarcane(Saccharum spp. hybrid cv. CoL-54 and cv. CP-43/33). Plant Cell Tissue and Organ Culture56(3): 155-162.

Bieberach C. 1995. Embriogénesis somática y regen-eración de plantas en cultivares de Musa spp.

Thesis submitted for the Magister Scientiaedegree. CATIE, Turrialba, Costa Rica. 84pp.

Cronauer-Mitra S. & A.D. Krikorian. 1983. Somaticembryos from cultured tissues of triploid plan-tains (Musa ABB) Plant Cell Reports (2): 289-291.

Côte F., R. Domergue, S. Monmarson, J. Schwendiman,C. Teisson & J.V. Escalant. 1996. Embryogenic cellsuspensions from the male flowers of Musa AAA cv.Grand Naine. Physiologia Plantarum 97: 285-290.

Dhed’a D. 1992. Culture de suspensions cellulairesembryogéniques et régénération en plantules parembryogenèse somatique chez le bananier et lebananier plantain (Musa spp.) Thèse PhD,KuLeuven, Belgique. 171pp.

Dhed’a D., F. Dumortier & B. Panis. 1991. Plantregeneration in cell suspension cultures of thecooking bananas cv. Bluggoe (Musa spp. ABBgroup). Fruits 46: 125-135.

Escalant J.V., C. Teisson & F. Côte. 1994. Amplifiedsomatic embryogenesis from male flowers oftriploid banana and plantain cultivars (Musa spp.).In Vitro Plant Cell. and Dev. Biol. 30: 181-186.

Escalant J.V., C. Bieberach, L.E. Pocasangre, L. delS. Espinoza, R.G. Kosky, J.L. Ortiz & C. Teisson.1995. Regeneration through somatic embryo-genesis from male flowers of banana and plan-tain: 1. Amplification by temporary immersion.(Abstract). in Simposio CIRAD/CATIE.Mejoramiento genético y desarrollo de los cul-tivos tropicales. Resúmenes. CATIE, Turrialba,Costa Rica. 54pp.

FAO. 1999. FAO Quarterly Bulletin of Statistics 12(3-4).

Ganapathi T.R., N. Higgs, J. Van Eck, P. Balint-Kurti& G.D.May. 1999. Transformation and regenera-tion of the banana cultivar “Rasthali” (AAB). P. 34in Abstracts of the International symposium onthe molecular and cellular biology of banana, 22-25/03/1999, Cornell University, Ithaca, NY,USA. Boyce Thompson Institute for PlantResearch, Inc., Ithaca.

Gómez-Kosky R., T. Gilliard., L.A. Barranco & M. Reyes.2000. Somatic embryogenesis in liquid media:maturation and enhancement of germination ofthe hybrid cultivar FHIA-18 (AAAB). INFOMUSA

9(l): 12-16.Grapin A., J. Schwendiman & C. Teisson. 1996.

Somatic embryogenesis in plantain banana. InVitro Cell. Dev. Biol. Plant. 32: 66-71.

Grapin A., J-L. Ortiz, R. Domergue, J. Babeau, S.Monmarson, J.V. Escalant, C. Teisson & F. Côte.1998. Establishment of embryogenic callus andinitiation and regeneration of embryogenic cellsuspensions from female and male immature flow-ers of Musa. INFOMUSA 7(1): 13-15.

Lazzeri P., D Hildebrand & G. Collins. 1987. Soybeansomatic embryogenesis. Effects of hormones andculture manipulations. Plant Cell, Tissue andOrgan Culture 10: 197-208.

Murashige T. & F. Skoog. 1962. A revised medium forrapid growth and biossays with tobacco tissue cul-tures. Physiol. Plant. 15: 473-497.

Novak F.J., R. Afza, D.M. Van, D.M. Perea, B.V.Conger & X. Tang. 1989. Somatic embryogenesisand plant regeneration in suspension cultures of


Figure 7. Scanning electron microscope photographs. a: viable cells, b: external surface of a cell.

Figure 8. Formation of embryos of banana Musa AAA cv, “Grande naine” on M3 medium; a: 55-day old embryo cluster. (*) indicates “torpedo-type” embryos. b: germination and growth of embryos on M3 medium.

dessert (AA and AAA) and cooking (ABB)bananas (Musa spp.) Bio/Technology 7: 154-159.

Reinert J. & M.M. Yeoman. 1982. Plant cell and tis-sue culture. A laboratory manual. Springer Verlag,Berlin-Heidelberg-New York.

Sannasgala K. 1989. In vitro somatic embryogenesisin Musa. PhD Thesis, KULeuven, Belgium. 189pp.

SAS Institute. 1990. SAS/STAT User’s Guide. Version6.4. SAS Institute, Inc., Cary, NC.

Schenk R.U. & A.C. Hildebrandt. 1972. Medium andtechniques for induction and growth of mono-cotyledonous and dicotyledonous plant cell cul-tures. Can. J. Bot. 50: 199-204.

Schoofs H. 1997. The origin of embryogenic cells inMusa. PhD Thesis, KULeuven, Belgium. 258pp. + annexes.

Schoofs H., B. Panis & R. Swennen. 1998.Competence of scalps for somatic embryogenesisin Musa. Acta Horticulturae 490: 475-483.Proceedings of the 1rst International symposiumon banana in the subtropics. ISHS.

Schoofs H., B. Panis, H. Strosse, A. Moyo, J. Lopez, N.Roux, J. Colezel & R. Swennen. 1999. Bottlenecksin the generation and maintenance of mor-phogenic banana cell suspensions and plantregeneration via somatic embryogenesis there-from. INFOMUSA 8(2): 3-7.

Skirvin R.M., M.C. Chu, M.L. Mann, H. Young, J.G.Sullivan & T.W. Fermanian. 1986. Stability of tis-sue culture medium pH as a function of autoclav-ing, time, and cultured plant material. Plant CellReports 5: 292-294.

Smith M.A.L., J.P. Palta & B.H. Mc Cown. 1984. Themeasurement of isotonicity and maintenance ofosmotic balance in plant protoplast manipula-tions. Plant Sci. Let. 33: 249-258.

Takeuchi Y. & A. Komamine. 1982. Effects of cultureconditions on cell division and composition ofregenerated cell walls in Vinca rosea protoplasts.Plant Cell Physiol. 23: 249-255.

Vardi A., P. Spiegel-Roy & E. Galum. 1982. Plantregeneration from Citrus protoplasts: variability

in methodological requirements among cultivarsand species. Theor. Appl. Genet. 62: 171-176.

Williams E.G. & G. Maheswaran. 1986. Somaticembryogenesis: factors influencing coordinatedbehaviour of cells as an embryonic group. Annalsof Botany 57: 443-462.

Sandra Liliana Lerma works in the Laboratorio deProtección de Plantas, Departamento de Biología,Universidad del Tolima, Ibagué (Tolima), Colombia, e-mail: [email protected]; Pablo Acuña is Assesorin Plant Biotechnology, Guápiles, Costa Rica, e-mail:[email protected]; Alba Stella Riveros isAssociate Researcher in the framework of theConvenio Universidad del Tolima-CATIE, Unidad deFitoprotección, CATIE, Turrialba, Costa Rica, e-mail:[email protected] and Jorge Arturo Sandoval isSubdirector of Research at CORBANA, Guápiles, CostaRica. e-mail: [email protected].


Introduction and multiplication of improvedbananas and plantains in Nicaragua and distribution to farmers

Improved varieties Partnerships for distribution in Nicaragua

K. Dens, M. Vargas, G. Matton, S. Coessens, I. Van den Houwe

and R. Swennen

Banana and plantain in NicaraguaUnlike most other Central-Americancountries, the production of banana andplantain in Nicaragua is low (Table 1).The major production zones of bananasand plantains are located in the coastalarea near the Pacific Ocean. In theChinandega region (North-West) an esti-mated area of 2000 ha Cavendish(Musa cv. AAA) is grown for export, whilein the Rivas region (South of Managua)13 000 ha plantain are cultivated for localconsumption. For many small- andmedium-scale farmers in Rivas, plantainis the most important crop. Gros Michel(Musa cv. AAA), Bluggoe (Musa cv. ABB)and Silk (Musa cv. AAB) bananas are cul-tivated all over the country, mainly bysmall-scale farmers in backyards. In thehigher regions of Central Nicaragua, withaltitudes up to 1300 masl, bananas aregrown in combination with coffee orcacao. Bananas and plantains are alsoimportant for the people at the AtlanticCoast. Pelipita (Musa cv. ABB) and Red

(Musa cv. AAA) bananas are found insome regions of Nicaragua.

Plantain (Musa cv. AAB) is most pre-ferred because it is an attractive cashcrop. The most common landraces areFalse horn plantain with an average ofonly 25 fingers. The price of plantain onthe local market is much higher than theprice of the other bananas (Gros Michel,Bluggoe, Silk) because of its much largerfinger size and longer green life.Cavendish only enters the local market asafter being rejected on the export planta-tions. Its price is even lower than that ofGros Michel. Over the last five yearsprices of plantain have continued toincrease, which reflects the high demandand insufficient supply of bananas and

plantains caused by poor cultivation prac-tices, drought and pests and diseases.

Diseases and pests are the main prob-lems; black Sigatoka (Mourichon et al.1997) and plant material contaminatedby weevils (Gold and Messiaen 2000) arethe major constraints affecting thesmall-scale plantain producer. Anotherimportant problem, especially in theLeon-Chinandega region, is the unevendistribution of annual rainfall. Withoutirrigation banana and plantain yields arereduced due to the long dry season.

Creating multipliying effectsThe objective of the intervention is tocontribute to food security and foodquality of resource-poor farmers by giv-

Table 1. Production, export and consumption data of banana and plantain in fiveCentral American countries.

Population in 1999 Production in 2000 Export in 1999 Consumption in 1999(millions) (metric tonnes) (metric tonnes) (kg/cap/year)

Guatemala 10.8 802 545 576 900 4.5

Honduras 6.1 702 578 155 200 63.9

Nicaragua 4.8 13 636 37 846 14.5

Costa Rica 3.8 2 790 000 2 557 000 29.5

Panama 2.8 918 382 596 900 43.7Source: FAO on INIBAP’s Web site (http://www.inibap.org/network/statistics_eng.htm).

ing support to banana and plantain cul-tivation. Food insecurity is very high inNicaragua and the number of under-nourished people increased from 1.2 million in 1991 to 1.4 million in 1998(FAO 2001). The project focuses on theLeon-Chinandega region (Figure 1),where the poorest farmers live andwhere bananas and plantains could bepart of more diversified agriculture sys-tems, now that the cotton monoculturehas disappeared.

In 1996, the Universidad NacionalAutónoma de Nicaragua (UNAN), basedat Leon, and the Flemish Office forDevelopment Aid and TechnicalAssistance (VVOB) started their inter-vention with technical assistance fromthe Katholieke Universiteit Leuven(KULeuven). Improved germplasm camefrom the Fundación Hondureña de

Investigación Agrícola (FHIA) and theInternational Institute of TropicalAgriculture (IITA) via the INIBAP TransitCentre (Diekmann and Putter 1996). Thecompany Bananic supported this inter-vention by covering the operational costsof the laboratory and field activities.

Tissue culture facilities were set up atUNAN to produce high-value banana andplantain varieties. Selected varieties areevaluated at UNAN’s test farm beforedistribution to small-scale farmers(Table 2). They are sorted in four maincategories by local farmers according totheir comparison with Bluggoe, thelocally known False horn plantain, GrosMichel and Silk. At harvest, palatabilitytests are conducted with the farmers.Extension personnel of the projectteach relevant cultivation and field mul-tiplication techniques. Partnerships

were built with about 20 national andinternational organizations operating inNicaragua (Table 3), to accelerate thedistribution of improved plants andtechnologies, and to obtain a maximumfeedback from the farmers.

AchievementsThe project started in mid-1996. Rootedplantlets were sent by KULeuven forweaning in the small nursery of the UNANfarm in Leon, located a few kilometresfrom Leon City centre. These plants wereused for the first test plots at theUniversity farm.

The tissue culture laboratory of UNANwas built in 1997. Tissue culture tech-niques were transferred from KULeuvento UNAN that produced plantlets toextend the test fields at the Universityfarm. In cooperation with the Centro deEnseñanza Técnica Agropecuaria(CETA), workshops were organized in sixcommunities in Chinandega.

In 1998, five extension brochures incartoon style were developed for distribu-tion to participating farmers (Figure 2).At the test farm of UNAN, a field collec-tion was established containing both theintroduced and the locally grown vari-eties (40 in total), and 2 plots of 36 plantsof each variety were evaluated (Table 2).

In 1999, two trained Nicaraguan tech-nicians of the tissue culture laboratoryproduced 6500 plants. One hundred andforty new test plots were planted in thenorthwestern region of Nicaragua, mostlyin Chinandega because of the coopera-tion with CETA and the larger agricultureactivity of that region.

In 2000, 20 000 plants have been dis-tributed to 370 new farmers, comprisingfarmers of the Leon region as well(Figure 1). Ten thousands plantlets wereimported from KULeuven to acceleratethe distribution of the new varieties.Oxfam-Belgium contracted the UNAN todistribute 25 000 plants of superior vari-eties to nearly 1000 families that wererelocated after the hurricane Mitch inOctober 1998 and urgently needed newplant material. Therefore the shadehouse was extended to 700 m2.

In 2001, a few test fields were alsoplanted in the Rivas, central Nicaraguaand Atlantic coast regions, where a selec-tion of farmers received in vitro bananaand plantain plants.

The number of plants produced anddistributed by UNAN’s tissue culture lab-oratory increased from 2000 in 1998 to 15 000 in 2001. The number of farmersparticipating also increased considerably– from 40 at the start of the project in1998, to a total of 820 having received


Figure 1. Operational area of the projectshowing the location of demonstration fields in the Leon-Chinandega region.● represents 10 farmers’ fields; ● represents 25 backyards.

improved varieties and participated inthe project in 2001. During 2001, theplanting material was sold to cooperatinginstitutes who distributed the plants intheir own development programmes.

A total of 2757 farmers were trained indifferent workshops and 1500 brochuresdealing with field selection and prepara-tion and plant material were distributed.Workshops for farmers were organized incollaboration with local NGOs, govern-mental and international organizations,which drastically increased the contactwith farmers (Table 3). The project alsoparticipated in the organization ofregional and national workshops forextension workers. Six new brochureswere developed about diseases and pestcontrol. A catalogue of the new acces-sions following the format of Musalogue(Daniells et al. 2001) was made available.

Close contact is being maintained withthe farmers who are growing the newvarieties (Figure 3) to assess their reac-tion and improve the efficiency of theintervention. Interviews are conductedto determine the acceptance rate of thenew varieties and to identify the under-lying reasons, e.g. appearance, taste, cul-tivation as a cash crop and/or food crop,etc. (Table 4). The most popular varietyso far is FHIA-03, because of its droughtresistance and large bunches that arecomparable to the local Bluggoe cookingbanana. Tasting sessions are organizedon a regular basis and the new varietiesare prepared according to prevailingNicaraguan customs i.e. fried, green andripe plantains, plantain chips, cookedgreen and ripe plantain, and banana as adessert. People are asked to comparethe new fruits with the local fruits (Falsehorn plantain, Bluggoe or Silk). Firstresults confirm the acceptability of mostvarieties but also show that palatabilitytests are absolutely necessary sincevisual aspects can determine consumers(Table 5).

Plans for the futureCurrently the laboratory has the capacityto produce 50 000 in vitro plants perannum. Plans are being made to furtherupscale the production capacity toensure the sustainability of the tissueculture laboratory by selling the plantmaterial. Small-scale farmers willreceive plant material at subsidizedprices while commercial growers willhave to pay a higher price.

The best accepted banana varieties willbe produced in large amounts as well asother food crops for which a demandexists in Nicaragua.


Table 2. Harvest characteristics of 23 varieties obtained from test fields during theirfirst and second cycles (C1-2) and third and fourth cycles (C3-4). Varieties are grouped according to consumers’ preference.

ITC Plant height Bunch weight No. No. of Number Genome (cm) (kg) of hands fingers

Name C1-2 C3-4 C1-2 C3-4 C3-4 C3-4

Cooking bananas

Cuadrado1 (Bluggoe) ABB 310 356 19.5 20.5 6.5 102

FHIA-03 0506 AABB 305 381 29.3 42.1 13.2 204

Pelipita 0396 ABB 420 392 22.9 23.8 10.0 152

Cardaba 0394 ABB 344 - 11.3 - *7.1 *90

Saba 1138 ABB 375 - 25.2 - *8.8 *131

Plantains

Cuerno1 (False horn) AAB 283 400 9.1 11.7 7.4 39

TMPx 1621 1205 AAAB - 352 - 15.8 6.0 88

TMPx 4479 (PITA 17) 1293 AAAB 325 361 12.7 14.8 6.3 89

TMPx 7002 1272 AAAB - 325 - 14.6 6.0 80

TMPx 7152 (PITA 14) 1294 AAAB 299 350 16.8 13.5 6.2 78

TMBx 5295 (BITA 2) 1297 AABB - 396 - 16.8 10.6 101

Dessert bananas

Patriota1 (Gros Michel) AAA 286 355 20.5 22.1 10.0 161

FHIA-01 0504 AAAB 254 342 26.4 30.2 10.5 162

FHIA-02 0505 AAAB 238 300 15.9 18.2 10.0 143

FHIA-17 1264 AAAA 334 - 37.5 - *12.5 *213

FHIA-23 1265 AAAA 339 - 20.1 - *10.3 *159

Dessert bananas

Rosa1 (Silk) AAB 332 358 17.0 18.6 8.5 151

Pisang ceylan 0650 AAB - 382 - 24.5 14.4 193

Yangambi km5 1123 AAA 259 339 15.4 19.5 9.9 171

TMBx 1378 (BITA 3) 1296 ABBB 382 418 20.5 22.0 10.7 151

Pisang mas 0653 AA 329 361 6.6 9.5 9.8 147

AA cv. Rose 0712 AA 265 289 6.9 11.1 12.3 199

Pisang lidi 0395 AA 267 306 5.0 8.6 7.4 1251 local varieties; * data of cycle 1-2; - data not available.

Table 3. National and international partners involved in the project.

Name Description

Project coordinating and executing institutes

INIBAP International Network for the improvement of Banana and PlantainKULeuven Katholieke Universiteit LeuvenUNAN Universidad Nacional Autónoma de Nicaragua

Organizations involved in plant distribution

Nicaraguan NGOsALISTAR Foundation for community developmentATC Asociación de Trabajadores del CampoBLOQUE Evangelistic Association for Education of FarmersCIPRES Centro de Investigación y Promoción para el Desarrollo Rural y SocialSGJRH Association of Garmendia Jiron with Limited Responsibility UNAG Unión Nacional de Agricultores y GanaderosUNAPA Unión Nacional Agropecuaria de Productores AsociadosXochilt Acalt Women Association of Malpaisillo

Governmental organizationsCETA Centro de Enseñanza Técnica y AgropecuariaINTA Instituto Nicaragüense de Tecnología AgropecuariaMAG-FOR Ministerio de Agricultura, Ganaderia y Forestales

International organizationsCARE North-American NGOCATIE Centro Agronómico Tropical de Investigación y EnseñanzaCLUSA (USAID) Cooperative League of USA EU European Union Project León-Chinandega FAO Food and Agriculture Organization of the United NationsOXFAM-Solidarity Oxford Committee for Famine Relief – BelgiumSI Solidaridad Internacional – Spain

Private companies

BANANIC Corporación Bananera NicaragüenseSETAGRO Servicios Técnicos Agropecuarios de Occidente

Distribution and extension work will beincreasingly coordinated by local organi-zations and NGOs. To facilitate this, theUNAN/VVOB staff participated in 2001 inthe foundation of a national Musa net-work, MUSANIC.

A baseline study about the socioeco-nomic situation of the collaborating farm-ers has been carried out to be able tomeasure the project-impact within a fewyears.

ReferencesDaniells J., C. Jenny, D. Karamura & K. Tomekpe.

2001. Musalogue: a catalogue of Musa germplasm.Diversity in the genus Musa. INIBAP, Montpellier,France.

Diekmann M. & C.A.J. Putter.1996. FAO/IPGRITechnical guidelines for the safe movement ofgermplasm. No. 15. Musa (2nd edition). Food andAgriculture Organization of the United Nations,Rome/International Plant Genetic ResourcesInstitute, Rome.

FAO. 2001. The state of food insecurity in the world2001. Food and Agriculture Organization of theUnited Nations, Rome, Italy. (Available on-line athttp://www.fao.org/SOF/sofi/index_en.htm).

Gold C. & S. Messiaen. 2000. The banana weevilCosmopolites sordidus. Musa Pest Fact SheetNo. 4. INIBAP, Montpellier, France.

Mourichon X., J. Carlier & E. Fouré. 1997. Sigatokaleaf spot disease: black leaf streak disease (blackSigatoka) and Sigatoka disease (yellow Sigatoka).Musa Disease Fact Sheet No. 8. INIBAP,Montpellier, France.

Koen Dens, G. Matton and S. Coessens work ascooperants of VVOB at the UNAN; M. Vargas isHead of the Musa project of the UNAN, Laboratoriode Cultivo de Tejidos; Iglesia la Merced 1/2 C al N;Facultad de Ciencias, UNAN-León, Nicaragua; E-mail: [email protected] ; http://www.unanleon. edu.ni/~vitro/;Ines Van den Houwe is germplasm conservation offi-cer at the INIBAP Transit Centre, Kasteelpark Arenberg13 – 3001 Leuven, Belgium. E-mail: [email protected] and Rony Swennen, Head of the Laboratory of TropicalCrop Improvement, Katholieke Universiteit Leuven,Kasteelpark Arenberg 13 – 3001 Leuven, Belgium. E-mail: [email protected];http:// www.agr.kuleuven.ac.be/dtp/tro/home.htm


Figure 2. Extensionbrochures distributed to farmers.

Figure 3. Farmer growing FHIA-03 in his backyard in the Leon region.

Table 4. Most preferred varieties and reasons for it (N = 80).

Variety Most important reason

False horn Plantain* (Cuerno) Market

FHIA-03 Drought-resistant, bunch size

TMBx 5295 Good taste

Bluggoe* (Cuadrado) Drought-resistant, firmness of fruit, taste

TMBx 1378 Shape of fruit, taste

Pelipita Firmness of fruit, taste* local varieties.

Table 5. Acceptability of the fruit taste and visual aspect (N = 80).

Fruit taste Fruit visual aspect

Mode of Compared % better Compared % better Variety preparation with or the same with or the same

FHIA-03 Ripe Bluggoe 95 Gros Michel 44

FHIA-01 Ripe Gros Michel 62 Bluggoe 68

Pisang lidi Ripe Silk 37 Silk 13

TMBx 1378 Ripe Silk 99

TMBx 5295 Ripe fried False horn 86 False horn 57

TMBx 5295 Green fried False horn 67

TMPx 4479 Ripe fried False horn 75

TMPx 4479 Green fried False horn 85 False horn 49

Pelipita Green fried False horn 80 False horn 12

Pelipita Ripe fried False horn 37

Pisang ceylan Ripe Silk 72 Silk 91

Nguyen Xuan Thu, Le Thi Lan Oanhand Ho Huu Nhi

Banana is an important plant intropical countries. Banana origi-nates from Musa acuminata

(AA) and Musa balbisiana (BB). Tengroups of cultivars with levels of ploidyranging from diploid (2n = 2x = 22) totetraploid (2n = 4x = 44) and differentgenomes are recognized. The followinggenome configurations are known toexist: diploid AA, BB and AB; triploidAAA, AAB, ABB; and tetraploid AAAA,AAAB, AABB, ABBB (Simmonds andWeatherup 1990). Therefore, a wide vari-ety of classification and identificationsystems exists for banana. Up to now, thetraditional classification and identifica-tion have been based only on morphol-ogy and quantitative traits. Recently,molecular markers have been used tostudy diversity on plants, animals andmicroorganisms.

The random amplified polymorphicDNA (RAPD) technique, which utilizespolymerase chain reaction (PCR) ampli-fication with single primers of arbitrarynucleotide sequence, has been deve-loped by Williams et al. (1990) and Welshand McClelland (1990) to produce mole-cular markers for genetic analysis.RAPDs have been shown to be useful ingenetic fingerprinting (Yang and Quiros1993, Orozco-Castillo et al. 1994, Lanhamet al. 1995). In this study, we have usedRAPDs for identifying and classifyingsome banana cultivars.


MaterialsIn this study, six indigenous banana culti-vars of Vietnam (Table 1), which wereobtained from the Institute ofAgricultural Genetics (Vietnam) werescreened for RAPD markers.

Isolation of DNADNA was isolated from banana leavesusing the method of Murray andThompson (1980) with some modifica-tions. Four grams of fresh leaf materialwere grounded in liquid nitrogen inpresence of glass-sand. The powderedleaf tissue was stored at -20°C for twohours. Ten milliliters of extraction buffer

[1,5% cetyltrimethylammonium bromide(CTAB); 100 mM Tris-HCl) (pH 8); 20mM ethylenediaminetetraacetic acid(EDTA) (pH 8); 1.4 mM NaCl; 0.2% mer-captoethanol] heated to 65°C wasadded, and the mixture was then incu-bated at 65°C for 30 min. The mixturewas shaken lightly with 1.5 volume ofchloroform: isoamyl (24:1) for 20 min atroom temperature. The sediment wasremoved by centrifugation at 3000 rpmfor 20 min. The DNA was precipitated bythe addition of 0.8 volume of freezingpropanol (or 1.5 volume of 96% ethanol).The pellet was washed 2-3 times in 70%ethanol. At the end, DNA was redis-solved in a minimum volume of TE(about 200 ml).

DNA amplificationTwelve primers from OperonTechnologies, each ten bases in length,were used to amplify the DNA (Table 2).PCR was carried out in 25 ml reactionscontaining 20 ng of template (genomeDNA), 200 mM of each dNTP, 2.5 units ofTaq-polymerase, 15 ng of primers, 10 mMTris-HCl (pH 8.3), 50 mM KCl, 1.5mMMgCl2, 0.001% (w/v) gelatine and 20 mlof mineral oil overlay. Forty-five amplifi-cation cycles were performed, each con-sisting of 94°C for 30 s, 36°C for 1 min,72°C for 2 min. Products were analyzedby electrophoresis in 1.1% agarose gelsat 100 V for 3 h, stained with 0.01% ethid-ium bromide and photographed underUV light.

Data analysis• Coefficients of similarity among cultivars

were calculated by using the formula ofNei and Li (1979):

Sij = 2Nij

Ni + Njwhere:Nij = number of bands in commonbetween cultivars i and j, andNi and Nj = number of bands for culti-vars i and j, respectively.

• The dendogram of cultivars studied wasproduced using the NTsyspc 2.0 compu-ter programme.


RAPD-PCRTwelve primers were used to amplify thebanana genome DNA. Nine of themamplified to give multiple PCR amplifica-tion products (Figure 1: example withprimer H08), and three primers (G6, Y14,Y15) did not.

There were two kinds of bands:monomorphic bands which were presentin all cultivars and polymorphic bandswhich were asynchronously present or absent in all cultivars. Nine primerswere amplified into 79 bands, of which 67 bands (84.81%) were polymorphic and12 bands (15.19%) monomorphic. Thehigh proportion of polymorphic bandswas due to the very different origin of thecultivars studied. Two primers (D07, G14)amplified 5 bands only, while H07 ampli-fied 17 bands. The size of the bandsranged from 360 Kb to 3200 Kb.

Genetic similarityThe Nei and Li formula allowed to calcu-late coefficients of similarity among cul-tivars based on RAPD data. Similaritycoefficients reflected the relationshipbetween cultivars. Similarity coeffi-cients between the original cultivarsfrom M. acuminata ranged between0.764-0.826, while those between theoriginal cultivars from M. balbisianaranged between 0.696-0.835 (Table 3).Cultivars belonging to the two groupshad low similarity coefficients, rangingbetween 0.317 and 0.461.

Specific RAPD markers of cultivarsSpecific RAPD markers are bands thatare present only in one cultivar. In thisstudy, we found 12 specific markers for4 cultivars (Table 4). These results sug-gest that RAPDs can be used for theselection of banana breeds in agriculture.

Table 1. Cultivars and genotypes employed in the study.

Cultivar Genotype Cultivar Genotype

1 Chuoi Tieu Xanh AAA (2n = 3x = 33) 4 Chuoi Tay ABB (2n = 3x = 33)

2 Chuoi Tieu Hong AAA (2n = 3x = 33) 5 Chuoi La ABB (2n = 3x = 33)

3 Chuoi Ngu AA (2n = 2x = 22) 6 Chuoi Hot BB (2n = 2x = 22)


Using RAPD technique for identifying andclassifying some banana cultivars in Vietnam

Genetic resources Classification of Vietnamese germplasm

Phylogenetic tree of banana cultivarsBased on RAPD data, a phylogenetic treeof banana cultivars was constructed usingthe NTsyspc 2.0 program. The phyloge-netic tree had two branches: the cultivarsoriginating from M. acuminata were onone branch, and the cultivars originatingfrom M. balbisiana were on the otherbranch (Figure 2). These results are inagreement with the cytological analysisof these banana cultivars.

AcknowledgementsThe authors thank the Program ofFundamental Researches for supportingthis research, and Inge Van den Bergh forreviewing this article. ■

ReferencesLanham P.G., R.M. Brennan, C. Hackett & R.J. McNicol.

1995. RAPD fingerprinting of blackcurrant (Ribes

nigrum L.) cultivars. Theor. Appl. Genet. 90: 166-172.

Murray M.G. & W.F. Thompson. 1980. Rapid isolationof high molecular weight plant DNA. Nucleic AcidsRes. 8: 4321- 4325.

Nei M. &W.H. Li. 1979. Mathematical model for study-ing genetical variation in term of restrictionendonucleases. Proc. Natl. Acad. Sci. USA 74:5267-5273.

Orozco-Catstillo C., K.J. Chalmers, R. Waugh &W. Powell. 1994. Detection of genetic diversity andselective gene introgression in coffee using RAPDmarkers. Theor. Appl. Genet. 87: 934-940.

Simmonds N.W. & S.T.C. Weatherup. 1990. Numericaltaxonomy of the cultivated bananas. TropicalAgriculture 67: 90-92.

Welsh J. & M. McClelland. 1990. Fingerprintinggenomes using PCR with arbitrary primers.Nucleic Acids Res. 18(24): 7213-7218.

Williams J.G.K., A.R. Kubelik, K.J. Livak, J.A. Rafalski& S.V. Tingey. 1990. DNA polymorphism amplified

by arbitrary primers are useful as genetic mark-ers. Nucleic Acids Res. 18: 6531-6535.

Yang X. & C. Quiros. 1993. Identification and classifi-cation of celery cultivars with RAPD markers.Theor. Appl. Genet. 86: 205-212.

Nguyen Xuan Thu and Le Thi Lan Oanh work at theInstitute of Biotechnology (IBT), National Centre forNatural Science and Technology of Vietnam (NCST),Hoang Quoc Viet Street, Cau Giay, Ha Noi, Vietnam.E-mail: [email protected]; Ho Huu Nhi works at theVietnam Agricultural Science Institute (VASI), VanDien, Ha Noi, Vietnam. E-mail: [email protected]


Figure 1. Results of RAPD-PCR with primer H08.1: 1Kb scale; 2: Chuoi Tay; 3: Chuoi Tieu Xanh; 4: Chuoi Tieu Xanh; 5. Chuoi Ngu; 6: Chuoi TieuHong; 7: Chuoi La; 8: Chuoi Hot.

Table 2. Primers used in the study.

Primer Nucleotide sequence Primer Nucleotide sequence

AA10 5’AGACGGCTCC 3’ H07 5’CTGCATCGTG 3’

AA14 5’AACGGGCCAA 3’ H08 5’GAAACACCCC 3’

B17 5’AGGGAACGAG 3’ U01 5’ ACGGACGTCA 3’

D07 5’TTGGCACGGG 3’ Y14 5’GGTCGATCTG 3’

G06 5’GTGCCTAACC 3’ Y15 5’AGTCGCCCTT 3’

G14 5’GGATGAGACC 3’ Y18 5’GTGGAGTCAG 3’

Table 3. Similarity coefficients among banana cultivars calculated using the Nei andLi formula.

Cultivar Tay Hot La Tieu Xanh Tieu Hong Ngu

Tay 1.00

Hot 0.826 1.00

La 0.764 0.829 1.00

Tieu Xanh 0.577 0.461 0.586 1.00

Tieu Hong 0.500 0.373 0.489 0.835 1.00

Ngu 0.477 0.317 0.422 0.782 0.696 1.00

Table 4. Specific markers of some banana cultivars studied.

Chuoi La Chuoi Hot Chuoi Ngu Tieu Hong

AA10-950 H07-500 H07-900 H07-800

H07-1270 H07-400

U01-1400 U01-700

Y18-800

Chuoitay

Chuoihot

Chuoila

Tieuxanh

Chuoingu

Tieuhong

Coefficient

0.35 0.49 0.63 0.76 0.90

Figure 2. Dendogram of banana cultivars produced using the NTsyspc 2.0 computer program.


Consumption and expenditure patterns of bananaand plantain consumers in Nsukka Urban, Nigeria

Marketing Consumer studies in Nigeria

A.R. Ajayi and M.O. Aneke

In Nigeria, banana and plantain havealways been very important tradi-tional staple foods for both rural and

urban population. They serve as a sourceof revenue for smallholders who producethem at the compound farms, mixed-cropping farms and small-scale sole crop-ping farms (Baiyeri 1996, Ajayi andBaiyeri 1999).

Nsukka Urban is heavily populated. Ithas a large market centre, operated on adaily basis. Men, women and the youthswithin Nsukka Urban and neighbouringcommunities converge at the market cen-tre to buy and sell. Agricultural productssuch as banana, plantain, vegetables,pepper, mangoes, palm oil, other fruits,honey, yam, livestock, etc., are sold. Inthe area, banana and plantain are foundin compound farms and they are mixedwith other crops. Each of the bananaand/or plantain growers in the area hasless than 50 stands and a greater propor-tion of them grow more banana thanplantain (Baiyeri and Ajayi 2000).However, banana and plantain marketingis most prominent among women, espe-cially within Nsukka Urban, Universitycampus and the neighbouring communi-ties. The sale of banana and plantain pro-vides means of livelihood for many house-holds in the area.

In view of the significant contributionsof banana and plantain to the economic,health and nutritional well-being of bothrural and urban households in Nigeria, itis very important that efforts should bemade continually to improved their mar-keting and consumption patterns. Inplanning such a national banana andplantain marketing and consumptionimprovement programme, data based onthe consumption and expenditure pat-terns of their consumers in both ruraland urban areas would be necessary. Therole of Agricultural Extension (AE) inthe gathering of data, planning, imple-mentation, monitoring and evaluation ofsuch a programme cannot be overem-phasized. AE is a primary processthrough which the households can learnthe reasons for change, the value of

change, the results that can be achieved,the process through which change isachieved, and the uncertainties inher-ent in change (Williams 1978).

Expenditure patterns of households inNigeria vary from place to place. Apartfrom the income of the households, fac-tors such as preference of a particularproduct by a member of the household,quality and quantity of the product sold,environment under which the productwas processed and sold, and the relativeprice of the products, also influence theexpenditure patterns of the households(Anyanwu 1985).

Food consumption pattern, in a broadsense, means not only what the peopleeat or consume, but also the quantities aswell as the forms in which these foods areconsumed (Dury et al. 1999). Accordingto Olagoke (1989), food consumption pat-terns vary from one place to another dueto factors such as household size, educa-tional levels of members of the house-hold, relative prices of the food items,environment in which the consumers areliving, social values attached to some fooditems, nutritive values of the food items,type or status of job performed by mem-bers of the household, household’s tastesand preferences, season/period of theyear, and culture/religion of the house-hold members.

The study was designed to assess theconsumption and expenditure patterns ofbanana and plantain consumers inNsukka Urban in Enugu State, Nigeria.Specifically, the study was designed to:1. determine the consumption patterns of

bananas and plantains among house-holds in Nsukka Urban;

2. determine the expenditure patterns ofbanana and plantain consumers inNsukka Urban;

3. determine the decision-making role ofhousehold members in banana andplantain consumption in NsukkaUrban;

4. determine the major problems militat-ing against effective consumption ofbananas and plantains in the studyarea; and

5. draw implications for extension pro-gramme on improved and efficientpreservation, processing, marketing

and consumption of bananas and plan-tains in the study area.

MethodologyNsukka Urban lies within the centre ofNsukka Local Government Area, EnuguState, Nigeria. The land area of NsukkaUrban is about 45.38 km2 (Oformata1995). It is made up of the following dif-ferent sections (clusters): the Universityof Nigeria campus, Onuiyi, Odenigbo,Government Reserved Area (GRA),Odenigwe, Ugwoye, Umuyo, Ngwuru,Owerre, Makashi and Isiakpu.

Out of the above-listed 11 clusters, fivewere selected through simple randomsampling. From each of the five clusters,12 households were selected, using clus-tering and simple random sampling tech-niques. In all, a total of 60 householdswere involved in the study, and the head ofeach of the households was interviewed.A structured questionnaire schedulewas developed and used in obtaining rel-evant information from the consumersof bananas and plantains. The data collected were analysed through the use of percentage distribution and barcharts.

Results of the surveyConsumption and expenditure patternsof banana and plantain among house-holds in Nsukka Urban are presented inthe following figures and tables:

Consumption rate of banana and plantain Figure 1 shows that the consumption rateof bananas is higher than the consump-tion of plantains.

Sources of banana and plantain for consumption Most of the consumers depend on themarket for their banana and plantainsupply. A very small proportion of con-sumers produce their fruits regularly(Figure 2).

Period of the day that bananas and plantains are mostly consumedIt clearly appears in Figure 3 that peopleprefer to eat plantain (consumed boiled,roasted or fried) in the morning and at

night, and banana as a ‘snack’ in theafternoon.

Common forms of plantain meal among householdsRespondents preferred fried plantain forbreakfast. For lunch, pounded androasted plantains are the most eaten.For dinner, plantain accompanied with rice, beans or yams is preferred(Table1).

Expenditure rate of banana and plantain consumersIt is to be noted that plantains are moreexpensive than bananas (N121 or N15 perplantain finger and N5 per banana fin-ger). This could explain why the majorityof consumers surveyed buy banana moreregularly than plantain (Figure 4).

Proportion of monthly income spent on banana and plantainconsumptionMost of the respondents (Figure 5)spend only 1% of their income onbananas and plantains. The main factorsthat determine the percentage of theirmonthly income spent on the purchaseof bananas and plantains are the avail-ability of money, closely followed by thefamily interest, and then the price of thefruits (Figure 6 – NB: more than one fac-tor was given).

Forms in which bananas and plantainsare purchased in the marketBananas are mostly purchased ripe,whereas the respondents prefer to buyunripe plantains (Figure 7).

Response of households to changes in banana and plantain pricesTable 2 shows that most of the respon-dents do not change their habits in buying bananas when the priceincreases, but they buy more if the pricedecreases. In the case of plantain, morethan half of the respondents would buy


0

5

10

15

20

25

30

35

40

Daily Once a week

Twicea week

Thricea week

% o

f re

spo

nd

ents

Banana

Plantain

0

10

20

30

40

50

60

70

Purchased Produced Both

% o

f re

spo

nd

ents

Banana (%)

Plantain (%)

Figure 1. Frequency of consumption of banana and plantain. Figure 2. Percentage distribution of respondents on the basis of theirsource of banana and plantain fruits.

0

10

20

30

40

50

60

70

80

90

100

Morning Afternoon Evening Night

Banana (%)

Plantain (%)

Figure 3. Percentage distribution of consumers on the basis of the time ofthe day that banana and plantain are mostly preferred.

0

5

10

15

20

25

30

35

40

45

50

Daily Weekly Monthly Occasionally

% o

f re

spo

nd

ents

Banana

Plantain

Figure 4. Expenditure rate of banana and plantain consumers.

Table 1. Forms of plantain meal among the households.

Form of meal Breakfast (%)* Lunch (%)* Dinner (%)*

Fried plantain + pap 28.5 4.3 1.7

Pottage plantain 19.2 3.7 11.0

Plantain + beans 3.5 10.6 18.9

Plantain + rice 2.3 9.6 21.1

Boiled plantain + stew 15.1 5.3 13.3

Pounded plantain + soup 1.2 25.5 5.6

Plantain with yam (pounded) 4.7 10.6 17.8

Roasted plantain 3.5 24.0 5.0

Boiled plantain 22.0 6.4 5.6*Multiple responses

1 10 N (Nigerian Naira) = 0.085 USD, March 2002.


fewer plantains in case of increasedprice and 75% would buy more in case ofdecreased price.

Household decision-making role in banana and plantain consumptionprocesses According to Table 3, wives play the mostimportant decision-making role inbanana and plantain purchasing andconsumption process. On the otherhand, the husband plays the major rolein the utilization of the peels, while chil-dren play the greatest role in determin-ing the purchasing interval and storageperiod.

Major problems militating againsteffective consumption of bananas and plantainsThree major problems militating againstthe effective consumption of banana andplantain fruits were identified by therespondents (Figure 8). These includedstorage problems such as pest (house ratsand insects) attacks, over-ripening andmould formation due to sustainedbruises; processing problems such as lack

Table 2. Percentage distribution of respondents according to their response to change in banana and plantain price.

Response to change in price 10% increase in price 10% decrease in price

Banana (%) Plantain (%) Banana (%) Plantain (%)

Buy same quantity 75.0 41.7 25.0 20.0

Buy more quantity 0.0 0.0 75.0 80.0

Buy less quantity 25.0 58.3 0.0 0.0

Total 100.0 100.0 100.0 100.0

65

18,3

6,710

about1%

about2%

about3%

4 to 5%

Quality of fruits13%

Familyinterest31%

Price of fruits20%

Availability ofmoney (cash)33%

Availability offruits 3%

Figure 5. Percentage distribution of respondents on the basis of theproportion (%) of their monthly income spent on banana and plantainconsumption.

Figure 6. Percentage distribution of the respondents on the basis of thefactors determining the proportion of their monthly income spent onbanana and plantain consumption.

0

10

20

30

40

50

60

70

80

90

Ripe Unripe Over-ripe

Banana (%)

Plantain (%)

Form

Figure 7. Percentage distribution of respondents on the basis of the formsin which bananas and plantains are purchased.

Storage80%

Processing17%

Transportationwithout anybruise 3%

Figure 8. Major problems militating against effective consumption ofbanana and plantain fruits.

Table 3. Household decision-making role in banana and plantain consumptionprocesses.

Decision-making role Banana Plantain

H (%) W (%) C (%) H (%) W (%) C (%)

Proportion of monthly income 43.3 56.7 0.0 30.0 70.0 0.0

Quantity purchased 21.0 70.7 8.3 35.0 50.0 15.0

Form in which fruits are purchased 8.3 75.0 16.7 30.0 60.0 10.0

Processing 0.0 85.5 14.5 10.0 64.6 25.4

Storage 0.0 75.3 24.7 0.0 86.2 13.8

Utilization of peels 50.0 11.7 38.3 70.0 4.7 25.3

Storage period 16.7 25.0 58.3 11.7 21.7 66.6

Purchasing interval 33.3 41.7 25.0 13.3 33.3 53.4

Quality purchased 18.0 82.0 0.0 27.0 73.0 0.0H = Husband, W = Wife and C = Children

of technological know-how, unfavourableweather conditions, unavailability of pro-cessing mills and poor supply/lack ofelectricity, etc.; and transportation with-out any bruise. Chukwu (1996) observedthat inadequate storage, insufficient dis-tribution and lack of processing tech-niques result in large proportions ofbanana and plantain being transformedinto wastage.

ConclusionsThe analysis of these results leads to thefollowing conclusions:1. the consumption rate of bananas was

higher than that of plantain;2. a greater proportion of the respon-

dents depended on the market fortheir supply of bananas and plantains;

3. bananas were consumed mostly in theafternoon, while plantains weremostly consumed in the morning;

4. plantain meals were prepared andconsumed in various forms;

5. plantains were more expensive thanbananas;

6. the major factors that determined theproportion (%) of the monthly incomespent on bananas and plantainsincluded availability of physical cash,family interest, price, quality and avail-ability of fruits;

7. bananas were mostly purchased in aripe form, while plantains were mostlypurchased unripe;

8. households responded accordingly tochange in banana and plantain prices;

9. wives played the greatest decision-making role in banana and plantainpurchasing and consumptionprocesses than their husbands andchildren; and

10.the major problems militating againsteffective consumption of bananasand plantains in the area includedstorage, processing and transporta-tion problems.

Implications of the findings for extension programme on improved and efficientpreservation, processing,marketing and consumption of bananas and plantains1. Since fluctuation in the market price

of bananas and plantains affected theconsumption patterns of the house-holds, there is a need for the establish-ment of a consumer cooperative orga-nization for bulk purchase and retailsale of the fruits. Capable extensionagents should be attached to each ofthe so formed organizations for thepurpose of monitoring and evaluatingthe activities of members and giving

relevant pieces of information if andwhen necessary.

2. To ensure effective preservation, pro-cessing and utilization of bananas andplantains, the State AgriculturalDevelopment Project (ADP) shouldorganize workshops for the womenmarketers on methods of preservation,processing and efficient utilization ofbanana and plantain.

3. The fact that a greater proportion ofthe respondents purchased theneeded bananas and plantains fromthe market centres and hawkersshows that there should be efficientdistributing and marketing channelsto enhance consumers’ access to thefruits as of when due. Thus, the EnuguState ADP should embark on efficientMusa postharvest handling, distribut-ing and marketing strategy pro-gramme for the rural farmers and thesellers.

4. Even though the wives could be spe-cially targeted (because of theirlargest decision-making role inbanana and plantain consumption) bythe Enugu State ADP to improve onthe banana and plantain consumptionand expenditure patterns amonghouseholds through appropriate edu-cational activities, the importance ofthe household as a working unit inextension practice should not be over-looked. Therefore, all members of thehousehold should be intensivelyinvolved in any extension programmedesigned to improve consumption andexpenditure patterns of banana andplantain in the study area. ■

ReferencesAjayi A.R. & K.P. Baiyeri. 1999. Household decision-

making role in backyard banana and plantain pro-duction in the Nsukka agroecological zone insoutheastern Nigaria. Pp. 719-727 in Bananas andFood Security. Les productions bananières: unenjeu économique majeur pour la sécurité alimen-taire (C. Picq, E. Fouré and E.A. Frison, eds).Proceedings of an International symposium heldin Douala, Cameroon, 10-14 November 1998.

Anyanwu C.U. 1985. An evaluation of the relationshipbetween income level, expenditure and food con-sumption pattern in UNN. B. Agric. ResearchProject, Department of Agricultural Economics,University of Nigeria, Nsukka.

Baiyeri K.P. 1996. Characterization, correlation,path-analysis and selection indices of Musa geno-types under different environments. A PhDResearch Proposal, Department of Crop Science,University of Nigeria, Nsukka.

Baiyeri K.P. & A.R. Ajayi. 2000. Status and con-straints of Musa spp. production in a sub-humidzone of Nigeria. Pp. 73–77 in Proceedings of theFirst International Conference on Banana andPlantain for Africa (K. Craenen, R. Ortiz, E. B.Karamura and D.R. Vuylsteke, eds). ActaHorticulturae 540.

Chukwu U.E. 1996. Effect of post-harvest injury onshelf-life and extrusion processing of Musa spp.fruits. A PhD Research Proposal, Department ofFood Science, University of Ibadan, Ibadan, OyoState, Nigeria.

Dury S., N. Bricas, J. Tchango Tchango & A. Bikoï.1999. La consommation et les critères de qualitédu plantain à Douala et à Yaoundé. Pp. 507-523 in Bananas and Food Security. Les productionsbananières: un enjeu économique majeur pour la sécurité alimentaire (C. Picq, E. Fouré and E.A. Frison, eds). Proceedings of an Internationalsymposium held in Douala, Cameroon, 10-14November 1998.

Ofomata G.E. 1975. Nigeria in Maps. Eastern StateEthiope Publishing Company, Benin City, Nigeria.

Olagoke M.A. 1975. Food consumption patterns inthe Obafemi Awolowo University. BA ResearchProject, Faculty of Agriculture, Ile-Ife, Osun State,Nigeria.

Williams S.K.T. 1978. Rural development in Nigeria.Obafemi Awolowo University Press, Ile-Ife, OsunState, Nigeria.

The authors work at the Department of AgriculturalExtension, Faculty of Agriculture, University of NigeriaNsukka. Tel.: +234 042 770815 or 771019,Fax: +234 02 770664; E-mail: MISUNN @ aol.com


In Nigeria, False horn plantains are veryappreciated because of theit long finger size(Photo: R. Swennen, IITA).

Iris Engelborghs

Banana plants are the most impor-tant world fruit crop and show alarge diversity in shapes and sizes,

of which one is the dwarf type. Unlike thenormal type, the dwarf variant has ashorter pseudostem and wider leafs.Because these plants have the same num-ber of leaves as the normal variant, theirphotosynthetic ability is not reduced andtherefore the bunch size almost identi-cal. In addition, its reduced height pre-vents it from toppling during tropicalstorms. These characteristics make thedwarf variant a valuable plant for thetropical banana farmer. Different natu-rally occurring dwarf varieties existwhich have a normal variant, but thisphenotype is often obtained by in vitroculture too, e.g. in an in vitro germplasmcollection or during rapid in vitro multi-plication.

Vos et al. (1995) described the ampli-fied fragment length polymorphism(AFLP) technique as ‘a novel and verypowerful DNA fingerprinting techniquefor DNAs of any origin or complexity’.This DNA fingerprinting technique isbased on the selective PCR amplificationof restriction fragments from a totaldigest of genomic DNA and involves threesteps: (i) restriction of the DNA and liga-tion of oligonucleotide adapters, (ii)selective amplification of sets of restric-tion fragments, and (iii) gel analysis of

the amplified fragments. With thismethod, sets of restriction fragments arevisualized by PCR without knowledge ofnucleotide sequence. The power of thisDNA fingerprinting technique wasassessed on a few world species, but noton Musa spp. Therefore, it was here firstoptimized for banana. In addition, thetechnique was adapted for non-radioac-tive detection of the AFLP patterns usinga more recent detection method.Fluorescein labelled primers were usedin the PCR reactions which allow thecomputer based separation and detectionon a sequencing gel.

The assessment of the power of theAFLP technique and its variants three-endonuclease-(TE)-AFLP, cDNA-AFLPand the methylation sensitive amplifiedpolymorphism (MSAP) technique for thecharacterization and the early detectionof the dwarf type was performed in thisstudy on a range of dwarf-normal bananapairs. The dwarf ‘Curare enano’ and its invitro generated normal-sized off-typewere first used for the optimization of theAFLP technique. Later, the analysis wasextended to three naturally occurringdwarf varieties ‘Cachaco enano’, ‘Figuerose naine’ and ‘Prata ana’, which have anormal variant called ‘Cachaco’, ‘Figuerose’ and ‘Prata’, respectively. In addi-tion, the extra dwarf ‘Dwarf parfitt’, thenormal ‘Cavendish’ and the giant ‘Giantcavendish’ were analyzed.

Differential AFLP, TE-AFLP, cDNA-AFLP, cDNA-TE-AFLP and MSAP patternswere obtained and different levels ofpolymorphisms were observed betweenthe dwarf and normal type depending onthe technique, the primer combinationand the variety. For each variety, a dis-tinction could be made between thedwarf and the normal type. However, nodwarf specific fragment was found to becommon for all dwarf varieties, which isan indication that (i) either the observedpolymorphisms are not related to thephenotype, (ii) or the different varietiesoriginated in a different way, (iii) or that

several genes are involved in the process.Differential fragments were cloned andsequenced. Primers were designed on theobtained sequences and used withgenomic DNA of the respective variety toconfirm the differential and uniquenature of the fragments. However, speci-ficity was lost.

Besides the above-described analysesat the DNA level, some physiologicalassays were performed on the dwarf-nor-mal banana pairs. The relation betweendwarfism and gibberellic acid (GA) isdescribed for several mutant dwarfspecies, e.g. rice and wheat, as well assome Musa spp. Two categories of dwarftypes are described, i.e. a GA-(in)sensi-tive group and a GA-deficient group. Theinfluence of GA was tested in this studyon the in vitro growth of the dwarf-nor-mal banana pairs. The (in)sensitivityappeared to be variety dependent, sug-gesting that the different dwarf varietiesoriginated in a different manner and thatother mechanisms may be involved thanthe GA pathway alone. When grown onancymidol (a GA-synthesis inhibitor) invitro growth of all the tested dwarf vari-eties was inhibited, which indicates thatthese plants are not GA-deficient, and thesignal transduction after the synthesismust be impaired.

From these results we can concludethat the AFLP technique allows a fast andearly fingerprinting of these particulardwarf banana types, that the mechanismbehind dwarfism is complex and seems toinvolve gibberellic acid, (de)methyla-tion… and that the dwarf varieties hereanalyzed probably originated via differ-ent mechanisms. ■

ReferenceVos P., R. Hogers, M. Bleeker, M. Reijans, T. van de

Lee, M. Hornes, A. Frijters, J. Pot, J. Peleman, M. Kuiper & M. Zabeau. 1995. AFLP: a new tech-nique for DNA fingerprinting. Nucl. Acids Res. 23: 4407-4414.


Molecular characterization of dwarf banana plants(Musa spp.) using AFLPKatholieke Universiteit Leuven, Faculty of Agricultural and Applied Biological Sciences, Laboratory of TropicalCrop Improvement, Leuven, BelgiumDissertationes de Agricultura No. 515, 2002

Thesis

Annemie Elsen

Diseases and pests are the majorconstraints to the productivity ofbananas and plantains. Nema-

todes cause important yield losses inLatin America, West and East Africa andAsia. Usually, banana nematodes are con-trolled by nematicides. These are not onlyvery expensive but also extremely toxicfor non-target organisms, including theuser, and they pollute the environment.Arbuscular mycorrhizal fungi (AMF) areobligate symbionts that biotrophicallycolonize the root cortex and develop anextramatrical mycelium which helps theplant to acquire water and mineralnutrients from the soil, in exchange forcarbon as an energy source. In addition,AMF increase the ability of the plant to control the spread of soil-bornepathogens. In Musa the associationoccurs naturally when plants are trans-planted into the field. The association ofAMF with plant-parasitic nematodes andthe beneficial effect of the mycorrhizalsymbiosis on plant growth and nematoderesistance/tolerance led to investigationsinto the potential of AMF to limit yieldlosses due to nematodes.

In the first part of our study, the rela-tive mycorrhizal dependency (RMD) andthe AMF-nematode interaction werestudied in four Musa genotypes, selectedfor their known host plant response tonematodes (i.e. ‘Grande naine’, ‘GrosMichel’, ‘Pisang jari buaya’ and‘Yangambi km5’). Mycorrhization withGlomus mosseae (AMF) resulted in a sig-nificant better plant growth, even in thepresence of Radopholus similis andPratylenchus coffeae. No differences inRMD were observed among the four geno-types. Glomus mosseae could protect‘Grande naine’ and ‘Pisang jari buaya’against R. similis and P. coffeae, since thenematode reproduction was suppressed.Only in the case of R. similis (Indonesianpopulation with low pathogenicity) in

‘Pisang jari buaya’, no suppression wasobserved. However, when reproduction isalready very low (due to low reproductivefitness of the nematode population and/orthe resistant host plant response of thetested genotype), the presence of theAMF has no effect on the nematode repro-duction. The AMF reduced the root necro-sis, caused by P. coffeae. For R. similis, noreduction was observed. The nematodesreduced the frequency of mycorrhization,without reducing the intensity of themycorrhizal association.

In the second part, the RMD and theAMF-nematode interaction were studiedin Musa genotypes differing in root mor-phology. The influence of the AMF on theroot system and the influence of thealtered root system on the nematodereproduction were examined. Mycorrhi-zation with G. mosseae resulted in a sig-nificant better plant growth, even in thepresence of P. coffeae. The effect of AMFon the root system was related to theRMD of the genotype. Musa genotypeswith a low RMD will not experiencechanges in the branching of their rootsystem in response to mycorrhization.But in genotypes with a high RMD, theroot system will be more branched. Weshowed that P. coffeae also affects theroot system, by reducing the branching.The effect of AMF on the nematode repro-duction was not very clear. The nematodepopulation density tended to be reduced,but was not significant in the experimentwith ‘Obino l’ewai’. In the root system, itappeared that the decreased branchingcaused by the nematodes was counterbal-anced by the increased branching causedby the AMF. Therefore application of AMFcould be used as a strategy to decreasesusceptibility to nematodes.

In the third part of our study, AMF-nematode interactions were studiedunder in vitro conditions. Firstly, asepticnematode cultures were establishedusing alfalfa callus as a host tissue. Untilnow the lack of completely sterile culturesystems limited in vitro nematode-host

studies and AMF-nematode interactionstudies. Secondly, three model systemswere developed: Ri T-DNA transformedDaucus carota roots, in vitro Musaplants and in vitro Arabidopsis thalianaplants.

Finally, the transformed D. carota rootswere used to study the AMF-nematodeinteraction under sterile conditions. Theresults reported in this study confirmedthe suppressive effect of AMF on nema-tode reproduction. Glomus intraradicescould suppress the R. similis, P. coffeaeand to a lesser extent M. javanica popu-lation in the roots. The internal andexternal developments of the AMF werenot affected by the presence of theseplant-parasitic nematodes.

Although this in vitro system has sev-eral limitations, there are still many legit-imate reasons to use this system to studythe AMF-nematode interaction. The AMFdevelops appressoria, arbuscules andvesicules in the root cortex, produces pro-fuse extraradical mycelium and sporesand is completing its life cycle in vitro.The early colonization occurs in a similarway as under in vivo conditions. Thenematodes, R. similis and P. coffeae, caninfect and reproduce in the roots, andcause similar damage in the in vitro rootsas in in vivo roots. In addition, the effectsof the interaction reflect those observedin vivo. Although the dixenic system usedis artificial, it may represent a valuabletool for studying the AMF-nematodeinteraction, complementary to classicalexperimental approaches. ■


Study of the interaction between arbuscularmycorrhizal fungi and plant-parasitic nematodes in Musa spp.Katholieke Universiteit Leuven, Faculty of Agricultural and Applied Biological Sciences, Laboratory of TropicalCrop Improvement, Leuven, BelgiumDissertationes de Agricultura No. 517, 2002

Thesis

MusaNews

World

Farmer-participatory evaluation and dissemination of improved Musa germplasmINIBAP is the executive agency of animportant four-year project of theCommon Fund for Commodities (CFC)which started in November 2001. The aimof this project is to contribute toimproved food and income security forsmall-scale farmers in banana-basedfarming systems, through the distributionand evaluation of improved Musa hybridssuitable for local consumption and mar-keting. The project involves seven coun-tries namely Democratic Republic ofCongo, Ecuador, Guinea, Haiti, Honduras,Nicaragua, and Uganda.

The project will be implemented in twophases. The first phase includes the mul-tiplication of planting material of at leastten improved Musa varieties in eachcountry and the distribution of theseplants to farmers for on-farm evaluation.At least 150 farmers per country partici-pate in the trials. The second phase willconsist of funding support in the form ofloans to small-scale farmers to enablethem to purchase planting material andessential inputs for the more wide scaleproduction of improved hybrids. The pro-ject will also include market studies onthe improved hybrids and the training offarmers in improved production tech-niques, focusing on the integrated man-agement of pests and diseases. The mainresult from the project will be theincreased production of improved Musahybrids by small-scale farmers. Thesevarieties will produce higher yields andwill not require chemicals for pest anddisease control. In addition, farmers andentrepreneurs will be assisted to set upbanana-related businesses (the produc-tion of planting material for sale etc.),thus contributing to increased incomegeneration for rural communities. Themajor beneficiaries will be small-scalebanana farmers.

For more information on the project, pleasecontact Suzanne Sharrock, project coordinator,at INIBAP Headquarters.

Africa

The 3rd International bacterial wiltsymposium took place in South Africa – 4-8 February 2002 The 3rd International bacterial wilt sym-posium was attended by 110 scientists

from all over the world who presentedmore than 100 papers either orally or asposters. Aspects discussed were: epi-demiology, disease management, breed-ing and deployment for disease resis-tance, host response and diseasedevelopment, pathogen genetics, diver-sity and diagnosis.

Bacterial wilt caused by Ralstoniasolanacearum is reported to be one ofthe major constraints for many importantcrops such as potato, tomato, groundnut,banana, tobacco and ginger. In manycases, the disease causes very significantyield losses.

There is still a big gap in the researchprogress between developed and devel-oping countries. In developed countries,scientists are generally more interestedin the molecular aspects of the pathogensuch as pathogen genetics, diversity anddiagnosis. Except for diagnostics, otheraspects under study are not directlyrelated to control of the disease.Therefore, research being carried out ondisease control by scientists in develop-ing countries, where the disease is moreserious and widespread, needs to bestrengthened. Some work on breedingand deployment for disease resistancehas been carried out and good resultshave been obtained for groundnut andpotato. However, very little has so farbeen done for many other importantcrops such as banana and ginger.

Pathogen geneticsSignificant progress has been made instudies of the genome of R. solanacearum.The pathogen has a 3,716,413 base pair(bp) chromosome and a 2,094,509 bpmegaplasmid, which taken togetherencode over 5000 proteins. The chromo-some harbours all essential genes,whereas the megaplasmid is involved inthe biosynthesis of various amino acids,cofactors, and fitness to environments.There are about 200 candidate genes forpathogenicity distributed both in thechromosome and megaplasmid. Thisinformation is essential in understandingthe biodiversity in R. solanacearum inrelation to host specificity. Traditionally,R. solanacearum strains are groupedinto five races, based on host range, andfive biovars based on the oxidation oncertain carbon sources.

A new classification scheme has beenproposed based on the molecular analy-ses of R. solanacearum. Strains of R. solanacearum are classified into fourphylotypes such as Phylotype I ‘Asia’(include biovars 3 and 4, race 1, 4 and 5), phylotype II ‘America’ (biovar 1and 2, race 1, 2,and 3), phylotype III

‘Africa’ (biovar 1, 2), and phylotype IV‘Indonesia’ (biovar 1, 2, Pseudomonassyzygii, and blood disease bacterium –BDB). This shows that strains ofIndonesia, including P. syzygii whichcauses Sumatra disease of clove, and theBDB on banana, are separated fromother strains.

DetectionVarious diagnostic kits have been devel-oped, especially for quarantine purposesand for monitoring the pathogen in symp-tomatic and latently infected plant mate-rials, surface water, soil, vegetable wash-ings, and processing waste. Methods usedare selective isolation and enrichment,bioassay, immunofluorescence, serology,and polymerase chain reaction (PCR).For use in developing countries, serologymethods such as ELISA are more appro-priate as they are cheaper.

The 4th Symposium will be held in 2007,possibly in the UK.

The American Plant Pathology Societywill publish the papers presented in theSymposium.

Further information about the symposium is available from Dr Supriadi, Research Institute for Spice and Medicinal Crops, Jalan TentaraPelajar No. 3, Bogor 16111, Indonesia. Fax: (0251) 327010.

Asia and the Pacific

Musa acuminata in Northern BorneoA preliminary report on the status ofMusa acuminata in Northern Borneohas recently been prepared by MarkkuHäkkinen and Edmond De Langhe. Thisreport is based on a survey of Musa inSabah, Sarawak and Brunei carried outby the first author in August 2001.Although the main focus of the surveywas the section Callimusa, a large num-ber of photographs of Musa acuminatawere also taken. With the expertise ofEdmond de Langhe, a tentative taxo-nomic identification of the plants hasbeen made.

The photographs showed that all theplants exhibited the basic characteristicsof M. acuminata, with the typical top-likemale bud, the horizontal-to-oblique inflo-rescence and bunch, and rather slenderfingers. The flowers appeared to bewhite-to-creamy, but the details were notvisible in the photos.

The specimens in question were foundto fall into four main categories:• M. acuminata ssp. microcarpa or

truncata• M. acuminata of uncertain status• Edible AA diploids• Unclassified accessions.


A microcarpa-truncata cluster?The most important result of theHäkkinen visit to Northern Borneo, anarea previously little explored for wildbananas, is the domination of a wild M. acuminata population with a combi-nation of characteristics typical of thesubspecies microcarpa and truncata.The occurrence of yellow-green male budtypes is the first time this character hasbeen recorded for these subspecies. It ispossible that the two supposed sub-species may actually form one large andcompound population, and further stud-ies on this material in a field genebankare required to confirm the status of theaccessions in this group.

M. acuminata of uncertain statusA number of accessions were character-ized by a moderately to strongly imbri-cated male bud and a trend for the bractcolour to be pink/red/purple. Male budswith visibly imbricate bracts are charac-teristic for the subspecies siamea andburmannica, but are not expected forthe acuminata’s in Borneo or theIndonesian islands, with the exception ofJava. Further studies are required to con-firm if these are edible diploids or indeedtruly wild plants.

Edible AA diploids A number of plants are recorded as edi-ble AA diploids. Amongst these are plantsthat were found to be populating largeareas along the sides of roads, as trulywild populations do. Since there were novillages in the proximity, these may be theremnants of human population fromremote time.

ConclusionsThe findings presented in this report areclearly of a preliminary and tentativenature, as they are based on studies of pho-tographs only. However, it does provide abasis for further studies, which the authorshope will be stimulated by this report.

Copies of the report, including colourphotographs of all the accessions, are availablein PDF format from the INIBAP web site(http://www.inibap.org/publications/borneo.pdf)or in printed form from INIBAP Headquarters,Montpellier.

Study of the association betweennematodes and bananas in VietnamInge Van den Bergh, a VVOB/INIBAPVisiting Associate Scientist, was based at the Agro-biotechnology Department of the Vietnam Agricultural ScienceInstitute (VASI), Hanoi, Vietnam fromOctober 1997 to December 2001 to carryout a study on the association betweennematodes and bananas in Vietnam.

The project was funded by ACIAR(Australian Centre for InternationalAgricultural Research), INIBAP andVVOB (Flemish Association forDevelopment Cooperation and TechnicalAssistance)

The project had two main goals:1. capacity building: to improve the local

infrastructure for nematologicalresearch and to train local scientists inthe field of nematology;

2. scientific research: to gain moreinsight into the different aspects of theassociation between nematodes andbananas in Vietnam, in order toimprove local banana production.Specifically:

• to obtain a more detailed picture of theoccurrence of different nematodespecies on bananas in various regionsof Vietnam;

• to increase the knowledge about popu-lation-dynamics and the damage andyield loss potential of the most impor-tant nematode species on bananas;

• to screen Vietnamese Musa germplasmfor resistance to the most importantnematode species.

Activities undertaken and resultsachieved

Capacity buildingThe general infrastructure of the labora-tories was improved and various items ofequipment were bought. An e-mail/inter-net connection was established in orderto improve the communication betweenthe different project partners and withother nematologists in the world, and tohave access to information from theworldwide web.

During the time of the project, two staffmembers of the AgrobiotechnologyDepartment of VASI followed the post-graduate international nematologycourse in Belgium. In this respect:

Duong Thi Minh Nguyet defended herMSc thesis entitled: «In vivo and in vitrostudies of Radopholus similis andPratylenchus coffeae associated withbanana» in 1999 (Promotor: Prof. D. DeWaele).

Nguyen Thi Tuyet defended her MScthesis entitled: « In vitro and in vivoscreening for Radopholus similis resis-tance in Musa» in 2000 (Promotor: Prof.D. De Waele).

Scientific research carried out by theVVOB/INIBAP Visiting Associate Scientist

1. Assessment of the occurrence and dis-tribution of nematodes on wild Musa

species in natural habitats in NorthVietnam

Three survey trips were undertaken innatural habitats in North Vietnam androot samples from three species of wildbananas were taken. With the exceptionof R. similis, the most important Musanematode species, i.e. Meloidogyne spp.,P. coffeae and Helicotylenchus multicinc-tus, were found. This indicates that thenatural soils in Vietnam are infested withthese nematode species and that thethree wild banana species are suscepti-ble to these species.

2. Assessment of the occurrence and dis-tribution of nematodes on Musa culti-vars in North and Central Vietnam

Five survey trips were undertaken in sixprovinces in North and Central Vietnamand root samples of three commonly culti-vated banana genotypes were taken.Again, Meloidogyne spp., P. coffeae and H. multicinctus were found, but not R. similis. Damage parameters showed a clear relation with the presence of certain nematode species in the roots. For example, root-knot galling was posi-tively correlated with the number ofMeloidogyne spp. in the roots, while rootnecrosis was positively correlated with thenumber of P. coffeae found in the roots.

3. Influence of a Pratylenchus coffeaepopulation and Meloidogyne spp. onplant growth and yield of banana

A field was planted with over 150 bananaplants, of which one third was inocu-lated with P. coffeae, one third withMeloidogyne spp. and one third was keptnematode-free (control plants). The pre-liminary results showed that infectionwith P. coffeae and Meloidogyne spp. can reduce the plant height and thenumber of standing leaves in compari-son with the uninfected control plants.

4. Population-dynamics of a Pratylenchuscoffeae population collected on Musain North Vietnam

The reproduction of P. coffeae on carrotdiscs under in vitro conditions could bedescribed by the Gompertz equation: log(nem + 1) = 0.725 + 2.561 exp [-exp(1.742 (5.044 - time))].

From a greenhouse experiment thatwas repeated monthly over a period ofone year, it could be seen that tempera-ture has a strong effect on the reproduc-tion rate of P. coffeae on bananas. Duringthe winter months, the reproduction wasvery low, while during the summermonths, the population increased signifi-cantly. The extent of root-necrosis fol-lowed more or less the same pattern.


A greenhouse experiment was set up toassess the influence of irrigation on thereproduction of P. coffeae. A shortage ofwater had a very strong negative effect onthe plant growth, while the nematodescould still reproduce well. A very highapplication of water reduced the generalplant growth slightly, but the nematodescould barely reproduce. An intermediatewater volume was best for the growth ofthe plants, but also favourable for thenematode reproduction.

The reproduction of a P. coffeae popula-tion on banana plants in the field was fol-lowed for more than one year. From pre-liminary results, it can be seen thattemperature and rainfall have an effect onthe reproduction rate of the nematodes.

5. Screening of Vietnamese Musagermplasm for resistance to aPratylenchus coffeae population in thegreenhouse

Twenty-four Vietnamese banana geno-types were screened for resistance to P. coffeae in the greenhouse. The mostpromising genotypes are ‘Tieu xanh’,‘Tieu mien nam’, ‘Com chua’, ‘Com lua’,‘Man’ and ‘Ngu thoc’.

6. Screening of Vietnamese Musagermplasm for resistance toMeloidogyne spp. in the greenhouse

Twenty-two Vietnamese banana geno-types were screened for resistance toMeloidogyne spp. in the greenhouse. Nosources of resistance were found.

7. Screening of Vietnamese Musagermplasm for resistance toMeloidogyne spp. in the field

Eight Vietnamese banana genotypes,‘FHIA-01’, ‘FHIA-02’ and ‘Yangambi km 5’were evaluated for their host plantresponse to Meloidogyne spp. under fieldconditions. ‘FHIA-01’, ‘Ngu thoc’, ‘Tay’and ‘Com lua’ were found to be less sus-ceptible to Meloidogyne spp. ‘FHIA-01’,‘Ben tre’ and ‘Bom’ were less sensitive tothe knot-forming activity of Meloidogynespp. The number of juveniles recoveredfrom the roots was strongly influencedby the weather. During the cold and dryseason, the numbers dropped very signif-icantly. The number of egg-layingfemales in the roots (ELF) was muchless influenced by the environmentalconditions: there was a stagnation dur-ing the cold and dry season but nodecline. Meloidogyne spp. seem to over-winter as eggs in egg-masses. Root-knotgalling and ELF can be used as easyparameters to estimate the infection ofMusa with Meloidogyne spp. No effectsof the nematodes on plant growth were

found. The number of nematodes in theroots seems to be related to the physio-logical stage of the plants. The highestnematode numbers were found duringflowering.

Scientific research by Duong Thi MinhNguyet and Nguyen Thi TuyetDuong Thi Minh Nguyet started aresearch programme on the occurrenceof Radopholus similis in Vietnam and itsmorphological and biological aspects.Two surveys were carried out in TayNguyen (Western Highlands) to assess theoccurrence of R. similis on coffee, durian,bananas, etc. One R. similis populationwas collected from durian roots and isbeing maintained on carrot discs underin vitro conditions. Since R. similis isstill a quarantine pathogen in Vietnam,Duong Thi Minh Nguyet went to Belgiumfor a period of three months to study thecollected population. She determinedthat the optimal temperature for repro-duction of R. similis from Vietnam oncarrot discs was 25°C. She also comparedthe reproduction of the Vietnamese popu-lation with populations from Indonesiaand Uganda.

Nguyen Thi Tuyet is studying the biodi-versity of Pratylenchus coffeae inVietnam. She is collecting P. coffeae pop-ulations from various crops and places inVietnam to study the morphological, bio-logical and genetic diversity between thedifferent populations.

Integrated management of Panama wilt disease of banana in KeralaBanana wilt caused by Fusarium oxys-porum f. sp. cubense is one of the seriousthreats to banana cultivation in Kerala.The acidic soils of the state and the sus-ceptibility of the major commercial vari-eties offer an easy spread of the diseasethroughout the region, causing a yieldloss of 10-15%.

The symptoms of the disease appearwith the yellowing of older leaves whichextends rapidly from the margin towardsthe midrib. These leaves hang witheredaround the pseudostem and the infectionspreads to all leaves except the top,which hang down. The heartleaf alsowithers after 3-4 weeks. The plantexhibits longitudinal splitting withbulging and elongation of the pseu-dostem. When the rhizome is cut open,the discolouration of the vascular bun-dles can be seen and the cut stem smellsof rotten fish.

A survey conducted by Estelitta et al.revealed that the disease was prevalentin all the districts in Kerala causing

serious damage to the crop. It was alsonoticed that ‘Nendran’, the most impor-tant commercial variety in the State wassusceptible to Panama wilt whileCavendish group, ‘Palayankodan’,‘Karpooravally’ and cooking varietiessuch as ‘Monthan’, ‘Kanchikela’, etc.were not seen to be affected by the disease.

Several studies were conducted at theBanana Research Station, Kannara underKerala Agricultural University on inte-grated management practices forPanama wilt disease. The pathogen wasfound to be soil borne and its entry to thehost plant was through roots. Since theconidia can survive in the soil for as longas seven years, a package of agronomicpractices are recommended based on thefindings of the studies.

Field preparation should be carried outin a systematic way. In disease affectedlocations, weathering the pits for a weekor more and burning the soil with dryleaves is recommended. Field sanitation,especially removal of grass weeds is nec-essary as they become critical alternatehosts.

Cultivation of tolerant varieties is sug-gested for disease prone areas. In othercases, suckers should be selected fromdisease free areas. Dipping pared suckersin 0.2% solution of carbendazim was alsofound to be an effective prophylacticmeasure.

It was also found that the applicationof lime at 1 kg per plant as a soil amend-ment at the onset of the monsoon and


Longitudinal splitting of a banana pseudostemdue to Panama wilt.

good drainage were helpful in checkingthe disease.

The studies further indicated that useof organic manures in banana cultivationcould give a better stand of the cropagainst the disease, probably due toimproved soil structure with more aera-tion.

In the case of disease occurrence,removal and destruction of the diseasedplants is recommended to check its further spread. Application of lime at 0.5-1 kg in the diseased plant pits and inthe basins of surrounding plants also gaveencouraging results in checking the fur-ther spread of the pathogen.

From experiments carried out on dif-ferent chemicals to control Panama wilt,it was found that corm injections with 2% solution of carbendazim at 3ml/cormduring the 5th, 7th and 9th month afterplanting could help to control the dis-ease. Drenching the soil with 0.2% car-bendazim was also found to be effective.

Since the commercial banana varietiesin Kerala are often cultivated in exten-sive wetlands, studies indicated that croprotation, intermittent fallowing or flood-ing followed by fallowing are also effec-tive ways of reducing the spread of thedisease.

Further information is available from S. Estelitta,Associate Professor, Kerala AgriculturalUniversity, Mannuthy, Thrissur, Kerala, S. India.

Cross compatibility of some bananaclonesBefore initiating a hybridization pro-gramme in banana breeding, cross-com-patibility between desirable parents hasto be assessed. Such work is presently on-going at Tamil Nadu AgriculturalUniversity (TNAU) in India. Seventeen

varieties, including commercial triploids,diploids and TNAU-bred synthetichybrids were included in the study (seeTable 1). Anthers were collected from themale parents just prior to dehiscence andthe pollen grains were extracted andsmeared on the stigma of the female flow-ers of female parents on the day of open-ing in the early morning between 6.00 to9.00 a.m., when the receptivity of stigmawas good and ensured by stickiness bytouch. After pollination, the flowers werecovered with perforated paper bags. Onceripe, the fingers were longitudinally cutand the seeds, if present, were extractedcarefully.

Among the 74 cross combinationstested, compatibility was found only ineight combinations (Table 2), thus indi-cating the existence of compatibilitybetween clones. The successful crosseswere between diploid x diploid andtriploid x diploid. Out of the 10 male parents tested, ‘Pisang lilin’ and‘Anaikomban’ were compatible with allfemale parents. ‘Nendran’ which was ear-lier reported as female sterile (Alexander1970) was confirmed as female sterile inthis investigation. The study indicatedthat with the exception of ‘Karpooravalli’,the clones of commercial importancehave a very low percentage of female fer-tility, and diploids provide the bestfemale fertile clones. Seed set was how-ever good in the triploid x triploid crossof Karpooravalli x Robusta, which indi-cates the possibility of new line in bananabreeding, bringing the ‘Cavendish’genome into new hybrids. H-201(Pedigree: Bareli chinia x Pisang lilin xRobusta) is a good female parent and hashybridized with diploid as well as triploidparents (viz. Robusta). Sathiamoorthyand Balamohan (1993) reported that H-201 was a potential female parent par-

ticularly in the synthesis of triploids ofbispecific origin. Seed production wasmaximum in H-201 x Pisang lilin followedby H-201 x Anaikomban and Karpooravallix Robusta. In other successful combina-tions, seed production was very lowdespite the crosses being compatible.

ReferencesAlexander M.P. 1970. Mega and microsporophyte fer-

tility of some banana varieties. Pp. 27-28 in

Proceedings of the 3rd International Symposiumon Tropical and Subtropical Horticulture. Todayand Tomorrow Publishers, New Delhi.

Sathiamoorthy S. & T.N. Balamohan. 1993.Improvement of banana. in Advances inHorticulture Vol. I - Fruit Crops Part I. (K.L.Chadha and O.P. Pareek, eds). MalhotraPublishing House, New Delhi, India.

More information about this work is availablefrom: V. Krishnamoorthy, Dept of Fruit Crops,Horticultural College and Research Institute,TNAU, Coimbatore-641003, Tamil Nadu, India.

Collecting banana germplasm innortheastern IndiaThe northeastern states of India, namelyAssam, Arunachal Pradesh, Megalaya,Tripura, Mizoram and Manipur are a richsource of natural diversity in Musa. Since1998, INIBAP has been supporting aseries of Musa collecting missions in thisregion. These have been conducted by theNational Research Centre for Banana(NRCB), Trichy. Specimens of the wildspecies Ensete glaucum, Musa bal-bisiana, M. acuminata, M. ornata andother Rhodochlamys species have beencollected, together with a range of culti-vated varieties. All the collected materialhas been established in the NRCBgenebank for formal identification andcharacterization.

In Arunchal Pradesh, it was noted thatwhere Eumusa and Rhodochlamys

Table 2. Mean seed set in successful crosses.

Name of cross No. of flowers No. of seeds Mean seeds / fruitpollinated obtained

3x x 3x

Karpooravalli x Robusta 150 187 1.250

3x x 2x

Karpooravalli x Pisang lilin 185 5 0.027

Karpooravalli x H-110 120 8 0.067

2x x 2x

Matti x Pisang lilin 30 30 1.000

H-201 x Anaikomban 102 152 1.490

H-201 x Pisang lilin 79 427 5.405

H-201 x H-110 53 90 1.700

H-201 x Anaikomban 11 24 2.182

H-201 x Pisang lilin 12 4 0.333

H-201 x Robusta 11 1 0.091

Table 1. Details of parents used.

Female parents Male parents

Triploids Triploids

Karpooravalli (ABB) Robusta (AAA)

Red banana (AAA) Red Banana (AAA)

Rasthali (AAB)

Nendran (AAB)

Diploids Diploids

Nivediyakadalli (AA) Nivediyakadalli (AA)

Matti (AA) Pisang lilin (AA)

Sannachengadalli (AA) Sannachengadalli (AA)

Anaikomban (AA) Anaikomban (AA)

Ambalakadalli (AA) Ambalakadalli (AA)

Ney poovan (AB) Erachivazhi (AA)

Synthetic hybrids Synthetic hybrids

H-59 (AA) H-59 (AA)

H-97 (AA) H-97 (AA)

H-66 (AAA) H-66 (AAA)

H-201(AB) H-201(AB)


bananas grew together, the Rhodochlamyshad a closer eco-compatibility with M. acuminata than M. balbisiana,although M. acuminata was clearly domi-nant over the Rhodochlamys species. InAssam, Bhimkol, a seeded balbisianaclone is widely grown in backyards for itsmedicinal qualities. Although seeded, theseeds are soft enough that the fruit can beconsumed along with the seeds.Throughout the region, the unusual prac-tice of selling male buds was noticed. Theimmature male buds of wild bananas areharvested even before shooting and areused for the preparation of special dishes.

A summary of the wild species col-lected during the missions is provided inTable 3.

INIBAP News

New staffHélène Laurence, an intern funded bythe Ministère des Relations interna-tionales du Québec joined INIBAP as aResearch Assistant in January 2002. Sheis based at the Regional Office of INIBAPin Kampala and will begin work on a 3-year study of the impact of improvedbanana varieties (Musa) on the liveli-hoods of households in Eastern Africa.She will work closely with INIBAP scien-tists and regional NARS partners. Hélènehas a BSc in geography and a MSc inAgrometeorology.

Olivier Guinard, also funded by theMinistère des Relations internationalesdu Québec, joined the INIBAP pro-gramme in Montpellier as an intern inApril 2002. During his 6-month internshiphe will be working on a project using theMGIS database (Musa GermplasmInformation System) to carry out a seriesof tracer studies on important germplasmaccessions as well as visualizing geo-graphical assignments for the differentaccessions. Olivier comes from Québecwhere he studied for his BSc in Biology,specializing in molecular biology/biotech-nology at the University of Quebec inMontréal and has just completed his MSc.

Training course on MGIS in Africa

22–27 April 2002, CARBAP, Nyombé,CameroonA training course on the use of the MusaGermplasm Information System (MGIS)for the management of informationrelated to genetic resources of bananasand plantains (Musa spp.) was heldrecently for Musa germplasm curatorsfrom Africa. The objective of this trainingcourse was to provide these curators withthe expertise and tools to better manageinformation related to the accession intheir collections. The use of MGIS willalso allow them to exchange geneticresource information with otherresearchers and curators throughout theworld. This training course was heldthanks to funding support provided by theTechnical Centre for Agricultural andRural Cooperation (CTA).

The training course gathered 23 partic-ipants from West, Central, Eastern andSouthern Africa (see list below). Thecourse was held in French and English,with translation ensured by the trainers.All documents and training materialswere provided in both languages.

The course included both field andcomputer-based training. Exercises onthe taxonomical and botanical identifica-tion of varieties were held in the fieldusing the list of descriptors published byIPGRI, INIBAP and CIRAD. The largegermplasm collection maintained byCARBAP provided an excellent resourcefor the field exercises. This collectionconsists of over 400 accessions, repre-senting a very large range of African vari-eties, especially plantains, but alsoincluding some East African highlandvarieties.

With regard to the computer-basedtraining, participants learnt how toinstall the MGIS software on their com-puters, how to create users accounts,enter new records, and carry out informa-tion searches in the global database. Theywere also trained in the procedures fordata exchange through the global data-base.

Musaid.win, a software developed byCIRAD to assist in the identification ofunknown varieties, was also explained byXavier Perrier from the Biometry Unit ofCIRAD. This software is provided to allparticipants in MGIS.

Participants agreed that the trainingprovided a useful tool for the manage-ment of genetic resource information andhighlighted the importance of collectingand managing this information using astandard format. The workshop also pro-vided a valuable opportunity for curatorsto make contact with their colleaguesfrom the region as a whole, and also toidentify resource people who can helpthem in their future work.

Following the MGIS training course, aworkshop on the “Names and synonymsof plantains” was held with the partici-pants from West and Central Africa. Dr Kodjo Tomekpe conducted the work-shop using data and photos from MGIS. Afirst draft list of variety names has beenestablished that should be confirmed byfurther studies of the varieties in the field.

INIBAP gratefully acknowledges thevaluable support of CTA and CARBAP inthe organization of the training course.

List of participants

From national organizationsSylvestre M. Rogers, Rice ResearchStation, Rokupr, Sierra Leone;Guilavogui Zeze, Institut de rechercheagricole de Guinée (IRAG), Guinea;Simplice Koffi Kouassi, Centre nationalde recherche agronomique (CNRA), Côted’Ivoire;Lawrence Aboagye, Plant GeneticResources Centre, Niaouli, Ghana;Flore Sindemion, Institut national derecherches agricoles du Bénin (INRAB),Benin;Antoine Mputu Kena Kudia, Institutnational pour l’étude et la rechercheagronomique (INERA), DRC;Clotilde Ngnigone Ella, Institut derecherches agronomiques et forestières(IRAF), Gabon;Fernand Mouketo, Centre de rechercheagronomique de Loudima (CRAL),Congo;Selome Y. Dogbe, Institut togolais de larecherche agronomique (ITRA), Togo;Olagorite Adetula, National HorticulturalResearch Institute (NIHORT), Nigeria;Robert Muhwezi, National AgriculturalResearch Organization (NARO), Uganda;Mkulila Shaban, Agricultural Researchand Development Institute (ARDI),Tanzania;Margaret Onyango, Kenya AgriculturalResearch Institute (KARI), Kenya;


Table 3. Details of wild Musa species collected during the exploration innortheastern India.

Genus Section Species Site of collection No. of accessions Use

Ensete E. glaucum Assam, Tripur, Mizoram 1 Fibre, vegetable, ornamental

Musa Eumusa M. balbisiana Assam, Tripur, Mizoram 1 Fruit, vegetable

M. acuminata Assam, Tripur, Mizoram 9 Fruit, vegetable

Rhodochlamys M. ornata Assam, Mizoram 1 Ornamental

Unidentified Assam, Tripur, Mizoram 5 -

Antoine Nsabimana, Institut des sciencesagronomiques du Rwanda (ISAR),Rwanda;Ferdinand Ngezahayo, Institut derecherches agronomique et zootechnique(IRAZ), Burundi;Dickson L.N. Banda, Department ofAgricultural Research and TechnicalServices, Malawi;Dejene Abera, Ethiopian AgriculturalResearch Organization (EARO),Ethiopia;Connie Fraser, Institute for Tropical andSub-Tropical Crops (ITSC), South Africa.

From regional/internationalorganizations William Nguefack, Centre africain derecherches sur bananiers et plantains(CARBAP), Cameroon;Chyka Okarter and Perpetua Udu,International Institute of TropicalAgriculture (IITA), Onne research sta-tion, Nigeria;Emmanuel Njukwe, InternationalInstitute of Tropical Agriculture (IITA),Mbalmayo, Cameroon;Deborah Karamura, INIBAP-ESA,Uganda.

Resources persons:Kodjo Tomekpe CARBAP, Cameroon;Ekow Akyeampong, INIBAP – MUSACO,Cameroon;Elizabeth Arnaud and Suzanne Sharrock,INIBAP Headquarters, France;

Xavier Perrier, CIRAD, France.

Geographical InformationSystems (GIS) and Musa diversityINIBAP is investigating the possibility ofusing a newly developed GIS software(DIVA) for the spatial analysis of Musagenetic resource information. DIVA hasbeen developed by the InternationalPotato Centre (CIP) with the support ofFAO, the CGIAR’s System-wide geneticresources programme (SGRP) andIPGRI. A training course was heldrecently at INIBAP’s headquarters inMontpellier to train staff in the use of thesoftware and to assess the potential touse this software in association with dataavailable in the Musa GermplasmInformation System (MGIS), in particu-lar with data recorded during collectingmissions. INIBAP hopes to use the DIVA-GIS software for the following tasks: • Prediction of where particular

species/varieties can be found;• Prediction of where germplasm with

specific traits is likely to be found;• Identification of high-diversity areas at

genetic and species level;

• Identification of where gaps exist inthe coverage of collecting missions(comparison with production areas);

• Documentation of the potential effectsof climate change and other potentialgenetic erosion factors on wild speciesdistribution;

• Comparison of species distribution inrelation to pests and diseases;

• Predict and assess impact, e.g. of newvarieties.More information about the DIVA soft-

ware is available from the CIP website(http://www.cipotato.org/diva/). The soft-ware is freely available and can be down-loaded, along with the user’s manual,from the website.

Fifth meeting of the regionalsteering committee of MUSACOFrom 11 to 12 February 2002, the regionalsteering committee of the banana andplantain research network for west andcentral Africa, MUSACO, met in Cotonou,Benin for its annual meeting. In atten-dance were representatives from Benin,Cameroon, Congo Republic, Côte d’Ivoire,Gabon, the Democratic Republic ofCongo, Ghana, Guinea, and Sierra Leoneas were those of CARBAP (formerlyCRBP) IITA and INIBAP. Togo was admit-ted as the 13th member of the network atthis meeting.

In a speech to officially open the meet-ing, Dr David Arodokoun, ScientificDirector of the Institut national desrecherches agricoles du Bénin acting onbehalf of his Director General stressedthat banana and plantain are crops thatcould contribute to food and nutritionalsecurity, alleviation of poverty, and thecreation of employment in Benin. Bananaand plantain are two of the crops thatBenin is now promoting to diversify thefood crop base. He reported that 33% ofhouseholds in Benin regularly consume

plantains and Musa production in Beninhas increased from 22 000 tonnes in 1998to 45 000 in 2001. He hoped that theresearch carried out in the network willhelp find solutions to constraints facingfarmers in Benin such as lack of cleanplanting materials, pests and diseasesand post-harvest transformation of thefruit.

Adoption of the report of the AccrameetingThe first order of business was the adop-tion of the minutes of the last regionalsteering committee meeting held inAccra in April 2001. Before memberswould do that, they wanted to know if therecommendations of that meeting hadbeen implemented. The President andsecretary provided the following informa-tion on the various recommendationsthat were made in Accra:• The training course on Mycosphaerella

leaf spot diseases was held in Malaysiain June 2001. Drs Kobenan Kouman ofCNRA, Côte d’Ivoire and EkowAkyeampong were the two partici-pants from West and Central Africa. Asecond training course on the post-laboratory handling of tissue-culturedplantlets and on rapid multiplicationof Musa planting materials was orga-nized at CARBAP, Nyombé from 2 to 7 December 2001 for francophonemember countries of the network.

• The nine countries which receivedfunds from INIBAP have collected andsent the secondary baseline informa-tion to the network secretariat. Areport is being prepared by a youngprofessional officer seconded to theoffice by the FAO.

• Concerning participation in PROMUSAworking groups by scientists from thesub-region, only Benin, Côte d’Ivoire,Gabon and Ghana sent names of


Participants to the 5th MUSACO meeting.

researchers to the PROMUSA secre-tariat through Dr Adiko, the West andCentral Africa representative on thesteering committee of PROMUSA.

• As at the time of the meeting, nothinghad been heard from CORAF regardingthe eight initiatives written in collabo-ration with CARBAP that were sent inresponse to its call for projects.

• On the question of IITA scholarships,members were informed that Mr BenBanful, former representative of Ghanaon the MUSACO steering committeehas received an IITA scholarship forPhD studies.

Country Musa newsEach country representative gave a briefreport on new activities that are takingplace in their countries. To popularizebanana and plantain production in SierraLeone, nurseries and demonstration plotshave been established in four districts.These will be extended to four more dis-tricts. Also, local varieties have been col-lected and planted for characterizationpurposes.

As in Sierra Leone, to re-launch plan-tain and banana production in Littoraland in the Forest regions of Guinea(Conakry), nurseries have been estab-lished to produce clean planting materialsthat will be distributed to farmers.Among the varieties to be given to farm-ers are hybrids of IITA and CARBAP

Studies are being planned in Côted’Ivoire to attempt to explain the obser-vation that Pratylenchus spp. seems tobe replacing Radopholus similis as themain nematode on Musa. IITA is inter-ested in the issue of changes in speciescomposition and will consider offering adoctoral fellowship for work on this incollaboration with KULeuven. It was rec-ommended that a nematological surveybe conducted in all member countries todetermine if the nematode diversity andthe relative abundance of the speciesremain the same as before.

The other new activity in Côte d’Ivoirewas the high planting density technologythat Ivorian scientists are testing on-sta-tion. The high planting density trialsbeing conducted in Côte d’Ivoire and alsoin Cameroon are the follow-up of a visitmade by ten scientists, farmers andextension agents from West and CentralAfrica to the Dominican Republic andCosta Rica to study the high density plan-tain plantation production technologiesbeing used there. The delegation wasimpressed by the up to 60% yieldincreases that have been obtained in theLatin America and Caribbean region frommanaging ‘False horn’ plantain as an

annual crop at densities from 2500 to5000 plants per hectare.

Ghana’s new representative on thesteering committee, Dr Anno-Nyakoinformed the meeting that Musa breed-ing has started in Ghana. A plant biologylaboratory with a tissue culture unit hasbeen established in Gabon.

Being the first time to participate inthe meeting, the Togolese representative,Dr S. Dogbe, gave an overview of theresearch on banana and plantain in hiscountry. Bananas and plantains are culti-vated in the cocoa/coffee zone as a shadefor the cash crops. The programme has afield genebank consisting of 32 acces-sions many of which originate fromGhana. A major problem confronting theproduction of the crops in Togo is the lackof good quality planting materials.

Updates on regional projectsMembers reported on the two regionalprojects, Musa germplasm evaluationand periurban Musa production.

Germplasm evaluation trials have beenestablished in almost all the networkcountries but it is only in Côte d’Ivoirethat the first crop has been harvested. Itwas reported that ‘FHIA-23’ is the mostproductive among the bananas (‘FHIA-01’,‘FHIA-18’ and ‘SH-3460’) that are beingevaluated in Côte d’Ivoire, but it also hasthe longest crop cycle. The inoculumpressure of black leaf streak disease(BLSD) was so high that at harvest,‘Orishele’, the susceptible plantain vari-ety had virtually no functional leaves.Consequently, bunch weights of ‘Orishele’were low compared to the resistant

hybrid, ‘CRBP-39’, which maintained sixgreen leaves to harvest. Tests to deter-mine consumer acceptance of the fruitsare being conducted but the results arenot yet available.

The periurban Musa production projectis making good progress in Benin with thevitroplantlets provided by CARBAP.Among the hybrids established in Beninare ‘FHIA-25’, ‘FHIA-18’, ‘FHIA-23’ and‘CRBP-39’. Survival of plants in the fieldwas very high so is the enthusiasm of the40 farmer participants. The visit to someof the farms generated a lot of interest asthe plants were doing very well. Theresearchers and farmers exchangedviews on farmer participatory researchapproaches. These discussions continuedwhen the scientists returned to the meet-ing place the next day. A survey on periur-ban Musa production conducted in Beninrevealed that 56% of Musa farmers culti-vate plantain on an average plot size of0.8 ha. In Benin also, inadequate supplyof planting materials is cited a bottleneckto expanded production

The periurban project has not reallytaken off in Ghana as the national insti-tution that was supposed to supply theplanting materials failed to do so.Materials imported from South Africahave been weaned and will be suppliedto farmers at the beginning of the rainyseason.

International and regional institutionsCARBAP and IITA each sent a delegationof several scientists to present the differ-ent areas of Musa research at their cen-tres. Dr Lutaladio representing FAOtalked about collaboration with the net-work.

CARBAP presented the advances oftheir programmes regarding plantainbreeding, agronomy and integrated sys-tems, plant pathology and pest controlwith an integrated pest managementapproach (leaf spot diseases, nematodesand weevil), post-harvest technologiesand socio-economics. Achievements ofthe centre include the development andtransfer of in vivo multiplication tech-niques to produce large numbers of cleanplanting materials, and ‘CRBP-39’, a plan-tain-like hybrid that has been releasedand distributed within the MUSACO net-work and to more than 40 countriesaround the world in the framework of the 3rd International Musa TestingProgramme coordinated by INIBAP.Secondary triploids with resistance todiseases have been created by differentbreeding schemes and short and earlyyielding plantain hybrids are also underevaluation. Banana and plantain-based

‘CRBP-39’, one of the hybrids selected for theperiurban project. (Photo: CRBP)


snack foods, infant formulas and flourhave been developed.

Presentations from IITA were on par-ticipatory research, integrated pest man-agement, Musa breeding and agronomy.In farmer participatory evaluations con-ducted in eastern Nigeria, farmers pre-ferred ‘PITA-14’ one of IITA’s plantain-type hybrids over ‘Agbagba’, the localvariety. ‘PITA-14’ gave higher financialreturns as well. The meeting wasinformed of a pilot project supported byUSAID in which hybrids developed atCARBAP, IITA and FHIA are being evalu-ated in farmers’ fields in Nigeria. It ishoped that after the pilot phase, the pro-ject will be expanded to other countriesin the West and Central Africa. IITA con-tinues to develop plantains hybrids withsuperior resistance or tolerance to dis-eases, including a practical physiological-genetic and biotechnological approach tocontrol banana streak virus, and goodagronomic characteristics such as earli-ness, short stature, and good rooting.Integrated pest management researchhas included developing and testingmethods such as using hot or boilingwater to clean suckers contaminatedwith pests. There are also studies todetermine the efficacy of nematicidalplants such as Flemingia interplanted inplantain fields.

The four projects of INIBAP, namelygermplasm management, germplasmimprovement, information and communi-cations and regional networks werebriefly described and activities in Africaunder each were mentioned. The objec-tives and modus operandi of PROMUSA,the global programme for Musa improve-ment, coordinated from INIBAP head-quarters were also described.

The FAO representative at the meeting,Dr Lutaladio, traced the history of thecollaboration between his departmentand INIBAP. In 1999 AGPC/FAO and INI-BAP discussed collaboration on the gath-ering and exchange of information andthe transfer of technology. A young pro-fessional officer (YPO) was recruited andposted to the INIBAP/MUSACO secre-tariat to develop instruments for the col-lection and compilation of baseline infor-mation and to incorporate collectedinformation into HORTIVAR, a databaseon performances of horticulture cultivarsin relation to environmental conditionsand cultivation practices. In addition, theYPO was to provide inputs in the urbanand periurban horticulture programme inrelation to food security and was to assistin project proposal development. DrLutaladio informed the meeting that theYPO has prepared preliminary reports on

the different tasks he was given at thebeginning of his assignment.

In the immediate future the FAO willassist at least two MUSACO membercountries in designing projects forimprovement of banana and plantain pro-duction for small-scale growers throughthe setting up of efficient and cost-effec-tive multiplication systems for produc-tion of disease-free planting materials.The FAO will collaborate with MUSACO,CARBAP and IITA to collect and charac-terize Musa germplasm in the Congobasin, to upgrade tissue culture and nurs-ery facilities in certain countries, and totrain researchers in handling tissue cul-ture plantlets, virus indexing, and rapidproduction of planting materials. Finally,the FAO will assist in the development ofa protocol for mass propagation and dis-tribution of quality planting materials.

Functioning of the networkThe delegates discussed ways to improvethe operation of the network. For a start,it was agreed that working groups basedon identified research priorities shouldbe set up. These will meet as often as nec-essary depending on available resources.Working groups are to be formed immedi-ately on (1) rapid multiplication of cleanplanting materials; (2) profitable plan-tain production systems and (3) farmerparticipatory research. The secretary ofthe network was asked to identify leadersfor each of the three working groups.Activities proposed by the working groupswill be approved and their implementa-tion monitored by the regional steeringcommittee. The current unsatisfactorycommunication links among membersand between members and the secre-tariat was attributed to the lack of infor-mation technology equipment in many ofthe research stations where the Musaprogrammes are based.

RecommendationsThe assembly passed the following rec-ommendations:1. A survey should be conducted in all

member countries to determine thediversity and prevalence of Musanematodes in West and Central Africa;

2. INIBAP, IITA and CARBAP shouldassist MUSACO to organize trainingcourses on germplasm evaluation andon participatory research methods;

3. All should wait for results on the high-density planting technology trials beingconducted by CNRA in Côte d’Ivoireand CARBAP in Cameroon before dis-seminating the technology to farmers;

4. Working groups on (a) rapid multipli-cation of clean planting materials; (b)

profitable plantain production systemsand (c) farmer participatory researchshould be created immediately withinthe network;

5. A grant proposal should be developedto look for funds to equip all nationalprogrammes with basic informationtechnology equipment and Internetaccess.

MUSACO 2003The next steering committee meeting willbe hosted by IRAG in Guinea (Conakry)during the first week of March 2003,under the presidency of Mr BernadinLokossou, the representative of Beninwho was elected to replace Madam AdèleSambo of Gabon.

2nd International workshop on Mycosphaerella leaf spotdiseases of bananas

San José, Costa Rica, 20–23 May 2002This workshop was organized by INIBAPin collaboration with the CorporaciónBananera Nacional (CORBANA), theEscuela de Agricultura de la RegiónTropical Húmeda (EARTH) and theCentro Agronómico Tropical deInvestigación y Enseñanza (CATIE).Coming 13 years after the last interna-tional meeting on this topic, it provided atimely opportunity to analyse the currentsituation regarding Mycosphaerella leafspot diseases at the global level. Themeeting also allowed new lines of investi-gation to be suggested and facilitated there-orientation of breeding programmesand biotechnology strategies for thegenetic improvement of bananas andplantains.

More than 60 scientists attended theworkshop from both the private and pub-lic sectors, representing more than 16 dif-ferent countries from Latin America andthe Caribbean, Europe, Africa, Asia andthe Pacific, including Australia.

In order to maximize the outputs of themeeting and to guarantee the developmentof new strategies in the control of the dif-ferent Mycosphaerella leaf spot diseases,participation in this workshop was by invi-tation only. The results of the meeting willhowever be widely disseminated throughthe publication of the proceedings.

The meeting was inaugurated by Dr Jorge Sauma, Director of CORBANA.Dr Emile Frison, Director of INIBAP, wel-comed all the participants and paid trib-ute to Ramiro Jaramillo Celis, former INIBAP regional coordinator for LatinAmerica and the Caribbean, in recogni-tion of his invaluable contribution andtireless efforts to the development of the


regional Musa research network. Theofficial opening was made by Dr SalvadorMonge, Executive Director of theSecretary of sector-based planning of theMinistry of Agriculture of Costa Rica.

The workshop was organized around five main topics: 1) Impact ofMycosphaerella leaf spot diseases ofbananas; 2) Population biology and epi-demiology; 3) Host-pathogen interac-tions; 4) Genetic improvement for amanagement of durable resistance and5) Integrated disease management.

During the workshop, participants had the opportunity to learn about the distribution and impact of the differentMycosphaerella leaf spot diseases in several countries around the world.Discussions were held at the end of theeach session to allow research prioritiesand corresponding activities required atthe global level to be identified or refined,in order to significantly reduce the impactof these diseases and thus make Musa amore sustainably productive crop.

Session 1. Impact of Mycosphaerellaleaf spot diseases of bananasIntroductory papers presented informa-tion on the global spread, current dis-tribution and impact of the threeMycosphaerella leaf spot pathogens - M. musicola, M. fijiensis and M. eumusae.Other papers described techniques devel-oped in Australia for the rapid diagnosisof M. musicola and M. fijiensis, theeffects of M. musicola and M. eumusaeon banana cultivation in South Africa andthe impact of M. fijiensis in Cuba, Braziland tropical Asia. The latest taxonomicwork undertaken on the anamorph of M. eumusae and on other Mycosphaerellaspecies was also described. M. fijiensiscontinues to spread to new areas. From2000 to 2002, the pathogen was identifiedfor the first time in Madagascar, theBahamas, and the Galapagos Islands ofEcuador and in the north Queenslandbanana growing area where eradicationis being attempted. M. eumusae leaf spothas also been observed on ‘Mysore’ (AAB)in Sri Lanka. As this clone has strongresistance to M. musicola and M. fijiensis,this is the cause of some concern.

It was agreed that more taxonomicinformation about Mycosphaerella spp. isneeded, as well as information on otherrelated genera that either form or occurin banana leaf lesions. A greater knowl-edge of Mycosphaerella pathogens/saprophytes and those in related generais a prerequisite to the development ofrapid diagnostic tests to distinguish leafspot pathogens. The exact distribution ofM. eumusae also needs to be investi-

gated. Further surveys in South andSoutheast Asia to determine where M. musicola, M. fijiensis and M. eumusaeoccur are necessary. More information onthe effect of M. eumusae on the growthand yield of banana clones is needed.Information suggests that Cavendish andPlantain cultivars are very susceptible.

Session 2. Population biology and epidemiologyPathogenicity and distribution variabil-ity, sources of resistance, epidemiologyand population structure of the mainspecies (M. fijiensis, M. musicola andM. eumusae) at the national, regionaland international levels were defined asfundamental information for the contin-ued success of banana production. Suchstudies are particularly necessary inAsia, which is the centre of diversity ofthe three pathogens and where littleresearch has so far been conducted. Astudy of the evolution of host–pathogenrelationships for the three pathogens,particularly involving resistant cultivars,is of a special concern, in order to iden-tify pathogen populations that couldbreak down plant resistance and to eval-uate selection pressure. Molecular toolssuch as microsatellites have been rec-ommended to monitor the genetic variability of the pathogen populationsand pathogenicity should be evaluatedconcurrently. Epidemiological studies,including disease dispersal, are neededto better understand the distributionand the spread of the pathogen and willcomplement, together with the studieson pathogenicity and genetic variability,all the information required to antici-pate the evolution of pathogen popula-tions and to define resistance manage-ment strategies.

Session 3. Host-pathogen interactionsSeveral cases of an unexpected level ofsusceptibility to black leaf streak disease(BLSD) have been reported. Althoughdifferent reasons have been offered toexplain the phenomenon (poor nutrition,environmental stress), the problem of theerosion of resistance cannot be ignoredand requires a precise characterizationof the pathogen population. A greaterunderstanding of the mechanismsinvolved in plant-pathogen interactionscontinues to be needed to ensure the longterm success of breeding programmes.Further studies are also needed to com-pare the effect of infection by each of thethree pathogens (M. fijiensis, M. musi-cola and M. eumusae) on the host plants.

Other pathosystems (such asMagnaporthe grisea) have shown thepowerful nature of the genetic approachto identify without any a priori the path-ogenicity factors. These approachesinclude the study of gene expression dur-ing production of pathogenicity mutants,comparative genomic and gene functionvalidation techniques. Consequently, thedevelopment of genetic and molecularbiology tools for M. fijiensis in collabora-tion with M. graminicola groups and thelaunching of a genomic initiative toaccess genomic tools and set up agenomic-wide comparison of M. fijiensiswith M. graminicola have been recom-mended.

It is also recommended that the differ-ent mechanisms of resistance (partial orvertical) should be studied.

Session 4. Genetic improvement for amanagement of durable resistanceDuring this session, progress that hasbeen made towards the creation of newvarieties resistant to BLSD, either


Sharing information on Mycosphaerella leafspot diseases during the visit at the CORBANA researchstation in Guápiles. (Photo: C. Picq, INIBAP)

through conventional and/or moderntechnologies, was presented. Newtetraploids hybrids resistant to BLSD arealready available and some of these arewidely grown around the world. However,because of the lack of new sources ofresistance and due to the presence of theactivable form of the banana streak virus(BSV) in interspecific hybrids (A x B),the production of a new generation oftriploid hybrids is seriously jeopardized.Good progress has been reported in thedevelopment of a molecular toolbox forbananas and plantains in the area of thegenetic transformation, allowing the pro-duction of transgenic banana plants.

The study of the diversity of the Musabalbisiana genome, using both morpho-logical and molecular characterizationwas the first recommendation from thissession. It was also recommended, that inanticipation of the needs of geneticimprovement programmes, the T and Sgenomes, Musa textilis and Musa schizo-carpa respectively, should also bescreened.

Mutation induction techniques shouldno longer be seen as an independentgenetic improvement strategy but moreas a tool that can contribute to cross-breeding programmes by increasinggenetic diversity of parental lines.Mutants could also help in understandingthe mechanisms of resistance (functionalgenomics).

Session 5. Integrated diseasemanagementYellow and black Sigatoka control strate-gies on banana can, according to thecountry and the scale of production,include not only chemical and culturalpractices but also the use of mixed cropsor resistant clones. The importantinhibitory effect of some natural sub-stances derived from microorganismsantagonistic to fungi, have also beenreported as effective in reducing thedevelopment of M. fijiensis in vitro.

The integration of various or specialistsfrom different disciplines has been rec-ommended to facilitate the developmentof an achievable integrated pest manage-ment (IPM) approach for banana leafspot diseases. The participants in theworkshop also recommended to investi-gate the potential of natural or syntheticsubstances able to promote or activatesystemic acquired resistance in its broad-est sense.

The proceedings of the workshop willbe published shortly by INIBAP. This pub-lication will include the full papers of allpresentations and a summary of the dis-cussions and recommendations. It is

expected that the proceedings willbecome a reference document ofMycosphaerella diseases for the next tenyears.

Books etc.

Strategy for the Global MusaGenomics ConsortiumReport of a meeting held in Arlington,USA, 17-20 July 2001ISBN: 2-910810-48-08The Global Musa Genomics Consortiumwas launched at a meeting held inArlington, Virginia, which was critical for

laying a solid foundation for future col-laboration in Musa genomics and allowedthe first important steps to be takentowards the development of a coherentstrategy for Musa genomics.

This document provides further back-ground information about the establish-ment of the Consortium and its aims andobjectives. It also provides a review of thecurrent status of Musa genomicsresearch and provides details of thenature and scale of the work to be carriedout by the Consortium members.Information is provided on an incremen-tal strategy developed by the Consortiumto achieve its goals and the proposedmodus operandi, as agreed during theArlington meeting. Further details areprovided in the Annexes.

Copies are available from INIBAPHeadquarters.

Addendum to the ‘Descriptors forBanana (Musa spp.)’ To complete the revised version of the‘Descriptors for Banana, Musa spp.’ andresponding to demand from East Africa,additional characters specific to the EastAfrican Highland bananas were incorpo-rated as an addendum in 2001. Thedescriptors for Musa are unique inincluding a colour chart. This helps to

remove the subjective nature of colourrecording, and leads to a common under-standing of such characters.

Copies of the descriptors and theiraddendum are available from INIBAPHeadquarters.

Resúmenes analíticos de la investigación sobre plátano en ColombiaEdited by D.G. Cayón and F. SalazarAlonsoISBN: 958-96885-1-9CORPOICA could certainly be proud forthe effort they made recently to identify,analyse and compile the existing infor-

mation on plantain research and technol-ogy transfer in Colombia under the title‘Resúmenes Analíticos de laInvestigación sobre Plátano enColombia’. This information product is aunique inventory of the major part of thescientific literature published on thistopic including grey literature. Itincludes 792 abstracts and authors- andthematic indexes which make the searcheasier for the reader.

This important document in Spanish isavailable in both electronic (database onCD-Rom) and printed (400 pages) for-mats and will certainly be highly appreci-ated by all those working on plantain atinternational level.

The document is available on requestat CORPOICA, Apartado Aéreo No 1087,Av Bolivar Sector Regivit 28 Norte,Armenia, Quindío, Colombia – Fax: (57-6) 7496331 and at the INIBAP Office forLatin America and the Caribbean, C/oCATIE, 6170 Turrialba, Costa Rica.

Banana varieties: The ACIAR years1987-1996J.W. Daniells and N.J. BrydeInformation series Q101013ISBN 0727-6273During the period 1987-1996, theQueensland Department of PrimaryIndustries (QDPI) was the lead agency ofthe ACIAR project ‘Banana improvementin the South Pacific’. In the course of theproject, banana varieties were collectedfrom all around the world. One hundred


and six varieties are reported in this pub-lication which represent significantcross-section of those available for evalu-ation. They include some hybrids fromconventional breeding programmes,selections originating as offtypes from tis-sue culture propagation, existing culti-vars and wild species.

The report put together the agronomicinformation collected along with colourphotographs of bunches which permit agood appreciation of each variety. Manyreaders will also find these photographsuseful for identification purposes.

Clean & green bananas – Where tofrom here?J.W. DaniellsInformation series Q101014ISBN 0727-6273Current sales for clean & green/organicbananas are very limited in Australia.Organic export is risky and uncertain.However, the market is shifting in thisdirection and in the longer term, theorganic niche will probably eventuallygrow to 10-15% of the market. Major salesof both products will be facilitated bysupermarket participation and the cur-rent price of the existing organic productwill need to come down. Efficiencies ofproduction must be improved by specificresearch on limiting factors such as soilfertility management, leaf disease con-trol and greater development/exten-sion/adoption of existing technology.Good progress has been made by thebanana industry to reduce pesticides usebut it is now necessary to pull togetherthe body of knowledge and develop anECO-OK type system implemented oncommercial farms which complies toauditable standards and market develop-ment so that producers are rewarded fortheir efforts.

The two publications mentioned aboveare available on request at Departmentof Primary Industries, GPO Box 46,Brisbane QLD 2001, Australia.


Announcements

3rd International symposium onthe molecular and cellular biologyon Musa9-11 September 2002, Leuven, BelgiumThe first symposium, held in March 1999 atCornell University in Ithaca, USA, wasorganized to open a forum where all thoseinterested in molecular and cellular biol-ogy had the opportunity to meet andexchange ideas about their research activi-ties. The meeting was a resounding suc-cess. It was therefore suggested that thisconcept be continued under the auspicesof PROMUSA. After the 2nd Internationalsymposium on the Molecular and cellularbiology of bananas held in October 2001 inByron Bay, Australia, INIBAP andKULeuven are pleased to announce the 3rd

International symposium on the Molecularand cellular biology of bananas, organizedby INIBAP and KULeuven, to be held inLeuven, Belgium from the 9th to the 12th

September 2002.The scientific programme includes five

sessions. During the event, more than 30 papers and 31 posters will be presented.

Twelve keynote lectures will also begiven by outstanding speakers: Prof. Francis Quétier, Dr Xavier Draye andProf. Guido Volkaert (Session 1: Genomics),Drs A. De Picker and D. Inzé (Session 2:Gene expression and transformation),Prof. G. Gheseyn, Drs Johan Nayts and D. Van Der Straeten (Session 3: Molecularplant pathology and disease/pest resis-tance), Drs Isabel Roland-Ruitz and PeterBreyne (Session 4: Biodiversity characteri-zation and conservation) and Drs EmmaSchofield and W. Peumans (Session 5:Biochemistry and physiology).

A workshop on Intellectual propertyand genetically modified organisms willalso be led by Dr Victoria Henson.

A scientific visit to KULeuven and theINIBAP Transit Centre facilities will beorganized.

To know more about the symposium,visit the INIBAP web site at:

http://www.inibap.org/actualites/actualites_eng.htm

Global conference on banana andplantainGrand Ashok Hotel, Kumara Krupa,High Grounds, Bangalore, IndiaOctober 28 to 31, 2002To address the new emerging agenda for growth and development of thebanana industry, the Association for theImprovement in Production andUtilization of Banana (AIPUB), India,

with support of INIBAP, the Food andAgriculture Organization of the UnitedNations (FAO), Department of Agricultureand Cooperation, Government of India andIndian Council of Agricultural Research, isorganizing a global conference on bananaand plantain focusing on ‘Banana produc-tion for nutrition and livelihood security’.

This Conference is being organizedwith the following objectives:• To bring together global players in

banana research, development andtrade for deliberation and discussionon various issues for sustainable devel-opment of banana.

• To deliberate upon the opportunitiesfor Indian banana and banana productsin international trade.

• To involve national and internationalexperts to develop and recommend pol-icy initiatives for nutritional and liveli-hood security through banana produc-tion. The Conference deliberations will focus

on the following topics:• Genetic resource management and

crop improvement; • Biotechnological advances; • Strategies in production technology; • Organic production of banana; • Integrated disease and pest manage-

ment; • Post harvest management, product

diversification and value addition; • Policy support and programmes; • National and international trade; • International cooperation.

Important dates Deadline for submission of abstracts -

31st July 2002.Deadline for submission offull papers - 30th September 2002

Registration fees

Before 30 August 2002Member of AIPUB: 50 US$No member: 75 US$Corporate: 100 US$

After 30 August 2002Member of AIPUB: 100 US$No member: 125 US$Corporate: 150 US$Registration form along with the

fees should reach the ConferenceSecretariate: Bagwani Bhavan, 47 JanakpuriInstitutional Area, Pankha Road, NewDelhi-110058; Tel (91-11) 5622150/5531211;Fax (91-11)-5531211/33849780.

For more information and registra-tion, visit the web site:

http://www.aipub.org/conferences.htm

• HeadquartersParc Scientifique Agropolis II34397 Montpellier Cedex 5 – FRANCEe-mail: [email protected]://www.inibap.orgDirectorDr Emile FRISONe-mail: [email protected] Genetic Resources ScientistDr Jean-Vincent ESCALANTe-mail: [email protected] Conservation ScientistMs Suzanne SHARROCKe-mail: [email protected] Information/CommunicationsMs Claudine PICQe-mail: [email protected] in charge MGISMs Elizabeth ARNAUDe-mail: [email protected] ManagerMr Thomas THORNTONe-mail: [email protected]

• Regional Office for Latin America and the CaribbeanRegional CoordinatorDr Franklin E. ROSALESAssociate Scientist, Musa technology transferLuis POCASANGRE

C/o CATIEApdo 60-7170 Turrialba, COSTA RICATel/Fax: (506) 556 2431e-mail: [email protected]

• Regional Office for Asia and thePacificRegional CoordinatorDr Agustín MOLINAC/o IRRI Collaborators Center3rd Floor, Los Baños, Laguna 4031PHILIPPINESFax: (63-49) 536 05 32e-mail: [email protected]

• Regional Office for West andCentral AfricaRegional CoordinatorDr Ekow AKYEAMPONGAssociate Scientist, Technology transferKim JACOBSENC/o CRBP – BP 12438Douala, CAMEROONFax: (237) 342 91 56e-mail: [email protected]

• Regional Office for Eastern andSouthern AfricaRegional CoordinatorDr Eldad KARAMURA

Associate Scientist, Technology transferGuy BLOMMEPO Box 24384KampalaUGANDAFax: (256-41) 28 69 49e-mail: [email protected]

• INIBAP Transit Center (ITC)Officer in chargeMs Ines VAN DEN HOUWEKatholieke Universiteit LeuvenLaboratory of Tropical Crop ImprovementKasteelpark Arenberg 13,B-3001 LeuvenBELGIUMFax: (32-16) 32 19 93e-mail: [email protected]

• Associate Expert, NematologyMr Thomas MOENSC/o CORBANALa Rita Research StationApdo 390-7210Guápiles, COSTA RICAFax: (506) 763 30 55e-mail: [email protected]

Typescripts should be prepared inEnglish, French or Spanish and submit-ted in duplicate to the Managing Editor.They should be double-spaced through-out. All pages (including tables figures,legends and references) should be num-bered consecutively. Include the fullname of all the authors of the paper,together with the addresses of theauthors at the time of the work reportedin the paper. Indicate also the authornominated to receive correspondenceregarding the paper.

If the typescript was prepared on acomputer, please send a copy on diskette(or by e-mail) along with the printedones, indicating the name and version ofthe wordprocessor used. • Abstracts: An abstract not exceeding

200-250 words should be sent in thesame language as the typescript, aswell as translations (including thetitle) into the two other languages, ifthis is possible.

• Acronyms: These should be written infull the first time they appear in the

text, followed by the acronym inparenthesis.

• References: All literature referencesmade in the text should be referred toby author(s) and year of publication(e.g.: Sarah et al. 1992, Rowe 1995). Alist of references, in alphabeticalorder, should be provided at the end ofthe text.

Please follow the style shown below:Periodicals: Sarah J.L., C. Blavignac &

M. Boisseau. 1992. Une méthode delaboratoire pour le criblage variétaldes bananiers vis-à-vis de la résistanceaux nématodes. Fruits 47(5): 559-564.

Books: Stover R.H. & N.W. Simmonds.1987. Bananas (3rd edition). Longman,London, United Kingdom.

Articles (or chapters) in books: Bakry F.& J.P. Horry. 1994. Musa breeding atCIRAD-FLHOR. Pp. 169-175 in TheImprovement and Testing of Musa: aGlobal Partnership (D.R. Jones, ed.).INIBAP, Montpellier, France.

Tables: These should be numbered con-secutively and referred to by these num-

ber in the text. Each table shouldinclude a title.Illustrations: These should be numberedconsecutively and referred to by thesenumbers in the text. Each illustrationshould include a clear and simple caption.Graphs: provide the corresponding rawdata with the graphs.Drawings: provide originals if this ispossible.Black and white photographs: providethem on bright paper and with good con-trast.Colour photographs: provide good qual-ity proofs and films or original slides.Note: When plant material used for theexperiments reported originates or is reg-istered in the INIBAP genebank, its acces-sion number (ITC code) should be indi-cated within the text or in a tabular form.

Thank you in advance for followingthese instructions

This will facilitate and acceleratethe editing work.

INIBAP addresses

Instructions to authors

The meeting started with a brief presentationby each participant of their research capaci-ty, in terms of human resources, researchfacilities, and future and ongoing projectsrelated to Mycosphaerella leaf spot dis-eases. Participants also commented on theirparticipation in PROMUSA, defining theirareas of interest where they would like todevelop partnerships with other participants.

The participants identified variousresearch priorities and defined the mainactivities that should be carried out.

RecommendationsDevelopment of a detailedunderstanding of the populationstructures of M. musicola, M. fijiensis and M. eumusae

Survey of geographical distribution of the three Mycosphaerella speciesThe survey of the distribution of the differentspecies requires wide sampling at the nationallevel of the different agroecological areaswhere Musa is found, and the morphologicalcharacterization of the species through theobservation of the anamorph stage (conidia),including molecular characterization usingPCR diagnostics.

The PROMUSA Sigatoka working groupratified the recommendation made during the“2nd International workshop on Mycosphaerellaleaf spot diseases of bananas” held from 20 to23 May 2002 in San Jose, Costa Rica: ‘Theexact distribution of M. eumusae needs to beknown. Further surveys in south and south-east Asia to determine where M. musicola,

M. fijiensis and M. eumusae occur are neces-sary. The name of the banana clone affected,an indicator of the severity of the leaf spot andlocal environmental data would be useful asthis may help explain distribution. IMTP trialsare seen as ideal locations for assessing thereaction of different clones to the different leafspot pathogens. The collection and diagnosisof specimens of leaf spot from IMTP trials sitesneeds to be continued. The cooperation andcollaboration of scientists in south and south-east Asia is viewed as essential. Identificationtools should be provided to enable diagnosesto be undertaken locally ‘.

Development of national collections of the different Mycosphaerellaleaf spot pathogensThe PROMUSA Sigatoka working group rat-ified the recommendation made during the“2nd International workshop on Mycos-phaerella leaf spot diseases of bananas”: Areliable, rapid test to distinguish M. musico-la, M. fijiensis, M. eumusae and possibleother Mycosphaerella pathogens/sapro-phytes needs to be developed to aid identifi-cation. Information on how to distinguish the3 pathogens on morphological characteris-tics also needs to be produced and circulat-ed to banana scientists. INIBAP was askedto address this need”.

The creation of a national collection of dif-ferent strains from different Mycosphaerellaleaf spot pathogens on Musa is of specialrelevance in the understanding of the popu-lation structure. The collection must bebased on single-ascospore cultures with anin vitro characterization of the anamorphstage (in vitro sporulation of conidia). It has

PROMUSA I

A global Programme for Musa Improvment

INFOMUSA — Vol 11, N° 1

PROMUSA N° 9

Contents3rd meeting of the PROMUSA Sigatoka working group . . . . . . . . . . . . . . . . . . . . .p.IPROMUSA: banana weevil working group inauguration . . . . . . . . . . . . . . . .p.VIAbstract of presentations . . . . . . . . . . .p.VIII4th and final FAO/IAEA research coordination meeting on Cellular biology and biotechnology including mutation techniques for creation of new useful banana genotypes . . . .p.XIVA summary report . . . . . . . . . . . . . . . .p.XIVAbstract of presentations . . . . . . . . . . .p.XV

What is PROMUSA ?The Global Programme for Musa

Improvement (PROMUSA) is a broad-basedprogramme which aims at involving all themajor players in Musa improvement. It wasdeveloped as a means to link the work carriedout towards addressing the problems of export banana producers, with those initiatives directed towards improving bananaand plantain production at the subsistence and smallholder level. The global programmebuilds upon existing achievements and isbased upon ongoing research initiatives. PROMUSA is therefore a mechanism to further maximize the outputs and acceleratethe impact of the overall Musa improvementeffort. The programme is an innovative mechanism to bring together research carriedout both within and outside the CGIAR, creating new partnerships between NationalAgricultural Research Systems (NARS) andresearch institutes in both developing anddeveloped countries. The formation of suchpartnerships will also contribute to strengthening the capacity of NARS to conduct Musa-related research.The major thrust of PROMUSA is to develop a wide range of improved banana varietiesfrom which growers worldwide can selectthose most suited to their needs. The programme brings together conventionalbreeding based on hybridization techniqueswith genetic engineering and biotechnological breeding approaches. Thisbroad-based genetic improvement effort issupported by research being carried out onspecific pests and diseases within the various PROMUSA working groups. An efficient mechanism for evaluating new varieties produced within the framework ofPROMUSA is also an essential component ofthe programme.

3rd meeting of the PROMUSA Sigatoka working group24-25th May 2002, EARTH, Costa Rica

been recommended to provide the partici-pants with a protocol to sample, establishand maintain the collection. INIBAP wasmandated to collaborate with CIRAD in thedevelopment and distribution of the technicalinformation required. The establishment of anational collection should be promoted andfacilitated through the organization of a train-ing course; especially for those countriesthat develop breeding programmes, but alsowhere disease resistant hybrids of bananaare used on an industrial scale, and wherethe high diversity of Musa would have origi-nated similar diversity in the pathogens.

Genetic population structureThe study of the genetic population structuresof Mycosphaerella leaf spot diseases isalready on going at national, regional andinternational levels. However, the group rec-ommends increasing the number of countriesinvolved at the national level, which will allowrefinement of both regional and internationalstudies. Both biological (morphological) andmolecular determinations have been recom-mended to improve the understanding of thedifferent population structures. The samplingprotocol and methodology should be stan-dardized and the recognition of the differentspecies facilitated through the development ofa technical factsheet to be widely distributed.INIBAP and CIRAD agreed to work togetherin the preparation of this information whichshould include several detailed illustrations ofthe different pathogens and their anamorphstages. This information will also becomepart of the IMTP guidelines. The develop-ment of more molecular markers as SSR andCAPS should allow the study of the differentpopulations to be refined. The recommenda-tion to include partners from south and south-east Asia made during the last global meetingof PROMUSA in Bangkok was reiterated bythe participants who strongly suggested thatthe INIBAP regional office for Asia and Pacificstrengthen and facilitate any exchangebetween Asian partners and the rest of thePROMUSA community.

Pathogenic characterizationThe pathogenicity of the different strainsshould be approached using either the invitro or in vivo inoculation systems.However, it is recommended that the differ-ent methodologies that currently exist bestandardized. The methodology for the invitro inoculation on leaf fragments devel-oped at CIRAD should be distributed,together with the methodology used to iso-late, cultivate and produce the inoculum ofthe different pathogens. INIBAP and CIRADhave been requested to compile, in a singletechnical document, all the different informa-

tion already published on these differentmethods.

Identification of new sources of resistance

The need of new sources of resistance toMycosphaerella leaf spot diseases hasbeen identified several times in the past.Collecting missions in Indonesia, northIndia and Vietnam have already takenplace but information has only been pro-vided on the characterization of the resist-ance of the different materials collected.The PROMUSA working group recom-mended that INIBAP help to gather anyinformation already available. The groupalso recommended to stimulate the char-acterization of existing collections whereM. eumusae has already been reportedtogether with other Mycosphaerella spp.e.g. MARDI, in Malaysia. In order to facil-itate the screening, the group suggestedusing the ‘severity index’ as the uniqueparameter to detect any source of resist-ance. This information should allow thedefinition of the different reference clonesneeded to evaluate the resistance toEumusae leaf spot disease.

The ‘severity index’ will also be used toevaluate the PROMUSA segregating popu-lations hosted at CORBANA.

DiagnosticsSeveral leaf fungal diseases have beenreported on Musa and other related species.The group recommended the development ofspecific diagnostic tools according to thethree main species of Mycosphaerellapathogen on Musa: M. fijiensis, M. musicolaand M. eumusae. The achievement of thesediagnostic tools will remain within the devel-opment of a worldwide collection ofMycosphaerella isolates; the morphologicaldescription of all the different Mycosphaerellasubspecies associated with banana leavesand the development of primers species-spe-cific as microsatellites and ITS-sequencesand their test on the worldwide collection ofMycosphaerella isolates.

It is therefore suggested:• to develop diagnostic tools to distinguish

the main pathogens and assess currentlyavailable molecular methods for speci-ficity,

• to develop a manual with descriptions ofsymptoms and morphological characters,

• to develop protocols for collection andanalysis of samples,

• to transfer to and train PROMUSA partici-pants on the different technologies

required (collection and sampling, mono-ascosoporing cultures and molecular markers).

Durability of the resistanceSignificant changes in the levels of resist-ance to Sigatoka and black Sigatoka dis-eases have been reported in Australia, Indiaand Cuba. However these may exist justbecause of high inoculum loads. Thus,changes in pathogen populations should bedistinguished from particular epidemiologicaleffects. Therefore, the group recommendedstudying the changes in pathogen popula-tions in response to selection pressure fromnew banana genotypes resistant to Sigatokadiseases. It is essential to monitor changesin pathogen populations in areas where newresistant hybrids are being grown on a largescale. A special recommendation towardsthe development of specific trials in Cubahas been made. Two different aspects ofthe durability of the resistance need to beaddressed: the genetic drift of the pathogenresistance and the selection effect within thepathogen population. Participants in PRO-MUSA Sigatoka working group recom-mended: • the selection of areas where resistant

hybrids have been grown for a long time(e.g. Cuba) and to follow the evolution ofthe pathogen populations, isolatingMycosphaerella strains on resistant andsusceptible cultivars or hybrids,

• the development of molecular markerslinked to the pathogenicity of the fungalstrains (molecular markers will inform onthe genetic drift when pathogenic evalua-tion will be related to the selection effect),

• the quantification of the selection pressureover the time, and

• the study the breakdown of resistance by invitro testing.

Dispersal of Mycosphaerellaleaf spot diseases on MusaM. eumusae is currently limited in extentthroughout most of Asia, although there issome evidence that the pathogen may havereached Africa. The dynamics of the dis-ease are not fully understood. Some projec-tions indicate that this disease will becomemore important than black Sigatoka. Inorder to prepare adequate disease controlstrategies, a detailed knowledge of the epi-demiology of this pathogen is urgentlyrequired. To address the epidemiology of thedifferent Mycosphaerella spp. pathogens onMusa, the group recommended:• the collection of disease incidence data

from the field and literature,

II PROMUSA INFOMUSA — Vol 11, N° 1

• the development of methodologies tounderstand the mechanisms of sporerelease and spore survival in the air at lab-oratory level, and

• the clarification of laboratory data at plan-tation level and to assess the potential forwindborne dispersal (as opposed to furtherspread of the disease by the transfer ofinoculum).

Host-pathogen interactionsThe genetic approach has been shown to beextremely powerful when studying host-pathogen interactions in some pathosys-tems (such as Magnaporthe grisea). Thisapproach does not require the identificationof pathogenicity factors a priori and includesthe study of gene expression during infec-tion (differential display, DNA chip, SSH,etc.), production of pathogenicity mutants,comparative genomic and gene function val-idation techniques.

Here again, the PROMUSA Sigatokaworking group ratified the recommendationmade in the framework of the “2nd

International workshop on Mycosphaerellaleaf spot diseases of bananas” to study: ”thedevelopment of genetic and molecular biolo-gy tools for M. fijiensis in collaboration withM. graminicola groups as well as to launch agenomic initiative to access to genomic tools(EST collection, physical map, genomesequence) and set up a genomic-wide com-parison of M. fijiensis to M. graminicola”.

International core collectionThe group recommended to develop aninternational core collection of M. fijiensis, M.musicola and M. eumusae. The differentstrains should be conserved as fungalmycelia and DNA. CIRAD was suggested to

INFOMUSA — Vol 11, N° 1 PROMUSA III

Institutional interest in the activities of PROMUSAResearch topics Institution willing to participatePopulation structure• Survey of the geographic distribution in Asia• Nigeria IITA• National collections INISAV, CORBANA, CORPOICA, FHIA, EMBRAPA, FABI,• QDPI, CIRAD• Genetic structure of populations INISAV, FABI, CATIE, CIRAD, CORPOICA, CARBAP• Pathogenic characterization CORBANA, CATIE, CIRAD, FHIA, EMBRAPA, CORPOI•

CA, CARBAP, IBPEvaluation of segregating population CORBANA, EMBRAPADiagnostics CRCTPP, CIRAD, CBS, FABIDurability of resistance INISAV, CIRAD, CATIE, EMBRAPA, CARBAP and FHIADispersal• M. eumusae , Asia NRI• M. fijiensis , Caribbean NRI, CIRAD, WIBDECO, CRCTPPHost-pathogen interactions FABI, CIRAD, IBP, CARBAP and CICY• Mechanism of pathogenicity• Mechanism of resistance

Institutional contribution to PROMUSA

Institution, Country Research facilities Human resources Research topics On-going activities Contact

CORBANA, Costa Rica Tissue culture lab. • Field evaluation on new clones • IMTP phase III J.A. Sandoval,Plant pathology lab. • Inoculation of M. fijiensis at greenhouse • Evaluation of Musa segregating R. VargasGermplasm field bank level populations. M. GuzmanExperimental fields • Evaluation of M. fijiensis

segregating populationsINISAV, Cuba Plant pathology labs • Epidemiology of M. fijiensis in hybrids • Population survey of M. fijiensis L. Pérez Vicente

Experimental stations resistant populations (variability and distribution)Coll. with CIGB • IMTP phase III • Durability of the resistance

• Diversity and distribution of M. fijiensis of FHIA hybrids to M. fijiensis(molecular and morphological characterization)

CRCTPP, QDPI, Plant pathology labs Principal plant pathologist (1) • Mycosphaerella leaf spot diseases • Molecular diagnosis of Oceanic R. Peterson (QDPI)Australia Glasshouse Technical officers (2) • Black leaf streak: Sigatoka isolates J. Henderson

Research stations Technician lab. (1) Cultivar evaluation & diagnostics • Isolate collection & preliminary ID (CRCTPP)• Sigatoka: Diagnostics & epidemiology by symptomatology and morphology K. Grice (QDPI)• Molecular diagnosis of black leaf • Sequence analysis of ITS regions streak and Sigatoka diseases of Oceanic isolates

• If needed, sequence of other suitablegenes will be undertaken

host the international collection using a sim-ilar mechanism as INIBAP developed withKULeuven to host the international Musagermplasm at the INIBAP Transit Center.INIBAP was asked to address this need incollaboration with CIRAD.

IV PROMUSA INFOMUSA — Vol 11, N° 1

Institution, Country Research facilities Human resources Research topics On-going activities Contact

CORPOICA, Colombia Molecular laboratory • Mycosphaerella leaf spot diseases • Characterization of the Colombian A. Gutierrez Rojas

Plant pathology lab. • Population structure and diversity: population of Mycosphaerella leaf spot S. Aponte

Field stations (different altitudes) • Morphological and molecular pathogens

characterization • Morphological and molecular

• Evaluation of germplasm characterization of germplasm

CICY, Mexico Biotechnology labs Researchers (4) • Genetic improvement using • Construction and characterization of 2 genetic A. James

Biochemistry & molecular Technicians (3) biotechnology BIBAC libraries of 2 diploid bananas, and D. Kaemmer

biology of plants MSc students( 4) development of a transformation protocol using L. Conde

Equipment facilities: PhD students (2) vacuum infiltration mediated by Agrobacterium, L. Peraza

CHEF MAPPER Undergraduates (13) CONACYT, 3 years (Resp.: Dr A. James)

Capillar Sequencer • Genetic and physical mapping of M. fijiensis,

CHEF DRII CONACYT; 3 years (Resp.: Dr D. Kaemmer)

Screening of Calcutta IV BAC library for Resistance

genes (submitted) (Resp.: Dr D. Kaemmer)

• Construction of a BAC library of M. fijiensis

(submitted) (Resp.: Dr A. James)

• Agronomic evaluation of newly introduced

banana and plantain cultivars in Mexico,

and of mutant-induced plants (submitted)

(Resp. : Dr A. James)

CIRAD, Montpellier Phytopathology labs Researchers ( 2) • Taxonomy and identification of J. Carlier

Mycosphaerella spp. C. Abadie

Greenhouse & climatic chamber Geneticist (1) • Collection

In vitro evaluation facilities Technicians (3) • Molecular diagnostic

Access to in vitro laboratory Students (2-3) • Distribution of Mycosphaerella spp. in Asia

Collection of about 2500 isolates • Pathogen populations structure at different scales

Laboratory of molecular biology • Collection

(CAPS & microsatellites, sequencers, • Molecular markers

genomic) • Pathogenicity

• Population structure of Mycosphaerella leaf spot

pathogens in Asia

• Efficacy and durability of partial resistance

• Characterization of new resistance sources

• Genetic of resistance

• Evolution of pathogen population on resistant

cultivars (large area of culture)

• Other

• Genomic and host-pathogen interaction studies

• Collection (core collection, network? data base?)

CATIE, Costa Rica Molecular biology Lab. Researchers (2) • Biological control and resistance On-going: A. S. Riveros

Plant pathology Lab. Technician (2) induction • Studies of population structure of M. fijiensis G. Rivas

Biologic control Lab. • Bank of antagonistic bacteria and fungi • Biotechnology of Musa: tissue culture,

Tissue culture lab. • Microbiological products with bombardment protocols

Particles bombardment antifungal potential or elicitor molecule Submitted:

Shade house • Screening of plants with antifungal or • Population structure of M. fijiensis

resistance induction in Dominican Republic.

Tools offered: • Botanical products with antifungal or • Presented by T. Polanco (IDIAF) to IAEA in

M. fijiensis: Isolating protocols, elicitor molecule January 2002. Partners: CATIE (Costa Rica)

DNA extraction, DNA amplification, • Adaptation/development of a rapid and CIRAD (France).

DNA electrophoresis and efficient screening method to • Population structure of M. fijiensis in

Use of molecular markers evaluate black Sigatoka resistance. Honduras and Dominican Republic.

for studies of populations • Genetic transformation of Musa to Presented by G. Rivas (CATIE) to FINNIDA.

Induction of resistance introduce resistance to black leaf May 2002. Partners: FHIA (Honduras), IDIAF

Pathogenesis tests streak disease (Dominican Republic) and CIRAD (France).

Musa tissue culture protocols • Transformation protocol in Musa Studies of population structure of M. fijiensis

Bombardment protocols • Biotechnology of Musa: tissue culture,

bombardment protocols Population structure

of M. fijiensis in Honduras. Project supported

by INIBAP/FHIA. Partners: CATIE (Costa Rica)

and CIRAD (France).

INFOMUSA — Vol 11, N° 1 PROMUSA V

Institution, Country Research facilities Human resources Research topics On-going activities ContactEMBRAPA, Brazil Cooperative network of 3 EMBRAPA Researchers (9) • Integrated control of Mycosphaerella On-going: Zilton Cordeiro

research centres including their leaf spot diseases in Brazil including • Evaluation of genetic and pathogenic variability Maria de Jesus B.laboratories and fields breeding in M. musicola Calvacante

• Evaluation of resistance to black and yellowleaf streak diseases• EpidemiologyNew proposal:• Creation and evaluation of segregating popu-lation of Musa acuminata (AA) for resistanceto black and yellow leaf streak diseases.

FHIA, Honduras Tissue culture lab. Plant breeder (1) • Breeding On-going: M. RiveraShade house Research assistants (3) • Field evaluation of new clones • IMTP phase III J.F. AguilarExperimental fields Plant pathologists (2) • Epidemiology • CFC Musa hybrid testing projectConventional plant pathology lab. Agronomist (1) • Banana breedingELISA capacity installed Technician (1) Perspective:PCR capacity (soon) • Population structure of M. fijiensis in Honduras.

Project supported by INIBAP/FHIA.Partners: CATIE (Costa Rica) and CIRAD (France)

IBP, Cuba Plant tissue culture lab. Biotechnologist • Early screening On-going Y. AlvaradoMolecular biology lab. Plant breeders • Evaluation of germplasm • Mutagenesis /breeding programPhytopathology lab. Plant pathologist (2) in screen-house • Standardized methods for early screeningCommercial plant tissue culture lab. Molecular biologist • Genetic transformation • Evaluation in greenhouseGreenhouse/ Shade house Microbiologist (plant/pathogen) • M. fijiensis transformationExperimental field Agronomist • Plant transformation

Perspective:• Use of similar test for pathogenesis evaluation• Development of methods to identify virulentand avirulent isolates

Plant Pathology Unit, Pathology lab. • Biological control against fungal P. LepoivreUniversity of Gembloux, Virology lab. diseases J.-P. BusogoroBelgium Greenhouse • Selection for resistance to plant diseases

Molecular biology • Study on resistance mechanismand serology facilities

NRI, University Plant pathology and virology Plant pathologist (3) • Epidemiology Perspective: P. Burtof Greenwich, UK laboratories Mycologist (1) • Airborne spread of M. fijiensis.

CT rooms and environmental cabinets Technicians (3)Electron microscopy Biometeorologist (1)Greenhouse Library

FABI, University Pre-graduate Plant Pathologist (2) • Fungal taxonomy • Morphological and molecular identification A. Viljoenof Pretoria, Post-graduate Mycologist (1) • Population genetics of Mycosphaerella spp.of Pretoria, BIOTECHNOLOGY: • Molecular biology • Population genetics of Mycosphaerella spp.South Africa Microarray facility • Disease management

SequencerLight cyclerMYCOLOGY:Electron and light microscopesCulture maintenanceGenetic and molecular analysis of fungiPLANT FACILITIES:Tissue culture laboratoriesTransformation and GMO facilitiesQuarantine facilities

BTI, Cornell University, • Molecular genetic tools to study host-USA Phytopathology labs. pathogen interaction

Greenhouse & climatic chamber • Genomics approaches to identify fungal andIn vitro evaluation facilities plant genes expressionAccess to in vitro lab. • Development of high throughput screening Laboratory of molecular biology methodologies for evaluating pathogenicity (CAPS & microsatellites, sequencers, and virulence in plant.genomic) Genetic transformation

Asian institutions, • It has been recommended to take the • Incidence/severity of Mycosphaerella represented by INIBAP opportunity of the next BAPNET species on Musa in Asia (morphological)Coordinator for Asia meeting to have a workshop with Asian • Evaluation and characterization of variousrepresented by INIBAP participants to the PROMUSA Sigatoka germplasm collections in Asia against and Pacific and working group to define their M. musicola, M. fijiensis and M. eumusae.Executive Secretary programme and activities. • Epidemiological studies on M. eumusae.of BAPNET. • Population structures of M. eumusae using

molecular tools and corresponding pathogenicity• Yield loss assessment due to the different Mycosphaerella leaf spot pathogen on Musa.

Origin of PROMUSAAs an introduction, Eldad Karamura, INI-BAP regional coordinator for easternand southern Africa, briefed participantson PROMUSA. He explained that PRO-MUSA was born out of the realizationthat genetic improvement is the mostsustainable strategy for addressing themajority of constraints limiting banana

production, particularly for small-scalefarmers who account for over 80% of theglobal production. Consequently, inter-disciplinary working groups have been created to generate complementa-ry information needed by the geneticimprovement working group. It wasclarified that participating in the PRO-MUSA research agenda did not precludeother research activities on the same topics.

Agenda and outputs of the meeting

The meeting agreed on the following dis-cussion agenda/outputs:• Structure, membership and financing of

working groups,• Research priorities for the Weevil working

group (inputs required for genetic improve-

VI PROMUSA INFOMUSA — Vol 11, N° 1

PROMUSA: Banana weevil working group inauguration2 March 2002, Tenerife, Canary Islands, Spain

Project proposalsThe group also worked on the developmentof different concept notes to achieve the dif-ferent recommendations made.

Airborne dispersal of Mycosphaerella pathogens of Musa - Monitoring of airbornespread of M. fijiensis intouninfected areas of the Caribbean

Potential partnersCatherine Abadie - CIRADPeter Burt – NRIHenry Fagan - WIBDECOJuliane Henderson - CRCTPPRonald Vargas – CORBANA

Development of diagnostic toolsfor Mycosphaerella species onbananaPotential partnersJean Carlier and Catherine Abadie - CIRADPedro Crous - CBSAltus Viljoen - FABIJuliane Henderson and Elizabeth Aitken -CRCTPPKathy Grice and Ron Peterson - QDPI

Investigation of the durability ofthe resistance of banana hybridsto M. fijiensis

Potential partnersCatherine Abadie -CIRAD

Mauricio Rivera - FHIALuis Pocasangre – INIBAP Luis Pérez Vicente – INISAV David Jones - Consultant, UK

Determination of the pathogenicvariability of M. fijiensis and M.musicola populations

Potential partnersYelenis Alvarado - IBPRonald Vargas - CORBANALaura Conde - CICYSergio Aponte - CORPOICAZilton Cordeiro - EMBRAPAMauricio Guzmán - CORBANAGalileo Rivas - CATIE

Name Institution CountryAlba .S. Riveros CATIE Costa RicaAlice Churchill BTI, Ithaca USAAltus Viljoen FABI South AfricaAndres Gutierrez Rojas CORPOICA ColombiaAndrew James CICY MexicoAristoteles Pires de Matos EMBRAPA BrazilBob Fullerton Hort Research New ZealandCatherine Abadie CIRAD-FLHOR FranceDavid Jones Consultant UKDieter Kaemmer CICY MexicoElizabeth Aitken CRCTPP AustraliaEric Foure CIRAD/ CARBAP CameroonFritz Elango EARTH Costa RicaGalileo Rivas CATIE Costa RicaGus Molina INIBAP PhilippinesIndra Ariyarathne ARS Sri LankaJean Carlier CIRAD-AMIS FranceJean-Vincent Escalant INIBAP FranceJorge Sandoval CORBANA Costa RicaJosé G. Garcia Lopez INIFAP Mexico

Name Institution CountryJuliane Henderson CRCTPP AustraliaKathy Grice QDPI AustraliaLaura Conde CICY MexicoLeticia Peraza CICY MexicoLorna Herradura BPI PhilippinesLuis Perez Vicente INIVIT CubaLuis Pocasangre INIBAP-CATIE Costa RicaMauricio Guzman CORBANA Costa RicaMauricio Rivera FHIA HondurasMoses Buregyeya NARO UgandaPeter Balint-Kurti DNA Plant Technologies USAPeter Burt NRI UKPhilippe Lepoivre Univ. Gembloux BelgiumR. Selvarajan NRCB, Trichy IndiaRon Peterson QDPI AustraliaRonald Vargas CORBANA Costa RicaSergio Aponte CORPOICA ColombiaW. Tushemereirwe NARO UgandaYelenis Alvarado IBP, Santa Clara CubaZilton Cordeiro EMBRAPA Brazil

ment from the Weevil working group),• Management of the Weevil working group.

Output 1: Membership • Agreement on the actual membership in

other working groups • Core group: scientists with active proj-

ects on aspects of genetic improve-ment,

• General membership: people workingon weevil biology, including policy mak-ers and those involved in technologytransfer.

• Convenor’s role: it was agreed that theconvenor is responsible for conveningmeetings, information exchange and liais-ing with PROMUSA secretariat.

Question 1. Who funds the convenor’s run-ning costs e.g. meetings?

The working group in collaboration withINIBAP, through PROMUSA, looks for fundsfor the operation of the core group. Usually,PROMUSA working group meetings areheld back-to-back with other internationalmeetings, a cost-effective way of organizingmeetings.

Output 2: Research priorities for genetic improvement

• Identify sources of resistance,• Develop screening methods and protocols,• Agree on references/checks.The following suggestions were made:

• To compile and exchange information onmethods and checks. Standardizing sam-pling methods is a prerequisite for develop-ing screening methods,

• To have standard protocols for screeninggermplasm and for identifying sources ofresistance,

• To compile information on mechanisms ofresistance,

• To assess the possibility of site specific dif-ferences regarding resistance,

• To develop research priorities that addressthe compatibility of genetic improvementwith other management practices.

• To consider developing IPM research pri-orities that contribute to the geneticimprovement of the banana,Certain institutions whose research

interests go beyond genetic ameliora-tion, raised the possibility of establishingthe working group as a separate entityfrom PROMUSA for fear of beingrestricted by the latter’s mission. In theend, everybody agreed there would be acore working group on activities relatedto genetic improvement, but that thegeneral membership would include poli-cy makers and all those working onbanana weevil (including its biology andstatus as a pest, control methods andtechnology transfer). It was also sug-gested to create a list-server to facilitateinformation exchange on all aspects ofbanana weevil research.

The fact that PROMUSA focuses ongenetic improvement does not mean that

other crop protection research activities, e.g.pheromones and entomopathogens, areless important. These should be addressedmore efficiently as researchers benefit fromthe multidisciplinary dynamics created byPROMUSA.

Way forward

Formation of the core group – This groupshould include scientists who actively con-tribute to genetic improvement, e.g. breed-ers and scientists working on host plantresistance, mechanisms of resistance,hybrids resistant to weevil, sources of resist-ance, relevant genetic studies, conventionalbreeding, biotechnological methods andscreening methods.

It was not felt necessary to split this groupinto scientists working on plantains, bananasor other banana types. For the time being,the group can include anyone working onany aspect of crop improvement of allbananas and plantains.

It was suggested that the group adopt thesame procedures for forming this group asthose used by other working groups.It was agreed that:• Members who were present at this meeting

form the working group, • A convenor should be elected to take

charge of the working group,• The convenor should organize a meeting

within the coming year to work out the wayforward.

INFOMUSA — Vol 11, N° 1 PROMUSA VII

Name Research focusCliff Gold – IITA IPM, microbial control, screening, resistance mechanisms, collaboration with breeders.Roger Fogain - CARBAP Integrated Management of the weevil (screening, resistance, biological control).Consuelo Castrillon - CORPOICA IPM, screening.Stijn Messiaen - KUL IPM, screening.Aurelio Carnero - ICIA IPM, genetic resistance.Gloria Lobs – ICID Protease inhibitors, post-harvest evaluation of genetically modified varieties.Schalk Schoeman - ARC-ITSC IPM of weevil, screening for ‘Cavendish’ sub-types.Douglas Cubillo - CORBANA IPM, screening.Thierry Lescot – CIRAD-FLHOR IPM application in diversified systems. Links between research and development.Fernando Garcia del Pino – Univ. Autónoma Barcelona Entomopathogenic nematodes for biological control.Angeles Padilla - ICIA Entomopathogenic nematodes for biological control, artificial diets.Dennis Alpizar - Costa Rica IPM in plantain, pheromones.Vincent Ochieng - ICIPE Use of genetics in banana weevil biotyping in relation to control and quarantine.Prem Govender - FABI IPM in commercial banana plantations, active plant pathology group, especially with biotechnology.Felix Ortego – CSIC Activity/ insecticidal protein in insects.Miguel Montesdeosca - ICIA Activity/ insecticidal proteases in banana weevil, pheromones.Pedro Castañera -CSIC Activity/ insecticidal proteins for insect control.Caroline Nankinga - NARO/IITA IPM, entomopathogenic fungi for biological control of weevil, on-farm screening. Andrew Kiggundu - NARO Use of foreign genes for resistance to weevil, protease inhibitors.

List of participants

Session 1. Status of Cosmopolites sordidus in the worldStudies on the banana borer weevil in Cameroon

R. Fogain, S. Messiaen & E. Fouré CARBAP (Centre africain de recherches sur bananiers etplantains) P.O.Box 832, Douala, Cameroon

In Cameroon, bananas and plantains are amajor staple food for a large proportion of thepopulation. A total of 1.7 million tonnes areproduced annually. These crops are threat-ened by a wide range of pests and diseasesamong which the banana borer weevil(Cosmopolites sordidus) is the major insectpest. For more than six decades, investigationshave been carried out on this pest but empha-sis was given to testing insecticides that satisfythe needs of large-scale commercial bananaplantations. It is only recently that studies onintegrated control options were initiated inorder to develop control strategies that couldalso be used by resource limited farmers. This report presents the activities carried outin Cameroon over the past ten years on thebanana borer weevil.

Distribution and population dynamicsFour weevil species are found in the bananaand plantain producing areas of Cameroon:Cosmopolites sordidus (Germar), Polytusmellerborgi (Boheman), Metamasius hemi-pterus sericeus (Olivier) and M. hemipterus(L.). C. sordidus seems to be the only weevil ofeconomic importance in banana and plantainplantations (Fogain 1994, Ysenbrandt et al.2000). The insect is found in all banana andplantain producing areas in Cameroon (Fogain2001). A survey carried out in all the banana

and plantain producing areas showed that thepercentage of occurrence of C. sordidus inCameroon varies between 50 and 90%, andthat 82.5% of the farmers are aware of theproblem and capable of recognizing weevildamage (Ngamo and Fogain 1998). Researchon the population dynamics of the weevil in twoof the most important production zones indi-cates that higher populations are observedbeween August and September. However, thisresult need confirmation.

Control methodsIn commercial banana plantations, chemicalcontrol, the use of clean planting material, andweevil habitat management are the most com-mon methods for controlling weevil popula-tions. Development of alternative controlmeasures and of an integrated pest manage-ment strategy are highly recommended forresource limited small-scale farmers, themajor producers of plantain.

Chemical controlAt the beginning of the 70s, weevil populationswere efficiently controlled with Kepone(Chlordecone) in Cameroon’s commercialbanana farms. Between 1975 and 1983, thewithdrawal of the product from the marketcaused a significant increase in weevil popula-tions because of its replacement with HCHand other less effective insecticides, likeDursban (chloropyrifos-ethyl) and Primicide(pyrimiphos-ethyl) (Kehe 1985). The rapiddecline of banana production was halted bythe arrival on the market of Curlone(Chlordecone) in the early 90s. With one ortwo applications per year, weevil populationswhere effectively controlled. But the productwas soon withdrawn from the market becauseof its limited degradability. Regent (fipronil)later came on the market, allowing efficientcontrol of weevil populations with two or threeapplications per year. In Cameroon, this prod-

uct is still the only efficient insecticide used incommercial banana farms. However, its con-tinuous application will probably produceresistant weevil populations in the near future.Therefore, it is recommended to use it alter-nately with nematicides, like Counter (ter-buphos) and Furadan (carbofuran), that haveinsecticidal activity and can be used whenpopulations are relatively low. In the Moungodepartment, the threshold for treatment inindustrial banana plantations is when 5% ofthe 20 sampled mats per hectare areattacked, based on the method proposed byVilardebo (1973). Other insecticides with inter-esting properties are: tebupyrimphos, athi-amethoxam, cartap and imidacloprid. Timely chemical control is an efficient way ofknocking down adult weevil populations incommercial farms, but is too expensive for themajority of resource limited farmers and hasunfavourable side effects on beneficial non-target organisms. According to a survey con-ducted in southwest Cameroon, 57% of small-holder farmers said they did not use pesticides(Chantelot 1993). Forty-three percent, mostlyin mixed plantain-cocoa plantations, treatedsuckers before planting and 87% used insecti-cides generally referred to as ‘gabaline’, insec-ticides used against timber or cocoa pestsand which include lindane (HCH), Dursban(chloropyrifos-ethyl) and methylparathion.Three percent of the farmers who treat suck-ers before planting use a nematicide withinsecticidal activity, such as Mocap(Ethoprophos), and 10% use other products(Chantelot 1993). Another survey in west,southwest, central and south Cameroonrevealed that only 11% of smallholders usepesticides, 57% do not use anything and morethan 32% use ashes because they believe itcontrols weevils (Ngamo and Fogain 1998).

Cultural controlIt is important to plant an uninfested field withclean planting material which can be obtained

VIII PROMUSA INFOMUSA — Vol 11, N° 1

Contributions of the various partners

CIRAD, Guadeloupe, expressed the desi-re to participate in agronomy and biotech-nology related activities and to coordinateactivities in Guadeloupe and Montpellier.

Spanish organizations will also providesupport to the working group.

CORPOICA, Colombia, will contribute tothe screening of cultivars for resistance tobanana weevil and nematode. CORPOICAwill provide support for screening methodsand could also provide a weevil biotechno-logy specialist (Consuelo Castrillon).

CORBANA, Costa Rica, will provide sup-port for screening methods and evaluationof germplasm.

EMBRAPA, Brazil, was not representedbut may be interested in conventional bree-ding, biotechnology and screening for localstress. Needs to be contacted.

FHIA, Honduras and EMBRAPA will becontacted to find out their interests (MarlineFancelli).

ITSC, South Africa, proposed to screennew banana varieties, especially‘Cavendish’ (Schalk Schoeman). TheUniversity of Pretoria will supervise stu-dents conducting research in banana bio-technology.

CARBAP will screen for resistance toweevils, nematodes and black Sigatoka(Roger Fogain).

IITA has a breeding programme on high-land bananas and plantains. IITA works

very closely with NARO and banana net-works in East and West Africa. IITA is inter-ested in mechanisms of resistance, as wellas conventional and biotechnologicalmethods for developing resistance (CliffGold).

Election of the convenorIt was proposed that Dr Cliff Gold of IITA beselected as convenor. He has worked exten-sively on banana weevil, speaks both Englishand Spanish and has access to communica-tion facilities. He can easily coordinate thepreliminary activities. Therefore Dr Cliff Goldwas nominated and unanimously approved.

Abstracts of papers presented during the meeting

from weevil-free plantations or tissue culturefacilities. Ninety-five percent of smallholderspractice paring of suckers before planting(Chantelot 1993), but since the availability ofgood planting material is a major limitation inCameroon, infested suckers of minor qualityare often planted. Residual corms in the soil should bedestroyed and post harvest residues slashedin order to prevent the multiplication of wee-vils. Weeding should be done regularly inorder to avoid development of a favourablehumid weevil habitat. In smallholder farms,habitat management is neglected becauselabour is limited or rented labour is not produc-tive enough. Weeding is minimal (two to threetimes a year) and herbicide application is rare. A minority of farmers prop with bamboo eventhough the practice can give good results withminimal investments. In a survey carried outbetween February 1997 and March 1998 of240 plantain plants in eight smallholder farms,plant losses due to nematodes, weevils, andwater and nutrient stress represented 60% ofthe losses, of which 37% were due to topplingor falldown (mainly at the beginning of therainy season, due to violent winds) and 29%were due to breakage of the pseudostem(mainly at the end of the dry season due towater stress) (Anonymous 1998).More than 30 % of smallholder farmers usehousehold ashes at planting because theybelieve it reduces damage to the corm(Ngamo and Fogain 1998). It is not clearwhether ashes have an insecticide or merely afertilizer effect. Under laboratory conditions,ashes have a repellent effect on adult C. sor-didus, but the toxicity to adults is quite low(Messiaen 1999).Commercial banana plantations are renewedevery five to six years. During the fallow peri-od, residual corms are usually destroyed bylocal women who use the fallow field for foodcrop production. Tissue cultured plants aretreated with Regent5G (fipronil) orCounter10G (terbufos) at planting and two orthree times a year. Crop hygiene (weeding,herbicide application, slashing of residualpseudostems and toppled mats) as well aspropping and guying are commonly practised.

Biological controlAt CARBAP, research on biological controlusing the entomophagous fungus Beauveriabassiana started in 1994 with the discovery oflocal strains in Cameroon (Fogain 1994).Since then, studies have been carried outunder controlled conditions to test the efficien-cy of the strains and the possibility of massproduction for field trials. Three strains ofBeauveria bassiana, isolated from infectedweevils caused 92% mortality after nine daysunder laboratory conditions. Research ispresently carried out on the maintenance ofviability with regards to delivery systems andthe feasibility of mass production for farmersor economic agents in Cameroon. Entomo-pathogenous nematodes have been isolated

from soil samples collected in Cameroonusing C. sordidus larvae.

Use of botanicalsDipping suckers in a 20% neem (Azadirachtaindica) seed solution at planting protects theyoung suckers from weevil attack for severalmonths, but a crown application three times ayear is not effective in reducing damage(Fogain and Ysenbrandt, 1998). It achievesthis result by reducing oviposition, through itsrepellent effect on adult weevils, and byblocking egg hatching (Messiaen 1999).

TrappingTrapping using pseudostem traps does notalways reduce weevil populations, dependingon the cropping system, the level of weevilimmigration from neighbouring infested plots,the number of traps placed and the initial pop-ulation. In Cameroon, trapping does not seemto be a viable control option in smallholderfarms because of the unrealistic amount ofpseudostem and labour needed, and becauseof weevil immigration from adjacent plots.Testing of a mass trapping system using ramptraps baited with sordidin, an aggregationpheromone, indicated that the traps were notattractive enough to constitute a viable controloption in industrial banana plantations, butadditional research is needed to assesswhether the attractiveness can be improvedwith another type of trap and kairomones. Insmallholder farms, pheromone mass trappingdoes not seem to be a viable option for con-trolling weevil populations because of prob-lems of storage and costs (Messiaen 2000a).

Host plant resistanceScreening for resistance to the banana borerweevil at CARBAP stated in the 1994 with thediscovery of the field resistant ‘Yangambi km5’and of the highly susceptible clones of theplantain subgroup (Musa AAB) compared tothe ‘Cavendish’ (Musa AAA) (Fogain andPrice 1994). Since then, techniques for earlyscreening in the field and under controlledconditions have been refined. In a recentscreening, more than 80 varieties were tested.Several varieties, including CARBAP hybrids,have been selected for enhanced screening inthe field. Results of preliminary screeningshow a large variety of responses to weevilattack between and within genomic subgroups(Messiaen 2000b). No genotypes are moresusceptible to weevil attack than the ones inthe plantain subgroup. Preliminary resultspoint to differences in larval development. Ifthe results are confirmed in the field, it will bepossible in the short or medium term to devel-op hybrids partially resistant to C. sordidus.

ConclusionsSignificant information has been gathered overthe past ten years on the distribution and popu-lation dynamics of the weevil. Despite gains inknowledge on the dynamics of C. sordidus inthe Fako and Mungo divisions (littoral and

southwest Cameroon), investigations are need-ed for other provinces, such as the major plan-tain producing areas of the centre and south(Anonymous 2000). The insect was found to bepresent everywhere in the country wherebananas and plantains are produced. A largerspectrum of insecticides is available, but mainlyto commercial growers. Several insecticidesfrom different chemical groups are now avail-able and can therefore be used in rotation toavoid the development of resistance to C. sor-didus. As for botanical and biological controlagents, neem (A. indica) and the entomopatho-genic fungus B. bassiana show great potentialfor weevil control. But field trials are needed toconfirm greenhouse results. Sources of resist-ance to the weevil have been identified andbreeding programmes can now use them todevelop genotypes resistant to the insect.Research on other non-chemical control meth-ods, such as cultural control and the use ofpheromones, continues.

ReferencesAnonymous 2000. Annuaire des statistiques du sec-

teur agricole, Agri-Stat No. 006, Direction desEtudes et Projets Agricoles, Ministère del’Agriculture, Cameroon.

Anonymous. 1998. L’observatoire des problèmes phy-tosanitaires. Plantinfo (34):9-11.

Chantelot E. 1993. Enquête diagnostic plantain dans laprovince du sud-ouest du Cameroun: description del’échantillon de parcelles. Document CRBP, 40 pp.

Fogain R. 1994. Les ravageurs des bananiers auCameroun. INFOMUSA 3(1):19-20

Fogain R. 2001. Nematodes and weevil of bananasand plantains in Cameroon: occurrence and hostsusceptibility. International Journal of PestManagement 47(3):201-205

Fogain R. & N.S. Price. 1994. Varietal screening ofsome Musa cultivars for susceptibility to the bananaborer weevil. Fruits 49(4): 247-251

Fogain R. & H. Ysenbrandt. 1998. Utilisation du neem(Azadirahta indica) et du champignon Beauveriabassiana contre le charançon noir des bananiers etplantains. Pp. 223-229 in Proceedings of the Vth

Annual Conference Bioscience and Food Security.Kehe M. 1985. Le charançon du bananier

(Cosmopolites sordidus Germar) en culture debananiers et plantains. Proceedings of the annualreunion of WARCORP, 2-6 December, Douala,Cameroon. 9pp.

Messiaen S. 1999. Neem (Azadirachta indica), woodashes, coffee husk and hot pepper (Capsicum spp.)for controlling the banana weevil (Cosmopolites sor-didus): investigations into their effect and mode ofaction. Technical report, CRBP, Cameroon.

Messiaen S. 2000a. Evaluation of a pheromone baitedmass trapping system of C. sordidus with B. bassia-na. Technical report, CRBP, Cameroon

Messiaen S. 2000b. Early varietal screening of Musavarieties for sensibility to the banana weevil: preli-minary results. Technical report, CRBP, Cameroon.

Ngamo L. & R. Fogain. 1998. Perception paysannedes problèmes phytosanitaires en culture de bana-niers au Cameroun. Document CRBP, 10pp.

Vilardebo A. 1973. Le coefficient d’infestation, critèred’évaluation du degré d’attaque des bananeraiespar Cosmopolites sordidus Germar, le charançonnoir du bananier. Fruits 28(6): 417-426.

Ysenbrandt H., R. Fogain, S. Messiaen & P. SamaLang. 2000. Infestation levels of weevil species onMusa cultivars Grande naine (AAA) and Frenchsombre (AAB) and subsequent plant mortality insouthwest Cameroon. African Plant Protection6(1):21-24.

INFOMUSA — Vol 11, N° 1 PROMUSA IX

The biology and management ofthe banana weevil, Cosmopolitessordidus in South Africa

P. Govender1 and A. Viljoen2

1 Department of Zoology & Entomology; 2 Department ofMicrobiology & Plant Pathology, Forestry and AgriculturalBiotechnology Institute (FABI), University of Pretoria, Pretoria,0002, South Africa

The banana weevil, Cosmopolites sordidus,introduced in South Africa about 30 years ago,is the most important insect pest of banana,causing economic losses in the Mpumalangaand the south coast of Kwazulu-Natal regions.Collectively this area represents about 78% ofthe total 12 078 hectares under commercialbanana production in the subtropical pocketsof South Africa. The weevil has a limitedpotential to migrate from its current distributionarea unless it is transferred with infectedplanting material. Information on the life cycleappears to be consistent with published litera-ture; the total developmental period beingabout 33 days. Adults emerge during springand late summer, and their nocturnal activityincreases during or after rainfall. Femalesgenerally lay one egg per week from lateAugust to February but this number canincrease during optimal environmental condi-tions and low pest densities. Adults have alife span of about two years. Although weevilnumbers are low in the winter months (May toJuly), they increase rapidly in spring and earlysummer (August to November). Weevils aremonitored using pseudostem traps at a densi-ty of 50 traps/ha. Economic threshold valuesare 1-2 adults/trap/week and 10 or more larvaltunnels/corm. Weevils are strongly attractedto the ‘Williams’ and ‘Chinese Cavendish’ cul-tivars. A tentative recommendation algorithmwas developed for the management of C. sor-didus in South Africa. It integrates standardcultural control, trapping, biocontrol and chem-ical control. Two local entomopathogenicfungi (Aspergillus flavus and Beauveriabassiana) have been isolated and tested inSouth Africa but their host specificity and path-ogenicity requires further investigation.Various nematicides with insecticidal proper-ties have been tested in limited field trials andcurrently only aldicarb 15% GR, at a rate of2.03 to 3.00 g a.i./planting station, is regis-tered for use against the banana weevil.

Overview of banana weevilresearch in Uganda

C.S. Gold1 and W.K. Tushemereirwe2

1IITA-ESARC, P.O. Box 7878, Kampala, Uganda; 2UgandaNBRP, P.O. Box 7065 Kampala, Uganda

The banana weevil is an important constrainton the production of East African highlandbananas and plantains. The larvae attack thecorm, reducing nutrient uptake and weakeningthe stability of the plant. Attack in newly plant-ed banana stands can lead to crop failure. In

established fields, weevil damage can result inreduced bunch weight, plant loss, mat die-outand shortened stand life. In Uganda andTanzania, it has been implicated as a primaryfactor in the decline and disappearance ofcooking bananas in traditional growing areas.Uganda has ranked banana weevil as themost important biotic constraint on highlandbanana production.Salient features of the weevil’s biology arerestricted host range (Musa and Ensete), longlife span (up to 4 years), low fecundity (1-3eggs/week), 1:1 sex ratio, nocturnal activity,uncommon flight and limited dispersal capabil-ity. The weevil most commonly enters newfields through infested planting material.Because of populations building up over time,pest problems are more pronounced in olderplantations. In one trial in Uganda, yield lossincreased from 5% in the plant crop to 47% inthe third ratoon. This loss was attributedequally to plant loss and bunch weight reduc-tions. By the 7th cycle of the second trial, 35%of the mats had died out in weevil-infestedplots compared to 2% in controls. Overallyield losses were 50% for the trial.Although pesticides can be an effective controlmethod, in Uganda the weevil has developedresistance to one chemical. IITA and theUgandan National Banana ResearchProgramme work closely together on culturaland biological controls, and on host plant resist-ance. Clean planting material is an importantmeans of keeping weevils out of new planta-tions, but the effect normally disappears withina few crop cycles. A one-year trapping studyshowed some positive effects on populationreduction but this control is beyond theresources of most Ugandan growers. Currentemphasis are on the use of neem, endemicants, microbial control (i.e. Beauveria bassianaand endophytes) and host plant resistance.Available data indicate that all highland bananaclones are susceptible to weevil. Howeverscreening trials suggest that many resistantMusa clones do exist and that antibiosis is thepredominant means of resistance in theseclones.

Cosmopolites sordidus in theautonomous region of MadeiraLuís Nuno and V. P. Ribeiro

Direcção Regional de Agricultura da Regional Autónoma daMadeira – Portugal

Direcção de Serviços de Produção Agrícola/Divisão deBananicultura, Centro de Bananicultura – Lugar de Baixo –9360-119 Ponta do Sol, Portugal

Located between 32°38’ north and 16°54’west, the island of Madeira lies about 800 kmfrom the coast of Morocco. Banana planta-tions occupy nearly 850 hectares of the islandand are exposed to a subtropical climate. In Madeira, the culture of banana started inthe 18th century. The most widely grown vari-ety on the island is the ‘Pequeña enana’, intro-duced in 1842.

Although its presence has been known forsome time, the banana weevil, Cosmopolitessordidus, is not a major source of problems.The first identification dates back to the 19th

century.In 1992, started the installation of spot irrigationsystems and the use of varieties produced invitro. In both instances, this has been accompa-nied by outbreaks of C. sordidus. In one case(spot irrigation), outbreaks increased becausefarmers stopped doing certain things like bury-ing plant residues, a practice which helped con-trol the pest. In the other case, available datasuggest that C. sordidus prefers plants pro-duced in vitro. Even though they occur in allplantations, these problems are more frequentin the above-mentioned situations.For some years, banana growers applied to thebase of the pseudostem an insecticide(Baytion) which has foxime as an active ingredi-ent. They also used a pirimifos-ethyl basedproduct (Bullit) but stopped when these insecti-cides were banned. They currently apply eto-profos (Mocap 10G) directly on the soil.A recent innovation is the use of pheromonetraps, produced by N.P.P. Calliope (France), tofight C. sordidus. The first results are promising.

Detection of Cosmopolites sordidus Germar on TenerifeIslandRuth Torres del Castillo and ClementeMéndez Hernández

ICIA, Tenerife, Canary Islands, Spain

The agricultural service of the government ofTenerife Island (Cabildo) did a series of sur-veys to determine the extent and severity ofdamages caused by Cosmopolites sordidusGermar. Carried out between 1996 and 2001,the surveys were repeated every three years,depending on the study area. Peeled off pseu-dostem double-disc traps and those with ahorizontal section of the corm were highly cor-related (R2=0.93). The distribution of C. sor-didus in relation to the type of irrigation, thealtitude of the plantation and the variety used(‘Pequeña enana’ or ‘Gran enana’) was alsostudied. Time trends were done to see theevolution in the distribution of this pest and thedamages it causes.

Management of banana and plantain weevil borer in Costa Rica

Douglas Cubillo y Mauricio Guzmán

Sección de Fitopatología y Entomología, Dirección deInvestigaciones de CORBANA S.A., Costa Rica

Chemical control and cultural practices are thebase for the management of banana weevil inplantain and banana plantations in CostaRica. Usually the damage of the pest is moreimportant in plantains (Musa AAB) than inbananas (Musa AAA), due to differences in

X PROMUSA INFOMUSA — Vol 11, N° 1

varietal susceptibility and in crop manage-ment. We are carrying out studies on theecology and biology of the pest, and on meth-ods for chemical, biological, ethological andcultural control. The combination of some ofthese methods could be the best alternativefor the management of the pest.

Session 2. Control of thebanana borer weevilCosmopolites sordidus

Overview of Beauveria bassianafor microbial control of thebanana weevil in Uganda

C.M. Nankinga1, C.S. Gold1, W. Tushemereirwe2

1International Institute of Tropical Agriculture, Eastern andSouthern Africa Regional Centre, P.O. Box 7878, Kampala,Uganda; 2National Banana Research Programme, KawandaAgricultural Research Institute, P.O. Box 7065, KampalaUganda

Collaborative research is being conductedin Uganda to assess the microbial controlpotential of Beauveria bassiana (Balsamo)Vuillemin (Hyphomycetes) for the bananaweevil, Cosmopolites sordidus (Germar),(Coleoptera: Curculionidae). Since theearly 1990s, isolation, characterization andpathogenicity studies have come up with aselection of indigenous isolates of B.bassiana that have good growth and pro-duction traits, causing 50-100% weevil mor-tality within 10-21 days after inoculation,depending on the isolate. In small-scalefield trials conducted at KawandaAgricultural Research Institute, one B.bassiana isolate (code G41), which showedhigh pathogenicity to C. sordidus as well assuperior growth and sporulation comparedto other isolates, was tested. Three meth-ods of delivering B. bassiana, namely (i)application of the fungus on topsoil aroundthe base of the banana mat (ii) applicationof the fungus with pseudostem and disc-onstump traps and (iii) application of the fun-gus to banana planting suckers, were evalu-ated. Treating banana suckers with a B.bassiana dry maize culture formulation anda soil-maize based formulation (2.3 x 1012

conidia/planting hole), reduced weevil dam-age by 20-30% within a period of eightweeks after planting in holes dug in a 2 to3-year-old banana field of local EAAH cook-ing cultivar. Dead C. sordidus adults andlarvae with B. bassiana fungal growth wereobserved in the treated suckers indicatingimmature stage infection. When the maizeand soil-based formulations of B. bassianawere applied beneath the pseudostem anddisc-on stump traps, it was observed thatthe moist conditions under the traps, inaddition to attracting weevils, also provideda favourable environment for extra sporula-tion of B. bassiana and this enabled the fun-gus to remain potentially infective. B.

bassiana cultures collected from the fieldtraps in the first five weeks after applicationwere highly infective causing 60-100% wee-vil mortality in 14 days, but the infectivity ofthe fungus was significantly reduced in thewet season; likely due to contamination byother soil micro-organisms. The maize cul-ture formulation (2 x 1015 conidial/ha) andmaize-soil based formulation (2 x1014 coni-dia/ha) applied at the base of the matsreduced the adult banana weevil popula-tions by 30-50% and kept them at lower lev-els than in the untreated plots. The treatedplant also showed reduced weevil damageand up to 16% B. bassiana disease infec-tion was observed in dead weevils in thefield.Previous studies have demonstrated thatgood potential exists for the use of B.bassiana for microbial control of the bananaweevil. The collaborative team, composedof scientists from the International Instituteof Tropical Agriculture (IITA), the NationalBanana Research Programme of theUganda National Agricultural ResearchOrganization (NARO), CABI Biosciences UKand University of Reading is undertaking fur-ther research into the mass production andformulation of B. bassiana and exploringother delivery systems of B. bassiana forintegration with other control measuresunder farmers’ conditions. Research isgeared to developing economically viablemass production, formulation and deliverysystems that will overcome the problemsassociated with field fungal efficiency, per-sistence and transmission. Further researchon the ecological relationships betweenbanana weevils and entomopathogens willalso be undertaken to understand the condi-tions under which B. bassiana is likely to bemost effective in controlling this pest. Thisinclude studies on the behaviour of the wee-vil, which might influence the likelihood ofthe insect contacting the pathogen; the bio-types in the Cosmopolites sordidus species,which might exhibit different susceptible lev-els to the pathogen; and pathogen viabilityand virulence under aerobic (ordinarly pseu-dostem traps) and anaerobic conditionssuch as in the semio-chemical-based trap-ping systems. We acknowledge fundingfrom the Rockefeller Foundation, DFID andBMZ in support of this work.

Entomopathogenic nematodesfor the control of insect pests. The outlook for the control of Cosmopolites sordidusFernando García del PinoUniversidad Autónoma, Barcelona, Spain

Entomopathogenic nematodes (Heterorhabditisspp. and Steinernema spp.) are used in biocon-trol against different insect pests in soil andcryptic habitats. They are symbiotically associ-ated with bacteria of the genera Photorhabdus

and Xenorhabdus, respectively. Nematodedauer juveniles, harbouring cells of their specif-ic bacteria in their intestine, search for insectsin the soil. After penetration in the host insect,they release their symbionts. The bacteria mul-tiply and produce suitable conditions for nema-tode reproduction in the dead insect. Afterabout two weeks, dauer juveniles emigrate fromthe cadaver and search for a new host.The use of entomopathogenic nematodesfor the control of Cosmopolites sordidusshould now be economically feasible.Production and formulation techniqueshave been improved to provide thesenematodes to growers at a cost equivalentto or lower than the one of chemical insecti-cides. Application of entomopathogenicnematodes requires less labour than insec-ticides, avoids the problems of insecticideresistance and has little or no adverseeffects on the environment. However, forreliable results, it is necessary to improvethe application technique, and to select theappropriate species and strains of ento-mopathogenic nematodes. Finally, variousstrategies for the control off Cosmopolitessordidus using entomopathogenic nema-todes are discussed.

The use of two insecticide-nematicides to control the weevilCosmopolites sordidus and thenematode Radopholus similisand their effect on someproduction variables of thebanana ‘Gran enana’ in relation to the environmental conditionsencountered in Costa Rica Dennis Alpízar M.

Estación Experimental Los Diamantes. Ministerio deAgricultura y Ganadería. Guápiles, Limón, Costa Rica

In Costa Rica, insecticide-nematicides are com-monly used to control Cosmopolites sordidus.Pseudostem or corm traps are also used whilethe utilisation of the aggregation pheromoneCosmolure®, introduced at the end of the 90sto control the banana weevil, is still a new agri-cultural practice in the country’s banana andplantain plantations.The objective of this study is to compare theeffect of using or not using, under the sameconditions over two years, two insecticide-nematicides: terbufos (four applications) andetoprofos (one application).After two years the results show, using theFisher t-test (0.05), that the ‘weight of thebunch’ was slightly but significantly higher inthe non-treated plots while the ‘number ofnematodes (Radopholus similis) in the func-tional root of the sucker’ was slightly lower inthe treated plots. The other variables werenot statistically different.

INFOMUSA — Vol 11, N° 1 PROMUSA XI

The cost of applying the insecticide-nemati-cide was $US 1200 per hectare for the two-year duration of the study.

Alternatives to the control of Cosmopolites sordidus(Germar) in banana plantainplantations

A. Padilla Cubas, F. García del Pino, L.V. López Llorca and A. Carnero Hernández

ICIA, Apartado aéreo 60, 38080 La Laguna, Tenerife, IslasCanarias, Espaa

In the Canary Islands, Cosmopolites sordidus(Germar) is the most important pest in planta-tions of banana plantain. Given the results ofchemical treatments, alternatives are sought tocontrol the weevil. We therefore sampled soils,cultivated and not cultivated, in the province ofSanta Cruz de Tenerife looking for parasitizedorganisms. Using ‘Galleria mellonella’ traps, weespecially searched for entomophagous nema-todes and fungi.Entomophagous nematodes were found in twosampling points from which we isolatedHeterothabditis spp. and Steinernema spp. Asfor entomophagous fungi, we isolatedAspergillus flavus, Beauveria bassiana,Metarhizium anisopliae and Paecilomycesspp. Verticillium lecanii was isolated formwhite flies collected in different localities.We characterized the morphology of the fungiand studied their germination and sporulationcapacity, their production of biomass and theirbehaviour under various naturally occurringconditions of humidity, temperature and pH.We also studied their enzymatic activity: quiti-no, amylo, proteo, lipo and pectinolytic.We also conducted biological assays using‘Galleria mellonella’ and, based on the results,we inoculated C. sordidus using two methods.Finally, we evaluated the interactions betweenthese isolates and Fusarium oxysporum, themain pest of banana.

Behaviour of entomophagous fungion vegetal tissueL.V. Lopez-Llorca

Departamento de Ciencias Ambientales y Recursos Naturales,Universidad de Alicante, Aptdo. Correos 99, 03080 Alicante,Spain

We studied the use of ornamental plant vege-tal residues from nurseries to produce inoculaof antagonistic fungi, including ento-mophagous fungi.Seeds of Phoenix dactylifera turned out to beexcellent for the production of Beauveriabassiana. Scanning electron microscopyrevealed a very porous substrate, a featurewhich facilitates fungal development andsporulation. A soil-based formulation of B.bassiana can sporulate and overcome fungis-tasis. Thanks to this type of seed, the fungus

can survive and maintain itself in the soil dur-ing at least three months. During bioessays,the mixture infected a pest of palm trees(Carpophilus dimidiatus) similar to the weevil.Moreover, B. bassiana can colonize the peti-oles of P. dactylifera. We think that such endo-phytic behaviour is useful in controlling pestslike the weevil.

Research on the Musa borerweevil (Cosmopolites sordidus) at CARBAP

Eric Fouré1, S. Messiaen1, Roger Fogain1,Thierry Lescot2

1CARBAP, B.P. 832, Douala, Cameroon; 2CIRAD-FLHORBPA, Boulevard de la Lironde, TA50/PS4, 34 398 MontpellierCedex 5, France

The banana borer weevil Cosmospolites sor-didus is the most important pest of bananaand plantain in African plantations. TheAfrican Center of Banana and Plantain (CAR-BAP) carries out researches on C. sordidus,with a focus on integrated pest management,namely:

• genetic resistance,

• population dynamics,

• biological (bio-insecticides and pheromones)and chemical control.

Results are presented on selection for weevilresistance; weevil evolution; effects on plan-tain production in southwest Cameroon; bestparameters for field infestation representation;efficiency and limits of chemical insecticides;use of pheromones in trapping system; effi-ciency of Beauveria bassiana in southwestCameroon; and use of insecticide plantsagainst weevils.

Research on the Musa borerweevil (Cosmopolites sordidus) at CIRAD

Christian Chabrier1, Thierry Lescot2

1CIRAD-FLHOR BPA, B.P. 153, 97202 Fort de France,Martinique, France, 2see above

The Banana, Plantain and Pineapple pro-gramme (BPA) of CIRAD-FLHOR carries outresearch in the French West Indies and in thecentral laboratories of Montpellier, France, aswell as collaborations with CARBAP inCameroon and applications in Indian Oceanislands.

The research focuses on integrated pest man-agement along the same lines as CARBAP.

The results presented focus on the combina-tion of two types of synthetic pheromones; theuse of antomopathogenous bacteria andnematodes; and the efficiency and limits ofchemical insecticides.

Session 3. Molecular biology

Resistance of diploid bananagenotypes to Cosmopolitessordidus (Germ. 1824)(coleoptera: curculionidae)M. Fancelli, A. Souza Do Nascimento, N. Fritzons Sanches, R. Correa Caldas and S. De Oliveira E Silva

EMBRAPA, Mandioca y fruticultura, Rua EMBRAPA s/n, CaixaPostal 007, 44380-000 Cruz das Almas, Bahia, Brazil

Plant resistance to insects is considered asecure and durable strategy for the control ofCosmopolites sordidus, especially in planta-tions where investments are low. Despite theexistence of a large number of varieties, thenumber of cultivars used in Brazil is small,hence the importance of evaluating the newgenotypes introduced and/or generated ingenetic improvement programmes. Althoughin the fields all varieties are infested, somestudies show differences among genotypeswith respect to development, survival andattractiveness for oviposition. Given the cur-rent expansion of banana plantations in Braziland the development of methodologies for theproduction of in vitro plantlets, there is a grow-ing interest for improved varieties, includingresistant ones. The tetraploid hybrids ofbanana (AAAB) are obtained by crossingdiploid (AA) genotypes with triploid (AAB) cul-tivars of types Prata (Silver) and Maçã(Pomme). A genetic improvement programmeof diploid genotypes is carried out, with theaim of increasing yield and resistance topests, one more reason for a close partner-ship between breeders and geneticists. The objectives of the present work are:• To evaluate diploid hybrids of banana in rela-

tion to Cosmopolites sordidus. • To study the mechanisms of resistance to

the weevil in diploid genotypes.

MethodologyThe following genotypes are being studied:0304-02; 0337-02; 0323-03; 1318-01; 2803-01; 4223-03; 5012-02; 4215-02; 4279-13 and4252-03. These materials are diploid hybridsgenerated by the Banana GeneticImprovement Programme, most of them pre-senting resistance to black Sigatoka. Plantletsfrom these genotypes were placed inscreened planting holes in the field and infest-ed with adults of the banana weevil using themethodology adopted by Seshu-Reddy andLubega (1993). Plants without the insect arekept under the same conditions to get informa-tion on the injuries caused by the pest. Thegenotype Terra is being used as susceptibilitypattern. The variables analyzed are: coeffi-cient of infestation, number of insects presentin the galleries, plant height, pseudostemdiameter, time period until inflorescence emis-sion, yield, bunch weight, number of hands,

XII PROMUSA INFOMUSA — Vol 11, N° 1

finger diameter and number of fingers/hand.Under laboratory conditions the developmentof the insect and its non-preference for feed-ing and oviposition will be studied for thesame genotypes in order to identify the typesof resistance involved in the interactionbetween the weevil and the banana plant. Theduration and viability of the larval and pupaphases, the weight of the pupa after 24 hoursand the number of adults with defects, will benoted. Tests for attractiveness and consump-tion will be carried out. Analyzes will be doneto identify the presence of attractive/repellentsubstances, as well as phago stimulantsand/or phago deterrents. Rhizome hardnesswill be evaluated using a penetrometer.

ReferenceSeshu-Reddy K.V & M.C Lubega. 1993. Evaluation of

banana cultivars for resistance to tolerance of theweevil Cosmopolites sordidus Germar. Pp. 143-148in Breeding banana and plantain for resistance todiseases and pests. (J. Ganry, ed.), CIRAD/INIBAP,Montpellier, France.

Aspects of banana weevilresistance in Musa and prospectsfor genetic engineering againstthe banana weevil

Andrew Kiggundu1 and Clifford S. Gold2

1National Banana Research Programme, Kawanda AgriculturalResearch Institute, P.O. Box 7065, Kampala, Uganda and theForestry and Agricultural Biotechnology Institute, University ofPretoria, 74 Lunnon Road, Hillcrest, Pretoria, 0002 SouthAfrica; 2International Institute of Tropical Agriculture (IITA). Eastand Southern Africa Regional Centre (ESARC) P. O. Box 7878,Kampala Uganda

The banana weevil (Cosmopolites sordidusGermar) is probably the most important pestaffecting the production of banana and plan-tain. Attack by the weevil results in severecrop losses from plant toppling, snapping,death and reduced bunch weights (INIBAP1986). Pesticides are effective, but uneco-nomical for small-scale subsistence farmers.In addition, the weevil is resistant to a widerange of insecticides and although culturalcontrols may help, labour and materialrequirements often limit their adoption (Gold1998). Host plant resistance shows potential for thelong-term control of the banana weevil onsmall-scale farms, within an integrated pestmanagement perspective (Seshu-Reddy andLubega 1993). However, the development ofweevil resistance in banana is still in its infan-cy and breeding programmes have onlyrecently included weevil resistance as a crite-ria for introgression into cultivated Musa. Thecumbersome nature of resistance screeningmethods has made work on weevil difficult,slow and at times expensive. Lack of under-standing of resistance mechanisms and theirassociated genes, coupled with long genera-tion times, triploid sterility of most edible culti-vars and poor seed set due to incompatibility,

have limited conventional breeding efforts onMusa.The literature on weevil resistance in Musareviewed by Pavis and Lemaire (1997),Kiggundu et al. (1999) and Kiggundu (2000)suggests that antibiosis is the key resistancemechanism. Sources of resistance have alsobeen found in the Musa germplasm tested byFogain and Price 1994, Lemaire 1996, andKiggundu et al. (in press). From a screening trial conducted over fourcrop cycles at the International Institute ofTropical Agriculture-East and SouthernRegional Center (IITA-ESARC), in Uganda, wefound that East African highland bananas(EAHB) (AAA-EA) and plantains (AAB) werethe most susceptible. These were followed byABB bananas (cvs ‘Pisang awak’ and‘Bluggoe’), diploid banana derived hybrids, ABbananas (cvs ‘Ndiizi’ and ‘Kisubi’), AAAbananas (cvs ‘Yangambi km5’, ‘Cavendish’ and‘Gros Michel’) and the wild AA type ‘Calcutta 4’being the most resistant. Several factors wereshown to contribute to weevil resistance. Drymatter content (representing corm hardness),resin/sap production of the corm and suckeringability were important for weevil resistance in allaccessions. Corm diameter (size) was alsoimportant in the EAHBs. Preliminary investiga-tions into the chemical basis of resistance,using high-performance liquid chromatographyprofiles of corm extracts, indicated that com-pounds present in some resistant cultivars(especially those with a B genome) negativelycorrelated with weevil damage. These com-pounds were absent in most of the susceptiblecultivars tested.Ortiz et al. (1995) studied the genetic inheri-tance of banana weevil resistance and found itto be under the control of more than one genewith partial dominance towards susceptibility.They found significant additive effects and mod-ifier genes plus dosage effects of susceptibilitygenes, causing higher susceptibility at higherploidy levels. Conventional crossbreeding in combination withmolecular biotechnology techniques, like mark-er assisted selection (MAS) and genetic trans-formation, appears to be an attractive option tofurther understand resistance to weevil andsimultaneously develop resistant cultivars.Sources of transgenes can include the Musagenome itself and others of plant or animal ori-gin (Carozzi and Koziel 1997). The NationalBanana Research Programme at KawandaAgricultural Research Institute, Uganda, in col-laboration with IITA-ESARC, is interested indeveloping a MAS programme to identify mark-ers and possibly resistance genes. At theForestry and Agricultural BiotechnologyInstitute (FABI), South Africa, we are involvedin studies to identify differentially expressedgenes during weevil infestation of resistant ver-sus susceptible Musa. Efforts are also expand-ed to investigate the potential of using proteaseand/or alpha-amylase inhibitors in the trans-genic control of banana weevil.

The beauty of genetic engineering is that genesfrom several sources can be exploited and thatthese can be transformed using a gene pyra-miding strategy. However, a great deal of infor-mation about the complex nature of weevilresistance is missing and molecular markeranalysis can assist in genetic analysis andmapping. Opportunities also exist for quicklydeveloping weevil resistance through genetictransformation.

ReferencesCarozzi N. & M. Koziel (eds). 1997. Advances in insect

control the role of transgenic plants. Taylor andFrancis, London. 301pp.

Fogain R. and N.S. Price. 1994. Varietal screening ofsome Musa cultivars for susceptibility to the bananaweevil, Cosmopolites sordidus (Coleoptera:Curculionidae). Fruits 49(4):247-251.

Gold C.S. 1998. Banana weevil: ecology pest statusand prospects for integrated control with emphasison East Africa. Pp. 49-74 in Proceedings of aSymposium on Biological Control in TropicalHabitats: Third International Conference on TropicalEntomology 30 October – 4 November 1994. (S.K.Saini, ed.). ICIPE, Nairobi, Kenya.

INIBAP 1986. Banana research in East Africa.Proposal for a regional research development net-work. INIBAP, Montpellier, France.

Kiggundu A., D. Vuylsteke & C. Gold. 1999. Recentadvances in host plant resistance to banana weevil,Cosmopolites sordidus (Germar). Pp. 87-96 inMobilizing IPM for sustainable banana production inAfrica. (E. A. Frison, C.S. Gold, E.B. Karamura andR.A. Sikora, eds.). INIBAP, Montpellier, France.

Kiggundu A. 2000. Host-plant interactions and resis-tance mechanisms to banana weevil Cosmopolitessordidus (germar) in Ugandan Musa germplasm.M.Sc. Thesis. University of the Orange Free State,Bloemfontain, South Africa.

Lemaire L. 1996. Les relations sémiochimiques chezle charançon du bananier Cosmopolites sordidusGermar (Coleoptera: Curculionidae) et la résistancede sa plante-hôte, le bananier. Thèse de doctorat,Université de Montpellier II, France.

Ortiz R., D. Vuylsteke, B. Dumpe & R.S.B. Ferris.1995. Banana weevil resistance and corm hardnessin Musa germplasm. Euphytica 86:95-102.

Pavis C. & L. Lemaire. 1997. Resistance of Musagermplasm to the banana borer weevil,Cosmopolites sordidus Germar (Coleoptera:Curculionidae) INFOMUSA 5(2):3-9.

Seshu-Reddy K.V & M.C Lubega. 1993. Evaluation ofbanana cultivars for resistance to tolerance of theweevil Cosmopolites sordidus Germar. Pp. 143-148in Breeding banana and plantain for resistance todiseases and pests. (J. Ganry, ed.), CIRAD/INIBAP,Montpellier, France.

Genetic biodiversity in bananaweevil (Cosmopolites sordidus)from different banana growingregions

Vincent OchiengInternational Center for Insect Physiology and Ecology(ICIPE), PO Box 30772, Nairobi, Kenya

Genetic variability in banana weevil popula-tions was analyzed in a large number ofsamples obtained from 15 banana growingtropical countries using random amplified

INFOMUSA — Vol 11, N° 1 PROMUSA XIII

Summary reportBy S. Mohan Jain

FAO/IAEA Joint Division, International Atomic Energy Agency,A-1400, Box 100, Wagramerstrasse 5, Vienna, Austria. Email:[email protected]

Bananas and plantains are grown in over100 countries throughout the world withannual production around 88 millions metrictons. Banana fruit production is severelylimited by several diseases and pests suchas banana bunchy top virus, burrowingnematodes (Radopholus similis), Moko dis-ease (Ralstonia solanacaearum), blackSigatoka or black leaf streak (Mycos-phaerella fijiensis), Fusarium wilt (Fusariumoxysporum f.sp. cubense). FAO/IAEA start-ed banana Coordinated Research Project(CRP) in 1994 with the general aim to inte-grate radiation induced mutations in vitroculture and molecular genetics methods into

the conventional breeding of banana toinduce desirable variation such as diseaseresistance, dwarfism and earliness, andalso to promote the development of meth-ods for large-scale and rapid multiplicationof the mutants/segregants through somaticembryogenesis and micropropagation. TheBelgium Government decided to fund thisCRP in 1996. Since than, three ResearchCoordinated Meetings (RCM) were held indifferent countries including Vienna,Malaysia and Sri Lanka. The fourth andfinal RCM of this banana CRP was held atthe Katholieke Universiteit Leuven(KULeuven), Leuven, Belgium, 24-28September 2001. A total of ten participantsattended it including from Austria(FAO/IAEA), Belgium, Cuba, CzechRepublic, Germany, Israel, Mexico,Philippines, and Sri Lanka. The results ofCRP will be published in a book entitled

“Banana improvement: Cellular and molecu-lar biology, and induced mutations”.

Overall achievementsResearch tools were developed forgermplasm characterization and improve-ment through induced mutations, cryop-reservation, somatic embryogenesis,somaclonal variation and genetic engineer-ing. Some of the existing cultivars havebeen improved for disease tolerance andimportant agronomic traits. Collaborationsamong participating laboratories wereestablished, including exchange of staff,training and technology transfer.

Practical achievementsResearch contract holders J. Lopez Torres(Cuba), Mak Chai (Malaysia), A. James(Mexico), and J. Dolezel (Czech Republic) didexceedingly well in this CRP. Nicolas Roux(FAO/IAEA) has been instrumental in dissoci-

XIV PROMUSA INFOMUSA — Vol 11, N° 1

Fourth and final FAO/IAEA research coordination meeting on Cellular biology and biotechnology including mutation techniques for creation of new useful banana genotypes

polymorphism DNA (RAPD). The studyincluded 46 RAPD markers. Poly-morphisms, as measured by percentage ofpolymorphic RAPDs, varied between78.3% and 97.8%. Genetic variability wasmeasured using Shannon’s informationindex and partitioned into inter and intra-population components. Overall, geneticvariation between C. sordidus populationswas 25.3% ((Hsp-Hpop)/Hsp). The geneticdiversity for the species (Hsp) was 83.4%.The mean of intra-population values (Hpop)for all populations was 62.3%. The totalgenetic diversity was explained by highvariation between populations (meanGst=0.213), which is consistent with lowgene flow between populations(Nm+0.811). The high inter-populationgenetic variation observed in this studycould be explained by limited migration,given to the restricted mobility andmonophagy of banana weevils. In a paral-lel study, PCR-RFLP analysis was used todifferentiate the CO1 mtDNA of 19 bananaweevil populations. The PCR-RFLPmethod produced unambigous profiles thatdifferentiated certain populations from oth-ers. In another study, the genetic variabili-ty in weevil populations was analysed insamples obtained from 15 sites in bananagrowing regions of Uganda. Although theweevil populations in this case were verysimilar genetically, some variability wasobserved.

Alternatives to control thebanana borer weevil,Cosmopolites sordidus Germar(Coleoptera: Curculionidae), onbanana P. Castañera, F. Ortego, M. Montesdeoca Montesdeoca

and A. Carnero HernándezConsejo Superior de Investigaciones Científicas (CSIC),Madrid, Spain

The banana borer weevil, Cosmopolites sor-didus Germ. is considered the main pest ofbanana in the world. In the Canary Islands, itwas first noticed in 1945 and eradicated. Itreappeared in 1987, but despite attempts tocontrol it by chemical and cultural means, thepest has spread causing increasingly severedamages.A project (RTA 02-100-C3) was recently initi-ated to develop an integrated pest manage-ment strategy against the weevil. Its aim is tomaintain the profitability of the culture by pro-ducing an environmentally friendly bananathat should have a competitive edge onEuropean markets over bananas from otherproducing countries.The objectives of the project are: 1) to optimize

the use of pheromone to control the weevil; 2) toevaluate the relation between the extent of thedamages caused by the weevil and yield losses;3) to determine weevil inter and intra-population

variability in relation to biological control; 4) toidentify and study the behaviour of the ento-mophagous organisms attacking the weevil.Our participation in this project consists in deter-mining weevil inter and intra-population variabili-ty among the populations affecting the bananaplantations of Tenerife, Gomera and La Palma,in order to provide information which could helpin controlling this pest, such as the origin andthe dispersal patterns of these weevils.Detecting DNA polymorphism will be done usingthe randomly amplified polymorphic DNA tech-nique, following the methodology used to studythe population genetic of another species ofCurculionidae, a pest of sugarbeet in Andalusia(Taberner et al. 1997, J. Mol. Evol. 45:24-31).We also plan to identify and characterize thedigestive enzymes of the weevil, and to con-duct bioassays with protease inhibitors in orderto determine their effect on the survival anddevelopment of this pest. This information isnecessary for the eventual production of trans-genic banana plants expressing defence pro-teins, a possibility which would offer newmeans of controlling the weevil. Preliminarystudies suggest that larvae and adults possessa complex proteolytic system which includesaspartyl, cysteine and serine endoproteases aswell as amino and carboxypeptidases. Oncethe characterization of digestive proteases isfinished, we will study their interactions withspecific inhibitors in order to determine whichinhibitors, or combination of inhibitors, could beincorporated by genetic manipulation in poten-tially resistant banana plants.

INFOMUSA — Vol 11, N° 1 PROMUSA XV

ation of chimerism and developing flowcytometry protocol.

Several young students benefited from thisCRP in completing their master and PhD pro-grammes in Israel, Czech Republic, andBelgium. Some of the participants presentedtheir results in international conferences. Atotal of 51 research papers have been pub-lished in conference proceedings and interna-tional refereed journals.

Many international trainees receivedtraining on several aspects of banana tis-sue culture, molecular cytogenetics andmolecular markers at KULeuven andFaculté universitaire des sciencesagronomiques, Gembloux (FUSAGx),Belgium; Institute of Experimental Botany(IEB); and University of Frankfurt,Germany. The trainees came from China,Cuba, Egypt, Mexico, and Rwanda. Theoutcome of these trainings was very suc-cessful. For example, a Cuban traineewas successful in establishing new somat-ic embryogenic banana cell suspensionsfrom Cuban plant material. In addition, hesuccessfully irradiated plant material inCuba. In Sri Lanka, 20 persons from coun-tryside were given training in tissue culturetechnology for mass production of banana.Post-graduate training on indexing ofbanana viruses was organized.

Flow cytometry facilities were estab-lished at the International Institute forTropical Agriculture (IITA, Nigeria) andThe Malaysian Institute for NuclearTechnology MINT, Malaysia). The transferinvolved staff training in the Institute ofExperimental Botany (IEB, CzechRepublic) and expert visit.

Specific achievements1. Detection of DNA methylation polymor-phism in banana micropropagated plantswith amplified fragment length polymor-phism (AFLP).2. Somatic embryogenic cell suspension cul-tures (ECS) were developed for severalbanana cultivars including plantains (AAB).Three cryopreservation techniques weredeveloped for long-term conservation ofmeristems. An INIBAP technical guidelinefor cryopreservation of banana was pub-lished in English, French and Spanish.3. Induced mutations generated a series ofimproved clones that were screened for dif-ferent traits such as early flowering, reducedheight, large fruit size, and tolerance toFusarium.4. Both Agrobacterium-mediated transfor-mation and particle bombardment methodswere used for banana transformation, andtransformation rate was cultivar dependent.

5. Virus indexing procedures were trans-ferred to Sri Lanka for indexing localbanana virus strains.6. An early screening technique was devel-oped for Fusarium wilt using tissue culture-derived plants in a double-tray system.7. A selection system was developed againstblack Sigatoka disease by using Mycosphaerellafijiensis crude extract, the semi-purified, and onepurified fraction (juglone).8. Screening techniques for nematoderesistance were developed in Musa undershade-house and field conditions. Asepticcultures of Radopholus similis andPratylenchus coffeae were establishedusing alfalfa calli, and their pathogenicitywas confirmed after greenhouse tests.9. DNA flow cytometry was used for detec-tion of polyploidy, monitoring of cytochimeradissociation, and analysis of karyologicalstability of ECS.10. Transposon mutagenesis was explored

for gene tagging, using maize Ac element,in banana genome. A substantial numberof distinct mutants were generated andcharacterized.11. Fluorescence in situ hybridization(FISH) protocol was developed for Musa fordetailed studying of karyotypes, providingdistinct chromosome landmarks, gene local-ization, analysis of long-range chromosomestructure, and linking to physical and genet-ic maps.12. A total of 28 allele-specific simplesequence repeat (SSR) markers were gen-erated for Musa and used to detect: poly-morphisms between the A and B genomes,identify hybrids, and trace back the Bgenome in hybrids. These markers are nowused within the CRP and worldwide. A totalof 24 locus-specific, highly polymorphicSSR markers were also produced forMycosphaerella fijiensis to discriminatethem from other species.

Detection of DNA methylationchanges in micropropagatedbanana plants (Musa AAA cv.‘Grande naine’) using the methylation-sensitiveamplification polymorphism(MSAP) techniqueA. James, S. Peraza-Echeverria, V. Herrera-Valencia and L. Peraza- Echeverria

Unidad de Biotecnología, Centro de Investigación Científicade Yucatán, Mérida, Yucatán, Mexico.

The extent of DNA methylation polymor-phisms was evaluated in leaf tissue of micro-propagated banana (Musa AAA cv. ‘Grandenaine’) derived either from the vegetativeapex of the sucker or the floral apex of themale inflorescence using the methylation-sen-sitive amplification polymorphism (MSAP)technique, which utilizes the restrictionisoschizomer pair Msp I and Hpa Il, whoseability to cleave at the sequence 5’-CCGG-3’is affected by the methylation state of thecytosines. In all, 465 fragments, each repre-senting a recognition site cleaved by either orboth of the isoschizomers were amplifiedusing eight combinations of primers. A totalof 107 sites (23%) were found to be methylat-ed at cytosine in the genome of micropropa-gated plants. The highest number of DNAmethylation polymorphisms was detected in

plants micropropagated from the male inflo-rescence with 14 (3%) and the lowest inplants micropropagated from the sucker with8 (1.7%). These differences were not statisti-cally significant. In leaf tissue of convention-ally propagated plants DNA methylation poly-morphisms were not detected.Micropropagated plants were relatively hyper-methylated in comparison to conventionallypropagated plants, with some bands beingmethylated in all micropropagated plants butnon-methylated in all conventionally propagat-ed plants. These results demonstrated theusefulness of MSAP to detect DNA methyla-tion events in micropropagated banana plantsand indicate that DNA methylation changesare associated with micropropagation.

Discovery of functional genes in the Musa genomeE. Khayat1, A. Ilan1, I. Regev2, S. Gepstein2,L. Sagi3, R. Swennen3 and H. Schoofs3

1Rahan Meristem, Dept of R&D, Kibbutz Rosh, Hanikra 22825Israel2Technion, Israel Institute of Technology, Faculty of Biology,Haifa 32000, Israel3 Laboratory of Tropical Crop Improvement, KULeuven,Kasteelpark Arenberg 13, B-3001 Leuven, Belgium

Development of stable and reproducibletransformation and regeneration technologiesopened new horizons in banana and plantainbreeding. Several transformation strategieshave been published in the last five years by

Abstracts of papers presented during the 4th and finalFAO/IAEA research coordination meeting

different banana biotechnologists. Diseaseresistance and improvement of fruit qualityhave been the focal points of most Musabreeders. However, despite growing interestin banana biotechnology, the pool of Musagenes in public databases is relatively small(of the approximately 300 accessions placedin the NCBI database less than 25% are anno-tated cDNA’s). Our laboratory is currentlyemploying several approaches for the identifi-cation of functional genes in the Musagenome. These include transposon tagging,‘high throughput’ random mutation byribozyme cleavage of mRNA, suppressionsubtractive hybridization (SSH) and bioinfor-matic annotation of clustered EST’s. We have introduced the maize Ac transpos-able element into the Musa genome and fol-lowed excision and insertion of the element innumerous transgenic lines. The goal was toinvestigate the frequency of transposition anddistribution of insertions along chromosomes.The constructs we have used include an Acelement fused to GUS reporter under the 35Spromoter. PCR analysis of a variety ofmutants revealed that most carried a chimericpattern with regard to expression of the for-eign genes. Consequently, only a few trans-genic lines (tissue cultured siblings) showeddetectable differences in the banding patternon Southern hybridization blots. Attemptswere made to stabilize the Ac element follow-ing a limited number of transpositions, bysilencing the gene encoding the transposaseenzyme after excision. Differentially expressed genes, which are acti-vated in the post climacteric phase of fruitdevelopment, were analyzed in the peel andpulp of banana fruit. Using suppression sub-tractive hybridization (SSH) we have isolatedover 200 partial cDNA’s encoding geneswhich are expressed during the final stages offruit development (senescence). Highthroughput screening by membrane hybridiza-tion was employed for preliminary selection ofcandidate genes involved in regulation of theonset of senescence. Sequence analysis andblasts against GeneBank databases revealedapproximately eighty non-redundant clones,which were up regulated in the post-climac-teric phase. Most, but not all of these geneswere up regulated, after exposure of greenfruit to 1000 ppm ethylene for 24 hours. Thesequenced pool of up-regulated cDNA’s fallinto one of three major categories: Genes involved in metabolic processes, main-ly carbohydrates and lipid components.Genes involved in cellular regulation (proteinkinases, transcription factors etc.).Genes involved in protection from pathogensand environmental stress conditions - metal-lothionein like protein, super oxide dismutase,osmotin-like protein, pathogen related proteinsetc. A significant number of sequences showed nosubstantial homology to functional genes inthe GeneBank.

Analysis of Musa genome using flow cytometry and molecular cytogenetics J. Dolezel1, M. Valárik1, J. Vrána1, J. Safár1,E. Hribová1, N. Gasmanová1, I. Van denhouwe2, M. Dolezelová1, R. Swennen2andH. Simková1

1Institute of Experimental Botany, Laboratory of MolecularCytogenetics and Cytometry, Olomouc, Czech Republic2Laboratory of Tropical Crop Improvement, KULeuven,Kasteelpark Arenberg 13, B-3001 Leuven, Belgium

This project focuses on the analysis of Musagenome at nuclear and chromosomal levelwith the aim to understand long-range organi-zation of Musa chromosomes and to charac-terize changes of chromosome structure dur-ing speciation and evolution of cultivatedclones.We have used flow cytometry to determineploidy levels of Musa accessions held at theINIBAP Transit Center (KULeuven). Flowcytometric ploidy assay involved preparationof suspensions of intact nuclei from smallamounts of leaf tissue and the analysis of fluo-rescence intensity after staining with DAPI.Chicken red blood cell (CRBC) nuclei wereincluded in every sample as an internal refer-ence standard (Figure 1). From the 890accessions analysed so far, 8.4% were classi-fied for the first time, and 7.6% accessionsexhibited other ploidy then reported previous-ly. In 2% of the accessions, plants of mixedploidy were detected. A reliable and high-throughput system for ploidy screening inMusa is an important outcome of the study.The use of CRBC nuclei, allowed high-resolu-tion analysis, and the results obtained so farindicated suitability of this system for rapiddetection of aneuploidy. As the materials foranalysis were sent by express mail, this work

demonstrates that it is possible to perform flowcytometric ploidy analysis in distant laborato-ries.In attempt to characterize DNA sequencescontributing to structure and evolution of Musachromosomes, we have constructed partialgenomic DNA libraries in M. acuminata and M.balbisiana and screened them for clones car-rying highly repeated sequences, andsequences carrying rDNA. Isolated cloneswere characterised in terms of copy number,genomic distribution in M. acuminata and M.balbisiana, and sequence similarity to knownDNA sequences (Table 1). In contrast tomany plant species where mobile elementsand their remnants contribute most of thenucleotide content, our observations indicatethat these elements do not represent a majorfraction of the Musa genome. All repetitivesequences were more abundant in M. acumi-nata. As the genome of M. acuminata is larg-er compared to M. balbisiana, the presentresults demonstrate that the increase ingenome size of M. acuminata was due to mul-tiplication of some repetitive sequences. Thefindings of this study improve the knowledgeof long-range organization of chromosomes inMusa. The availability of homologous probesfor fluorescence in situ hybridization (FISH)will allow more specific mapping of rDNAsequences.A novel protocol for isolation of high-molecu-lar-weight DNA in Musa has been developedand the work is in progress to construct bacte-rial artificial chromosome (BAC) library for theB genome of Musa. Availability of the BAClibrary will permit isolation of clones containinglow proportion of repetitive DNA. Such cloneswill be localized using FISH and will be usedto increase the number of already existingchromosome landmarks. Furthermore, BAClibrary will be screened for clones, which con-

XVI PROMUSA INFOMUSA — Vol 11, N° 1

M. acuminata ssp. banskii

CRBC

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tan

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nts

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Peak ratio: 0.534Diploid (2x)

Peak ratio: 1.089Tetraploid (4x)

Peak ratio: 0.813Triploid (3x)

Peak ratio: 1.620Hexaploid (6x)

Cavendish 901TMPx 2637-49

0 50 100 150 200 0 50 100 150 200 250Relative nuclear DNA content (channel number)

Figure 1. Examples of histogrammes of relative DNA content, which were obtained during ploidyscreening of ITC accessions using flow cytometry. The ploidy of individual plants was estimatedbased on the ratio of positions of peaks corresponding to G1 nuclei of Musa and CRBC. While theanalysis confirmed ploidy classification for M. acuminata ssp. banksii (ITC 0885), ‘Lakatan’ (ITC 0573),and TMPx 2637-49 (ITC 1196), the classification was not confirmed for ‘Cavendish 901’ (ITC 0738),which was found to be hexaploid.

tain molecular markers and/or genes of inter-est. This strategy should result in effectiveintegration of genetic and physical maps.Once developed, physically mapped molecu-lar markers will facilitate map-based cloning ofgenes of interest including those induced byirradiation and chemical mutagenesis.Ploidy screening of Musa germplasm wassupported in part by INIBAP.

Analyses of induced mutants of Philippine bananas (Musaacuminata, cvs ‘Lakatan’ (AAA)and ‘Latundan’ (AAB)) andgermplasm collection of Abaca(Musa textilis Nee) usingmorphological, RAPD, SSR and AFLP markers

D.M. Hautea1, N.O. Bituin1, H.F. Galvez1, C. Caspillo1, C.H. Balatero1, R.B. Quilloy1,E. Perez1, G.C. Molina1, J. Torrion1, A.J. Jamiri2, R.P. Laude3 and L. Gonzal4

1Institute of Plant Breeding, CA, University of the PhilippinesLos Baños, Philippines2College of Science and Mathematics, U.P. Mindanao,Philippines3lnstitute of Biological Sciences, University of the PhilippinesLos Baños, Philippines4National Abaca Research Center, Visayas State College ofAgriculture, Philippines

Banana (Musa acuminata) and abaca (M. tex-tilis Nee) are two of the most economicallyimportant Musa species cultivated in thePhilippines for fruit and fibre, respectively.These crops are both difficult to breed by con-ventional means and they share a number ofcommon diseases. This report summarizesour efforts during the past five years to gener-ate useful induced mutants of Philippinebanana cultivars and to evaluate the useful-ness of DNA markers in characterizinggenomic alterations in the advanced genera-tions of induced mutants. Furthermore, thisreport highlights the valuable contributionmade by this CRP project in analyzing genetic

variation in the related Musa species, M. tex-tilis, by utilizing some of the results of the DNAmarker techniques generated for banana. Thehighlights of our accomplishments are:• Advanced generations of induced mutants of

two Philippine banana cultivars ‘Lakatan’(AAA) and ‘Latundan’ (AAB) with promisingtraits were obtained from irradiation using40Gy gamma ray and 3Gy fast neutron andsubsequent in vitro culture manipulation andfield evaluation.

• DNA marker techniques such as RAPD,SSR and AFLP were successfully used tocharacterize genomic differences betweenthe two banana cultivars used. However,only RAPD and AFLP techniques were ableto detect genomic alterations between non-irradiated and induced mutants in the twocultivars. Due to better reproducibility andhigher multiplex ratio, AFLP technique is pre-ferred over RAPD technique. Hence, thistechnique was used to detect polymorphismbetween the original mutated clones andderived suckers. Silver staining procedurefor SSR and AFLP analyses were routinelyused.

• RAPD, SSR and AFLP techniques devel-oped for banana through this CRP projectwere found to be highly applicable to abaca.A number of SSR primers developed forbanana gave amplification products usingabaca DNA. With complimentary funds pro-vided by a grant from the Philippine govern-ment, RAPD, SSR and AFLP analyses weresuccessfully conducted to evaluate thegenetic variation in the abaca germplasmcollection of the Philippines. Comparisonbetween morphological and molecular analy-ses was also conducted.

Usefulness of embryogenic cell suspensions for theinduction and selection of mutants in Musa spp.N. Roux1, A. Toloza1, J. Dolezel2, R. Swennen3 , P. Lepoivre4 and F.J. Zapata-Arias1

1 Plant Breeding Unit, FAO/lAEA Agriculture and BiotechnologyLaboratory, International Atomic Energy Agency Laboratories,A -2444 Seibersdorf, Austria2 Laboratory of Molecular Cytogenetics and Cytometry,Institute of Experimental Botany, Sokolovska 6, CZ-77200Olomouc, Czech Republic3 Laboratory of Tropical Crop Improvement, KULeuven,Kasteelpark Arenberg 13, B-3001 Leuven, Belgium4 Plant Pathology Unit, Faculté universitaire des sciences agro-nomiques de Gembloux, Passage des déportés 2, B-5030Gembloux, Belgium

Induced mutation techniques are particularlyimportant for bananas and plantains (Musaspecies) where there is limited sexual repro-duction that could generate genetic variation,the basis for selection. Even though sponta-neous mutations have contributed to thegenetic diversity of Musa and significantlyincreased the variation used to breed Musaspp. their occurrence is too low. The use of invitro cultures for induced mutations in Musaspp. could be a method of choice if severalsteps of the mutation induction process couldbe optimized. The following aspects wereinvestigated: the possibility to detect geneticinstability in DNA content, the determination ofan optimal mutagenic dose, the elimination ofchimerism and the application of an earlymass screening for the selection of usefulmutants.With the increased use of embryogenic cul-tures in micropropagation of banana,somaclonal variation occurs among regenerat-ed plantlets. This variation may interfere withmutations, which could be obtained throughmutation techniques. Although the causes ofthis chromosome instability are poorly under-stood, chromosome instability itself is believedto be one of the most common causes of tis-sue culture-induced variation. Using flowcytometry, variation in chromosome numbercould be detected among plants regeneratedvia somatic embryogenesis from tissue cul-ture. The results obtained by flow cytometrywere verified by chromosome counting inmeristem root-tip cells. After standardizationof the method, the results indicated that flowcytometry was sensitive enough to detect ane-uploidy in Musa with ± 1 chromosome accura-cy. Abnormalities in DNA content could bedetected at an early stage, during in vitro cul-ture. For the first time, a banana embryogeniccell suspensions (ECS) with five chromo-somes missing was reported.To irradiate ECS, several preliminary studieswere performed. The first radio sensitivitytests of Musa ECS were performed and it hasbeen found that cell suspensions from Musacan tolerate up to 200 Gy. At 100 Gy thegrowth curve is only affected at 50% com-pared to the control.When irradiating cell suspensions, large popu-lations can be handled under controlled condi-tions and if embryos are of single cell origin,they overcome the problem of chimerism. Wesimulated this by treating ECS with colchicineand determined the ploidy of the regeneratedplants by flow cytometric analysis. Colchicinetreatment induced polyploidy and mixoploidy

INFOMUSA — Vol 11, N° 1 PROMUSA XVII

Homology to known DNA sequencesMusa repetitive DNA sequence Genebank SmallestDNA clone accession number sum probabilityRadka2 Hordeum murinum 5S ribosomal RNA gene AF096721 3x10-07

Radka1 Rice 26S ribosomal RNA gene M11585 0.0Radka7 Rice 26S ribosomal RNA gene M11585 0.0Radka14 — — —Radka5 M. acuminata repetitive element Y10144 6x10-16

Radka8 M. acuminata retrotransposon monkey AF143332 3x10-21

Radka9 M. acuminata retrotransposon monkey AF143332 3x10-21

Radka3 — — —Radka6 — — —Radka12 — — —Radka10 MUSA1 clone (cacao swollen shoot badnavirus) AF106947 10-108

Radka4 — — —

Table 1. Results of homology search of newly isolated repetitive DNA sequences.

(chimerism) if embryos are not of single cellorigin. To date no mixoploid regeneratedplants from colchicine treated ECS weredetected.An early mass screening method based on theuse of the toxin Juglone (5-Hydroxy-1, 4-naph-thoquinone), the main toxin to be responsibleof the global effect of the fungusMycosphaerella fijiensis, was used to screenfor resistance to the black Sigatoka disease.The test was applied when the acclimatizedplants reached the six leaves stage. The doseof 25 ppm permitted to differentiate betweenthe tolerant variety ‘Fougamou’ and the sus-ceptible variety ‘Grande naine’. To date, fromaround 4000 irradiated ‘Grande naine’ plantsscreened, eight putative mutants were select-ed for their tolerance to 25 ppm of Juglone.These plants are now being evaluated for theirtolerance to the inoculation of the fungus.

Towards basic genomics ofMycosphaerella fijiensis and M.musicola: DNA markers forgenetic diversity, populationstructure and genetic mapping of the banana pathogens

C.M. Molina1,2 and G. Kahl1

1 Plant Molecular Biology, Biocentre, University ofFrankfurt/Main, Germany.2 Corporación Colombiana de Investigación Agropecuaria(CORPOICA), Santafé de Bogotá, D.C., Colombia

Though much research is presently devoted tobanana genomics, its two most severe fungaldiseases black Sigatoka (causal agent:Mycosphaerella fijiensis) and yellow Sigatoka(causal agent: M. musicola) are definitelyunder researched. Yet both diseases led,lead, and will lead to major yield losses, espe-cially in banana and plantain plantations, butalso on smallholders fields. M. musicola, firstreported in Java in 1902, spread all over themajor production areas in Asia, Africa andSouth America during the first half of the lastcentury. It was regionally displaced by themore aggressive M. fijiensis, first reported onthe Fiji islands in 1962. Both pathogensforced to develop control measures, which inessence increased fungicide doses drasticallyand reduced the time interval between appli-cations, but also relied on the introduction ofnew, partly resistant host varieties. Inresponse, the pathogen populations becamemore aggressive and partly resistant to theprevalent fungicides.In spite of the economic importance of thefungi, systematic research on the pathway ofinfection, the crosstalk between the pathogenand the host plant, the molecules encounteredand the genes involved remained largely elu-sive. Therefore we started a more basic studyon both pathogens with the aim to understandthe molecular basis of the interaction(s).A set of elite, highly polymorphic microsatellitemarkers (sequence-tagged microsatellite site

markers, STMS) was developed for eachpathogen, using a microsatellite enrichmentstrategy. Microsatellites of the (CAA)n,(GAA)n, (GA)n, (CA)n and (TA)n type werespecifically captured, cloned, positive clonesidentified by hybridization, sequenced, andmicrosatellite-flanking primers designed withPrimer 3. Together with selective amplifica-tion of microsatellite polymorphic loci (SAMPL)and DNA amplification fingerprinting (DAF)markers, the STMS were used to study thegenetic variability of both pathogens in popula-tions from two continents, four Latin Americancountries and five different locations inColombia. First, all marker types generallydetected a high degree of polymorphism inboth species. Second, only few markers ofany type were sufficient to discriminate twoisolates unequivocally. Third, distinct popula-tions can be detected and differentiated fromeach other. For example, the number ofSTMS haplotypes turned out to be higher inNigerian as compared to Mexican M. fijiensis,and the M. musicola populations generally aremuch more clearly separated from each otherthan the M. fijiensis populations, pointing toless gene flow. Fourth, usually isolates fromone region grouped together. Fifth, about 10%of the primers designed for one pathogencould also be used for the other pathogen.The three marker techniques are used to con-struct and saturate a first genetic map of M.fijiensis and to tag a fungicide resistancegene.

Genetic and phenotypicvariability in Mycosphaerellapathogens of bananaA.C.L. Churchill

Boyce Thompson Institute for Plant Research, Ithaca, NY, USA

We are using fungal molecular biologyapproaches to study the interactions betweenMycosphaerella pathogens and their bananahosts. We have developed a DNA-mediatedtransformation system for M. fijiensis, M. musi-cola, and M. eumusae in which geneticallystable transformants constitutively expressgreen fluorescent protein in vitro and in vivo.Stable transformation is a first step in develop-ing tools for the genetic manipulation of thesepathogens, which will lead to the identificationof pathogenicity and virulence factors requiredfor plant infection and symptom development.Significant phenotypic variability exists in mostMycosphaerella banana pathogens. Whencultured on agar-based media, single coloniesare typically light to dark grey or black incolour. Stable spontaneous mutants altered inpigmentation are not uncommon. We haveisolated pigment mutants that appear to bedeficient in melanin production and havecloned a gene fragment with high similarity tofungal genes involved in melanin biosynthesis.Further characterizations of the pigmentmutants are in progress. Additionally, we are

interested in whether Mycosphaerellapathogens of banana have the potential toexhibit differential resistance to plant defenceresponses, such as the generation of reactiveoxygen species (ROS), or to themselves pro-duce ROS-generating toxins. We determinedthat M. musicola produces a singlet oxygen-generating anthraquinone toxin and exhibitssignificantly greater resistance to a wide rangeof light-activated, singlet oxygen-generatingdyes than does M. fijiensis. These resultssuggest possible differences in the way thetwo fungi interact with banana. In M. musico-la, the correlation between production of alight-activated toxin, high self-resistance toexogenous photoactivated singlet oxygen-generating toxins, and increased diseasedevelopment in the presence of light is intrigu-ing and will be examined further. Such a cor-relation is not evident in the M. fijiensis host-pathogen interaction.

Banana improvement throughgamma irradiation and testing for banana bract mosaic virus (BBrMV) in Sri LankaW.K. Hirimburegama, W.K.G. Dias and K. Hirimburegama Department of Botany, University of Colombo, Colombo 03,

Sri Lanka.

Banana is the most widely consumed fruit inSri Lanka, and is an attractive perennial fruitcrop for small-scale farmers. This is due to itshigh economic value throughout the year.Thus, lowland rice fields in some areas arebeing converted for banana cultivation.Among the local cultivars, ‘Embul’ (Mysore,AAB) has the highest demand for cultivation.The annual production of banana is around450 000 metric tonnes. Until recently, bananacultivation was limited to small plots, but largefields are now being established. More andmore rice farmers are changing over tobanana cultivation as the net profit is aboutfour times higher than for rice, and less labourand other inputs are required. During the lastsix years, 2500 ha have been converted tobanana cultivation (Anon. 2001, 2002). Inaddition, it was noted that the nutritional levelin farmers’ families has improved due to thehabit of consuming more fruit. Usually, farm-ers do not use pesticides for banana cultiva-tion and this has benefits for human healthand the environment. Since 1990, research on banana micropropa-gation through shoot-tip culture, inducedmutation through gamma irradiation, cell sus-pension cultures and somatic embryogenesis,and ploidy analysis for variation detection hasbeen conducted by the University of Colombo.From 1995, both ‘Embul’ and ‘Cavendish’ cul-tivars were included in the mutation breedingprogramme. After irradiation of in vitro shoottips with 45 Gy, two selections were madewith shorter height and early fruiting, six

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INFOMUSA — Vol 11, N° 1 PROMUSA XIX

month after planting (Hirimburegama et al.1997). Micropropagated plants of these selec-tions were tested for stability of the charactersuntil the second generation. The technologywas perfected and is being transferred to thefarmers (Laksiri and Hirimburegama 1999).Early fruiting and harvesting of micropropagat-ed banana plants saves at least one month incomparison to traditionally grown plants thatusually need eight months to flower. Thus,the number of ratoons in two years becomesthree instead of the usual two, therebyincreasing the income of farmers with 25%(equivalent to about US$350 per ha per year).Mass production of plants is in progress.Thus, indexing/testing of plants for viruses, i.e.banana bract mosaic (BBrMV) and bananastreak virus (BSV) has become essential,since virus-free, indexed mother stocks arerequired for micropropagation. The causativeagent of the banana bract mosaic disease wasconfirmed in the widely cultivated local variety‘Embul’ from Sri Lanka by Thomas et al.(1996, 1997). This disease is more prevalentin cultivations, which are not properly man-aged, but the impact on the yield appears tobe significant.

All the currently reported disease symptomswere observed in infected plants in the fieldbut in varying degrees. Spindle shaped purpleor dark red coloured streak patterns on thepseudostem in addition to dark spindle shapedstreaks on bracts were the most commonsymptoms of infection. Mature plants withinflorescence having black or reddish brownstreaks on the outer surface of the openbracts also had streaks on the pseudostem.The splitting of the base of young suckerscould also be due to other banana virus dis-eases.

A study was done with the objective of devel-oping a low-cost DAS-ELISA detection kit.Anti-serum for BBrMV (of QueenslandDepartment of Primary Industries - QDPI,Australia) was tested as the coating antibodyto replace the relevant component of theAgdia commercial kit. Results showed a rela-tively high efficiency with the QDPI antibody.Work is also in progress to make the alkalinephosphatase enzyme conjugated antibody tosubstitute the one of the test kit. Once thelocal antiserum is produced, it is expected thatan effective and low cost local diagnostic kitwill be developed for the routine indexing ofbanana plants for BBrMV. Purification of thevirus extract (Thomas et al. 1997) is a limitingfactor for obtaining the antigen for the anti-body production process.

ReferencesAnon. 2000. Annual Reports, Mahaweli Authority of Sri

Lanka, Colombo, Sri Lanka.

Anon. 2001. Annual Reports, Mahaweli Authority of Sri

Lanka, Colombo, Sri Lanka.

Hirimburegama K. & N. Gamage. 1997. Cultivar speci-

ficity with respect to in vitro micropropagation of

Musa spp. (bananas & plantains). J. of Hort. Sci.

72(2):205-211.

Laksiri B.D.P. & K. Hirimburegama. 1999. Banana

improvement in Sri Lanka through radiation induced

mutation and tissue culture. INFOMUSA 8(2):PRO-

MUSA 4:XII.

Thomas J.E. & L.V. Magnaye. 1996. Banana bract

mosaic disease. Musa Disease Fact Sheet No. 7.

International Network for the Improvement of

Banana and Plantain. Montpellier, France.

Thomas J.E., A. Geering, C.F. Gambley, A.F. Kessling

& M. White. 1997. Purification properties and diag-

nosis of banana bract mosaic potyvirus and its dis-

tinction from abaca mosaic potyvirus.

Phytopathology 87(7):698-705.

Advances and perspectives for the genetic improvement of banana (Musa spp.) via biotechnological and nuclear techniques at INIVIT

Jorge López Torres

Instituto de Investigaciones en Viandas Tropicales,Biotechnology Laboratory, Apdo 116 CP 53000 Villa Clara,Santo Domingo, Cuba

Bananas and plantains constitute an importantcarbohydrate source in the Cuban diet. This ismainly due to their food habits and bananaproduction all the year round. There is a greatneed to develop new banana cultivars due topoor yield and susceptibility to diseases(mainly black Sigatoka caused by the fungusMycosphaerella fijiensis). The application ofboth biotechnological and nuclear techniqueshas enabled to develop an efficient plant

regeneration system, and to induce geneticvariability for mutant selection. However, thesuccess in obtaining desirable mutants isrestricted to a few allele combinations andthus, more efficient alternatives are needed foran early mutant selection. Earlier, clonesobtained by gamma irradiation of meristematicapices, LD50 dose obtained in Seibersdorf(Austria) and the Centro de EstudiosAplicados al Desarrollo de la Energia Nuclear(CEADEN) in Cuba, were tested in field condi-tions. Somatic embryogenic cell suspensioncultures were established and the somaticembryo formation is being further modified toenhance further plant regeneration rate, espe-cially of AAB genotypes in collaboration withKULeuven. Preliminary evaluations were car-ried out to study the action of crude extract ofM. fijiensis on cell suspension cultures of‘Navolean’ in a solid ZZ medium. Filter paperdiscs were used to test different concentra-tions of crude extract on cell culture growth forselecting toxin tolerant cell cultures. Effect offungal crude extract on oxygen uptake studieswere made in ‘CEMSA ?’ clone, as well as invitroplants of different cultivars used in theIMTP for black Sigatoka studies. The vitro-plant leaves were treated with different con-centrations of crude extract solution of ethylacetate by placing them on different sizes ofdisks, e.g. starting from 0.28 cm2 size disks.Oxygen uptake was measured by Warburg’smanometers. Based on our results, a shortstature mutant (‘Parecido al Rey’ 6.44) wasobtained. Few more mutants were selected,which were tolerant to black Sigatoka withincreased yield, in some cases (‘Parecido alRey’ 6.32 and ‘Gran Enano’ 6.44 mutants andIII-2). However, these characters were unsta-ble, probably affected by the environment, orin vitro culture conditions. Therefore, welooked for other alternative approaches forimproving induced mutation rates, e.g. somat-ic cell suspension cultures and later mass ofsomatic embryogenic cells were established.From each batch of somatic embryogenic cellsuspension mass cultures, 3250 to 6625somatic embryos were obtained with 20.7%germination and 95% plant survival in ex vitroconditions. Their genetic stability is presentlybeing tested in field conditions. The action ofM. fijiensis extract on cell suspension cultureresulted in large number of oxidized cells athigher extract concentrations. Damaged cellswere characterized by a compact cytoplasmiccontent with a dark colour in its centre andleaving an empty space between it and thecellular wall. Forty-five days after incubation,more than 60% cells were found in the previ-ous described conditions due to toxin diffusionfrom discs. Oxygen uptake valves haddecreased normally in non treated control ofall IMTP cultivars, and the maximum decreaseobserved in treated plants in comparison withnon-treated plants was: ‘Yangambi km5’(38.7%); ‘Calcutta 4’ (27.0%); ‘Pisang berlin’(13.4%); ‘Pisang lilin’ (20.9%) while oxygenuptake was increased by 70% in ‘CEMSA ?’clone. The cultivars with higher resistance to

black Sigatoka showed a tendency todecreased oxygen uptake; this is probably dueto the damaged tissues, instead of a toxicityeffect of toxin treatment. At present, the firstsomatic embryos obtained from irradiated cel-lular suspensions are in the germinationphase and somatic embryogenesis in geno-types from the IMTP is being developed.

Agrobacterium-mediatedcotransformation of banana(Musa spp.)

K.Z. Ahmed*, S. Remy, R. Swennen and L. Sági

Laboratory of Tropical Crop Improvement, KULeuven,Kasteelpark Arenberg 13, B-3001 Leuven, Belgium*Permanent address: Department of Genetics, Faculty ofAgriculture. Minia University. El-Minia, ET-61517, Egypt

The introduction of foreign genes into the plantgenome is a basic technique to study geneexpression and physiological processes inplants and for breeding programmes.Improving the agronomic value of major cropsis likely to involve the introduction of multiplegenes, many of which will not provide directlyscreenable phenotypes among the initial prod-ucts of transformation. Major restrictions ofcurrent transformation techniques are thatonly a few genes can be transferred at thesame time and that selectable marker geneshave to be used, which frequently results intransgenic plants containing undesirableantibiotic resistance genes.

The objective of the present study is to deter-mine the efficiency of cotransformation withvisually scorable marker genes usingAgrobacterium tumefaciens and banana(Musa spp.). Cell suspension culture of fourcultivars were infected with two different A.tumefaciens strains each carrying a distinctdisarmed T-DNA containing one of threereporter genes [Luciferase (LUC), ß-Glucuronidase (GUS) or Green FluorescentProtein (GFP)] as well as the neo selectablemarker gene. Multicellular structures express-ing multiple genes were recovered, andcotransformation frequencies were measured.The cotransformation frequency was less thanthe sum of the frequency of each single trans-formation. Negative correlation was foundbetween the transient expression of two visualmarker genes introduced together for cotrans-formation. Significant differences in (co-)transformation frequency were detectedbetween the banana cultivars tested.

We anticipate that the simultaneous use ofmultiple reporter genes will provide a conven-ient method for the accurate determination ofcotransformation and will contribute to a strat-egy for multigene transformation.

Recent developments in early in vitro screening for resistanceagainst migratory endoparasiticnematodes in Musa

A. Elsen and D. De Waele

Laboratory of Tropical Crop Improvement, KULeuven,Kasteelpark Arenberg 13, B-3001 Leuven, Belgium

Among nematodes parasitizing bananasthroughout the world, Radopholus similis andPratylenchus coffeae are important migratorynematodes, causing severe yield losses incommercial and local consumption cultivars.Chemical control is currently the most usedmethod to manage the nematodes althoughnematicides are dangerous, toxic, and expen-sive. Therefore, nematode control throughgenetic improvement of banana is widelyencouraged. Many Musa cultivars have beenscreened to find resistance against these rootpathogens. Screening research is time con-suming because it most be carried out bothunder field and greenhouse conditions. Invitro screening could facilitate and hastenincorporation of genetic nematode control intobananas. However, an in vitro screeningmethod requires aseptic nematode cultures.In this paper, the development of aseptic cul-tures of R. similis and P. coffeae and an invitro screening method are discussed.Alfalfa callus on modified White’s medium hasproved to be a good aseptic culture system forboth R. similis and P. coffeae. Although thereproduction is significantly lower compared tocarrot disc cultures, this system has manyadvantages. The nematodes are not only cul-tured under complete aseptic conditions butthis system is also less labour intensive andoffers a more continuous inoculum production.In addition, culturing on alfalfa callus did notalter the pathogenicity of R. similis and P. cof-feae. Both R. similis and P. coffeae could infect andreproduce on the roots of in vitro grown‘Grande naine’ plants. For both nematodesnecrotic lesions were observed in the rootswithin 2-3 weeks after inoculation. In a lastexperiment, the reproduction of R. similis wastested in vitro on six different Musa cultivarswith a known host response to R. similis.Except for ‘Yangambi km5’, their hostresponse under in vitro conditions correspond-ed to their host response under greenhouse orfield conditions. The susceptible status of‘Grande naine’, ‘Gros Michel’ and ‘Cachaco’was confirmed as well as the resistant statusof ‘Pisang jari buaya’ and ‘SH-3142’.

Evaluation for nematode controlof transgenic plants expressingdifferent types of lectinsK. Carlens, A. Elsen, E. Van Damme1, L. Sági and R. Swennen

Laboratory of Tropical Crop Improvement, KasteelparkArenberg 13 and 1Laboratory for Phytopathology and PlantProtection, Willem De Croylaan 42, KULeuven, B-3001Leuven, Belgium

In the vast majority of African countries wherestarchy bananas and plantains are grown, Musaspp. are important staple food crops, oftenexclusively grown by small scale farmers. Thisespecially applies to the highland cooking andbrewing bananas of East and Central Africa.Over the last two decades, however, bananayields in the East African region have beensteadily declining partly due to migratory orsedentary endoparasites: the burrowing nema-tode (Radopholus similis), the root-lesion nema-todes (Pratylenchus coffeae) and root-knotnematodes (Meloidogyne spp.). With theadvent of transgenic methodologies, an attrac-tive method to control these nematodes is thetransfer into target Musa spp. of lectin encodinggenes. Prior to application in banana, thenematicidal effect of a number of lectins orlectin-related proteins is currently tested for theireffectiveness against banana nematodes intransgenic Arabidopsis thaliana and/or tobacco.The different in vitro test systems will be dis-cussed, together with the initial results.

Lectin binding to the plantparasitic nematode Radopholussimilis and its effect on hostfindingN. Wuyts, A. Elsen, E. Van Damme1, D. De Waele, R. Swennen and L. Sági

Laboratory of Tropical Crop Improvement, KasteelparkArenberg 13 and 1Laboratory for Phytopathology and PlantProtection, Willem De Croylaan 42, KULeuven, B-3001Leuven, Belgium

The effect of lectins on the plant parasiticnematode Radopholus similis was studied in aseries of experiments. FITC- or colloidal gold-labelled lectins of Canavalia ensiformis(ConA), wheat (WGA) and Helix pomatia(UPA) were found to bind the nematode in thehead region, at the excretion pore, the poresof the reproduction system and those of thephasmids. The viability and the chemotacticresponse towards plant roots, after treatmentof nematodes with lectins, were examined invitro by analyzing movement tracks left onagar plates. The assay included six plantlectins of five different classes and the bananathaumatin-like protein. A 1% concentration ofPhaseolus vulgaris agglutinin (PHA) had atoxic effect on R. similis females: 68% showedno or very little movement after inoculationcompared to an average of 30% for otherlectins and 5% for the control treatment. A0.05% concentration of PHA still reduced theviability of R. similis females by 75%. ConAand WGA did not alter the chemotacticresponse towards plant roots, despite of thedemonstrated binding of both lectins to R. sim-ilis. In contrast, Galanthus nivalis agglutinin(GNA) reduced orientated movement of R.

XX PROMUSA INFOMUSA — Vol 11, N° 1

similis females towards plant roots. Finally,the secretions of R. similis were stained withCoomassie Brilliant Blue R. These secretionsappear at the amphids, the excretion pore, thevulva, the spicules and the phasmids.Moreover, nematodes treated with GNA pro-duced less abundant secretions.

Early detection of dwarf off-typesof banana using AFLP, TE-AFLPand MSAP analysis

I. Engelborghs, L. Sági and R. Swennen


AFLP and several variant techniques wereperformed on banana in order to characterizedifferent dwarf phenotypes. The dwarf AABplantain ‘Curare enano’ and its normal-sized invitro generated off-type were analyzed byAFLP, TE-AFLP, MSAP, cDNA-AFLP, andcDNA-TE-AFLP. AFLP and TE-AFLP werealso performed on four pairs of naturallyoccurring dwarf/normal banana cultivars.Differential AFLP patterns were obtained and upto 25% polymorphism was observed dependingon the primer combination and the cultivar. TE-AFLP analysis generated shorter and a lowernumber of fragments resulting in only relativelyfew polymorphisms between the dwarf and nor-mal-sized cultivars. The somaclonal variantsobtained in vitro from the dwarf ‘Curare enano’might have been caused by methylation inducedby in vitro conditions. MSAP analysis, based onthe methylation (in)sensitivity of a pair of isoschi-someric restriction enzymes, appeared to be avaluable tool in revealing differential cytosinemethylation. Cloning and sequencing differen-tial fragments did not reveal significantly homol-ogous matches in public databases. cDNA-AFLP analysis between the dwarf and normal‘Curare enano’, revealed a normal-specific frag-ment, while cDNA-TE-AFLP analysis resulted ina dwarf-specific fragment. AFLP and the varianttechniques have shown the potential to differen-tiate between closely related genotypes. Moreprimer combinations and/or alteration of restric-

tion enzymes will increase the chance of findingmore dwarf-related sequences.

Establishment of embryogeniccell suspension cultures fromIndian banana cultivars

P. Suprasanna*, B. Panis, L. Sági and R. Swennen

Laboratory of Tropical Crop Improvement, KULeuven,Kasteelpark Arenberg 13, B-3001 Leuven, Belgium*Permanent address: Nuclear Agriculture & BiotechnologyDivision, Bhabha Atomic Research Centre, Trombay, Bombay400 085, India

Banana (Musa spp.) is an important and majorfruit crop in India, which is the largest produc-er of bananas in the world. However, dis-eases and pests such as black Sigatoka andPanama disease, bunchy top virus and nema-todes remain as major threats to production.Genetic modification using embryogenic cellsuspensions (ECS) appears to be a suitableapproach for integrated genetic improvement.Progress has been made in the developmentof protocols for the establishment of ECS, andimmature male flowers as well as proliferatingin vitro cultures have mainly been used.Especially in vitro proliferating meristems(‘scalps’) are an ideal starting material,because they can be generated all round theyear from most cultivars. The methodincludes a preculture of the proliferating meris-tems on high cytokinin medium which providesembryogenic competence.Several important Indian cultivars [(‘Robusta’(AAA), ‘Basrai’ (AAA), ‘Shrimanthi’ (AAA),‘Karpoora valli’ (ABB)] have been employed inthis study for the induction of good quality scalpsand embryogenic callus. Of these, ‘Robusta’have shown the formation of embryogenic cal-lus, which subsequently has been used for theestablishment of ECS. In addition, the effect ofalternative cytokinins such as meta-topoline(MT, thidiazuron (TDZ) and N-chloro-4-pyridyl-N’-phenylurea (CPPU) has been studied on iso-lated meristems of ‘Cachaco’ (ABB), ‘Williams’(AAA) as well as on scalps of ‘Robusta’. On iso-lated meristems (‘Williams’, ‘Cachaco’), CPPU

has only resulted in the development of singleshoots and roots, whereas MT resulted in theformation of watery callus and proliferation. TDZmainly induced swelling of the explants. TDZproved to be better cytokinin over MT in theinduction and maintenance of good qualityscalps. These are currently under evaluation forembryogenic induction. The established ECSwill be characterized and used in cryopreserva-tion and genetic transformation experiments.

RAPD, SSR and AFLPtechniques in detectingpolymorphism between irradiated parents and suckers in two ediblePhilippine banana cultivars D.M. Hautea, G.C. Molina, N.B. Coronado, H.F. Galvez, C.H. Balatero and R.B. Quilloy

Institute of Plant Breeding, College of Agriculture, University ofthe Philippines Los Baños, 4031 College, Laguna, ThePhilippines

This report summarizes the results of ourefforts to evaluate the usefulness of DNAmarker techniques, such as random amplifiedpolymorphic DNA (RAPD), microsatellites orsimple sequence repeat (SSR) and amplifiedfragment length polymorphism (AFLP), tocharacterize the genomic alterations ininduced mutants of the two Philippine bananacultivars. Mutants were induced in the twomost popular Philippine edible banana culti-vars by gamma and fast neutron irradiationfrom in vitro shoot-tip cultures. Promisingclones were selected and evaluated furtherusing molecular markers. In banana, severalDNA marker techniques were used to investi-gate genetic relationships between Musaaccessions and to determine differences insomaclonal variants and radiation inducedmutants. In this study, RAPD, SSR, and AFLPtechniques were successfully used under localconditions and found useful in characterizingirradiated and non-irradiated banana clones.RAPD, SSR and AFLP markers also showedsufficient polymorphism to differentiatebetween the two cultivars used. However,SSR and AFLP markers were found to bemore highly reproducible. One of the mostsignificant accomplishments of this CRP proj-ect is the development of a high quality, non-radioactive, silver-staining technique for AFLPanalysis, which can be easily adopted underlaboratory conditions in developing countries.AFLP markers showed both high reproducibili-ty and discriminating capacity. PolymorphicAFLP markers were identified (Table 2) andfound useful in fingerprinting bananas andother Musa species. AFLP was the onlymarker technique tested, which was able todetect variation in DNA profiles of inducedmutant clones, their first cycle suckers andnon-irradiated control clones (Figure 2), which

Selective No. of bands Number of % Polymorphismprimer pairs polymorphic bandsE-ACG/M-CTC 35 11 31.4 E-ACG/M-CTG 32 20 62.5 E-ACG/M-CTT 18 14 77.8 E-AGC/M-CAA 40 20 50.0 E-AGC/M-CAC 34 14 41.2 E-AGC/M-CAG 29 14 48.3 E-AGC/M-CAT 31 14 45.2 E-AGC/M-CTA 21 7 33.3 E-AGC/M-CTC 29 19 65.5 E-AGC/M-CTG 32 18 56.2 Mean 34 15.1 51.1

Table 2. Summary of polymorphism detected in AFLP markers used in the analysis of irradiated parentclones, first cycle suckers and non-irradiated clones of cultivars Lakatan and Latundan

INFOMUSA — Vol 10, N° 2 PROMUSA XXI

are otherwise morphologically indistinguish-able. The results indicate that AFLP is veryideal and useful for fingerprinting purposescompared with other marker systems becauseof its high multiplex ratio i.e. more bands(=loci) per gel can be resolved. While moreprimer combinations need to be tested, theseresults suggest the potential usefulness of thistechnique in detecting genome variationbetween cultivars and in detecting genomealterations in induced mutants of bananaincluding those showing very similar pheno-types. The detected variation between theirradiated parent clones and suckers suggeststhat the number of vegetative propagationcycles for the shoot-tip technique used (Novaket al. 1989) may not be sufficient to completelyeliminate chimeras in the mutated populations.The results obtained could provide a soundbasis for more successful application of muta-tion and molecular marker techniques forimprovement of banana in the Philippines.

ReferenceNovak F.J., R. Afza, M. Van Duren & M.S. Omar.

1989. Mutation induction by gamma irradiation of invitro cultured shoot-tips of banana and plantain(Musa cvs). Trop. Agric. (Trinidad) 67 (1):21-28.

Rat feeding tests of transgenicbanana expressing an antifungal peptide from onionS. Remy, G. Flo2, I. Deconinck, S. Lievens2.M. Cokelaere2, E. Decuypere1, L. Sági and R. Swennen

Laboratory of Tropical Crop lmprovement and 1 Laboratory ofPhysiology and Immunology of Domestic Animals, KULeuven,Kasteelpark Arenberg 13, B-3001 Leuven, Belgium2 Subfaculteit Geneeskunde KULAK, Sabbelaan 53, Kortrijk,The Netherlands

Transgenic plants of the dessert banana‘Williams’ containing a gene encoding theAce-AMP1 antifungal peptide of onion (Alliumcepa) were produced by particle bombard-ment for enhanced tolerance against attack bythe fungal pathogen Mycosphaerella fijiensis.ELISA assays on extracts from lyophilizedbanana pulp showed that the concentration ofthe Ace-AMP1 peptide reached up to 0.0316%of the total amount of soluble proteins or sixtimes above the background signal measuredin non-transformed banana pulp. We testedwhether this expression level had an effect onrats fed on a diet containing transgenicbanana pulp.While energetic content was comparable intransgenic and control pulp, dry matter andprotein content were lower and higher in trans-genic pulp than in control pulp respectively.Twenty per cent of lyophilized meal from con-trol or transgenic bananas were incorporatedin regular rodent food and supplied to maleand female Wistar rats. Feeding of the trans-genic meal during six weeks did not cause anydifference in food intake, growth rate andweight of internal organs in comparison tofeeding on control diet. Also, a complex bloodanalysis did not show any effect in rats con-suming the transgenic banana meal.

Cryopreservation for the elimination of cucumber mosaic and banana streakdiseases in banana (Musa spp.)

B. Helliot1 B. Panis2 R. Swennen2 and P.Lepoivre1

1 Plant Pathology Unit, Gembloux Agricultural University, 5030Gembloux, Belgium2 Laboratory of Tropical Crop Improvement, KULeuven,Kasteelpark Arenberg 13, B-3001 Leuven, Belgium

Bananas and plantains (Musa spp.) are threat-ened by various pests and diseases, includingimportant viral diseases which constrainbanana production and cross-bordergermplasm movement. This delays dramati-cally the distribution of high yielding and newlybred varieties to small farmers.INIBAP therefore established a system forthe safe international movement of Musagermplasm. This involves that all germplasm

held at the international Musa germplasmcollection of INIBAP (based at KULeuven,Belgium) is tested by different virus indexingcentres (South Africa, Australia and France).Currently about 25% of the collection com-prising a significant number of potentiallyimportant and improved varieties is infectedwith viruses. The most prevalent viruses inthis infected germplasm are BSV (bananastreak virus) but also with CMV (cucumbermosaic virus), BBTV (banana bunchy topvirus), BanMMV (banana mild mosaic virus)and BBrMV (banana bract mosaic virus).A programme of virus elimination is thereforecarried out by the Plant Pathology Unit(FUSAGx, Belgium) in collaboration with theLaboratory of Tropical Crop Improvement(KULeuven, Belgium). Different in vitro tech-niques, such thermotherapy, chemotherapy,meristem culture and cryotherapy are tested.Banana plants of the cultivar ‘Williams BSJ’(AAA) were either naturally infected with BSVor mechanically infected with CMV or BBTV.Proliferating in vitro cultures were producedfrom this material. Excised meristematicclumps were cryopreserved through vitrifica-tion using the PVS-2 solution. The healthstatus of regenerated material was firstchecked on in vitro plants through ELISA.Then, the putative virus-free material wastested again after greenhouse acclimatiza-tion. The virus eradication rates after cryop-reservation for CMV and BSV reached 30%and 90% respectively. In comparison, thefrequency of virus-free plants regenerateddirectly from highly proliferating meristems,which reflects spontaneous eradication,reached 0% and 52% for CMV and BSVrespectively. The conventional meristem cul-ture resulted in 0% CMV-free plants and 76%BSV-free plants.In conclusion, cryopreservation seems to be avery promising technique for virus eradicationfrom Musa germplasm enabling to faster dis-tribute germplasm of interest.

An ultrasensitive luminescentdetection system hi bananabiotechnology: from promotertagging to Southern hybridizationS. Remy, G. De Weerdt, I. Deconinck, R. Swennen and L. Sági


Digital imaging using a cooled CCD camera isbecoming an increasingly versatile tool inbiotechnology research. An ultrasensitivecamera system consisting of a liquid nitrogencooled slow-scan CCD camera connected topowerful image analysis software is able todetect very low levels of light emission fromseveral signal sources and is used for therecording of results for different applications inbanana biotechnology.

XXII PROMUSA INFOMUSA — Vol 11, N° 1

Figure 2. AFLP fingerprints of irridiated bananaclones (M), first cycle suckers (S) and non-irra-diated clones (N) on ‘Latundan’ (LT-3) and‘Lakatan’ (LK-40).

INFOMUSA — Vol 11, N° 1 PROMUSA XXIII

The most sensitive reporter system, biolumi-nescent luciferase (LUC) is used in T-DNAmediated promoter tagging. Since integrationof the promoterless luc gene is random, thelevel of LUC expression is suboptimal in mosttransformants to be screened requiring a high-ly sensitive detection. Preliminary results onin vivo screening of LUC expression in hun-dreds of putative promoter tagged cell cultureswill be presented.Chemiluminescence has been for severalyears the method of choice in our laboratoryfor non-radioactive hybridization analysis.Although exposure to and development of anX-ray film is a sensitive technique, it is timeconsuming and costly because multiple expo-sures are needed for the evaluation of results.In addition to increased flexibility and highersensitivity of detection, signals can be cap-tured faster with the CCD camera than withfilm. Good results can be obtained with a sin-gle exposure by adjusting the gray scale of thecaptured image as will be demonstrated.In addition to luminescence the camera canalso detect fluorescent signals, which isdemonstrated by the ability to monitor greenfluorescent protein expression in transgenicbanana cultures. Quantification of light inten-sity by software analysis will be demonstrated.

Agrobacterium- and particlebombardment transformation of a wide range of banana cultivars

G. Arinaitwe*, S. Remy, H. Strosse, R. Swennen, and L. Sági

Laboratory of Tropical Crop Improvement, KULeuven,Kasteelpark Arenberg 13, B-3001 Leuven, Belgium*Permanent address: Makerere University, Faculty ofAgriculture and Forestry, Department of Crop Science, P.O.Box 7062 Kampala, Uganda

Genetic transformation of banana (Musa spp.)by particle bombardment and Agrobacteriumis established only in a few laboratories world-wide. In general, transformation frequenciesare reported to be cultivar dependent. Thus,there is a need to optimize established trans-formation protocols for any particular type ofbanana. In this study, the two transformationmethods were compared and the effect ofphysical parameters on transformation fre-quency was investigated in four banana culti-vars: ‘Grande naine’ (AAA), ‘Obino l’ewai’(AAB), ‘Orishele’ (AAB), and ‘Three handplanty’ (AAB). DNA transfer frequency wasmeasured by monitoring expression of the b?-glucuronidase and the green fluorescent pro-tein gene. The results indicate major differ-ences between the two transformationsystems. Significantly higher transient andstable gene expression, in all banana culti-vars, were obtained with the Agrobacterium-based method. The effects of age and volumeof cell suspensions as well as the length ofinfection were optimized. The cultivars werecategorized on the basis of their competencefor transformation and their capacity for regen-

eration. Molecular and biochemical analysiswill be performed to confirm integration andexpression of the transgenes in the differentcultivars.

Cryopreservation of embryogeniccell suspensions of banana to support banana improvementB. Panis, H. Strosse, S. Remy, L. Sági and R. Swennen


The initiation of embryogenic cell suspensioncultures of banana is still difficult and time-consuming, irrespective of the starting materi-al used (immature male flowers, immaturezygotic embryos or proliferating in vitro meris-tems). The embryogenic response is very lowand slow. Indeed for most cultivars less than1% of initial explants give rise to an embryo-genic callus suitable for cell suspension initia-tion and 9- 26 months are needed before suchembryogenic cell suspension is established.Moreover, once established, these cell sus-pensions are subject to somaclonal variationand microbial contamination and a prolongedculture period may result in a lower and even-tually a total loss of morphogenic capacity.Up to now, transformation protocols of bananarely on embryogenic cell suspensions.Particle bombardment as well asAgrobactenum-based protocols resulted intransgenic banana plants. Somatic hybridiza-tion and protoplast electroporation are alsoboth depending on the isolation of regenerableprotoplasts from embryogenic cell suspen-sions. Finally, embryogenic cell suspensionscan be used for mass propagation as an alter-native to shoot-tip cultures.The safe preservation of this valuable sus-pensions through cryopreservation is thus ofoutermost importance. A cryopreservationtechnique was developed which involves cry-oprotection with 7.5% DMSO (dimethylsulphoxide) and 180 g/L sucrose, followed byslow freezing at 1°C/min to -40°C and plung-ing into liquid nitrogen. Currently, 651 cry-otubes containing embryogenic cell suspen-sions belonging to 48 independent cell linesand 14 different cultivars are safely stored inliquid nitrogen for the long term. Recently,banana cell suspensions were recoveredafter five years storage in liquid nitrogen.The ability to produce somatic embryosremained intact. Also their competencetowards Agrobacterium-mediated transforma-tion was screened and compared to a non-cryopreserved cell suspension of the samecell line. The transient expression of theintroduced marker gene as well as the regen-eration efficiency of transgenic plantlets wascomparable.

Oxidative events induced by Mycosphaerella fijiensismetabolites in banana (Musaspp.) black leaf streak diseaseand analysis of early selectionfeasibility for resistance to thisdisease

J.P. Busogoro1, B. Panis2, J. Messiaen3, P. Van Cutsem3, R. Swennen2

and P. Lepoivre1

1 Unité de Phytopathologie, Faculté Universitaire des SciencesAgronomiques de Gembloux, Passage des Déportés 2, 5030Gembloux, Belgium2 Laboratory of Tropical Crop Improvement, KULeuven,Kasteelpark Arenberg 13, 3001 Leuven, Belgium3 Unité de recherche en Biologie Végétale Cellulaire, FacultésUniversitaires Notre Dame de la Paix, Rue de Bruxelles 61,5000 Namur, Belgium

The mechanisms of action of M. fijiensis toxinsin black leaf streak (BLS) disease were stud-ied. The ethyl acetate crude extract (EaCE)from the pathogen culture filtrates and juglone(5-hydroxy-1,4-naphthoquinone), which is apurified metabolite from EaCE, were injectedin the leaves of two banana cultivars. The cul-tivars ‘Grande naine’ and ‘Fougamou’ servedas a susceptible reference and partially resist-ant reference respectively. These bioassaysinduced necrosis and showed decrease of thevitality index determined according to chloro-phyll fluorescence data (Lichtenthaler et al.1986). The ‘Grande naine’ cultivar was moresensitive than Fougamou whatever the bioas-say (induction of necrosis or chlorophyll fluo-rescence) taken into account. The light-dependence of the toxicity revealed by thesetests, the early effect on chlorophyll fluores-cence (Harelimana et al. 1997) and theswelling of ‘Grande naine’ chloroplasts afterinjection with EaCE, are indicative that chloro-plasts could be a potential target site for M.fijiensis toxins.A mechanical protocol (Leegood and Malkin1986) to isolate physiologically intact chloro-plasts from banana leaves was developed. Anew bioassay based on the addition of jugloneto banana chloroplast suspensions was usedto analyse the impact of M. fijiensis metabo-lites. By performing the Hill reaction (Allenand Holmes 1986) with the so treated suspen-sion to measure the ability of chloroplasts totransfer electrons, a direct inhibiting effect ofjuglone on this physiological activity was clear-ly demonstrated. Moreover, this effect wasagain higher with ‘Grande naine’ chloroplaststhan with those of ‘Fougamou’.Since chloroplasts constitute one of the sitesof active oxygen species production in plants(Sutherland 1991, Foyer et al. 1997), theirdirect interactions with juglone in bananas ledto a new hypothesis. Hence, oxidative eventswere suspected to be at the origin of the phys-iological damages in the isolated chloroplasts.In fact, involvement of fungal naphthoquinonemetabolites in oxidative process in not uncom-

mon (Medentsev and Akimento 1998). Theirauto-oxidative property responsible of the oxi-dation of NADH and NADPH leads to theremoval of these molecules from the oxidativephosphorylation system as potential sourcesof reduction equivalents for the respiratorychain.In the case of BLS disease, assessment ofthis hypothesis was performed by consideringpossible interactions between juglone andbanana antioxidant systems. We observedthat juglone causes an in vitro oxidation ofascorbic acid, the most abundant antioxidantin plants (Smirnoff 2000). The occurrence ofoxidative phenomena induced by this metabo-lite in bananas was also assessed byanalysing the superoxide dismutases (SOD)patterns at several intervals of time followingjuglone injection into leaves of the two refer-ence cultivars. In fact, superoxide dismutasesare assumed to play a central role in thedefence against oxidative stress (Beyer et al.1991, Scandalias 1993). Our preliminaryobservations showed that there was a repres-sive effect on one SOD isoform in ‘Grandenaine’ while a stimulating effect on anotherSOD isoform in ‘Fougamou’ seemed to occur.On the base of the results obtained with thetwo antioxidant systems analysed previously,juglone could be supposed to deprivebananas partly of their antioxidant capacity.Further investigations have to be done in thisarea in order to determine exactly all the

mechanisms affected by the pathogenmetabolites during BLS development.Finally, a first in vitro selection assay for BLSresistance was also performed. Therefore,different juglone concentrations were mixedwith embryogenic cell suspensions of twolines of THP (‘Three hand planty’ cultivar) aswell as with suspensions containing somaticembryos of the same cell lines. After anovernight incubation, the material was trans-ferred onto juglone-free fresh media. In gener-al, both plant materials necrosed and did notshow any weight increase during the period ofincubation following the treatment. However,from somatic embryos of one treated line,some plants were obtained from tissues thatdid not become necrotic with 50 ppm ofjuglone. These regenerated plants are goingto be evaluated for their eventual BLS resist-ance with the abovementioned bioassays aswell as by artificial inoculation of the pathogen.These plant regenerates will constitute a pre-cious material to further analyse the roleplayed by M. fijiensis toxins in development ofBLS disease.

ReferencesAllen J.F. & N.G. Holmes. 1986. Electron transport and

Redox Titration. Pp. 103-141 in Photosynthesis

energy transduction. A practical approach (M.F.

Hipkins and N.R. Baker, eds). IRL Press,

Washington, USA.

Beyer W., J. Imlay & I. Fridovich. 1991. Superoxide

dismutase. Prog. Nucl. Acid Res. 40:221-53.

Foyer C., G. Noctor & J.F. Morot-Gaudry. 1997.

L’oxygène: bienfait ou danger pour les plantes?

Biofutur 169:27-29.

Harelimana G., P. Lepoivre, H. Jijakli & X. Mourichon.

1997. Use of Mycosphaerella fijiensis toxins for the

selection of banana cultivars resistant to black leaf

streak. Euphytica 96:125-128.

Leegood R.C. & R. Malkin. 1986. Isolation of sub-cellu-

lar Photosynthetic Systems. Pp. 9-26 in

Photosynthesis energy transduction. A practical

approach. (M.F. Hipkins and N.R. Baker, eds). IRL

Press, Washington, USA.

Lichtenthaler H.K., Buschmann C., Rinderle U. &

Schmuck G. (1986) Application of chlorophyll fluo-

rescence in ecophysiology. Radiation and

Environemental Biophysics 25: 297-308.

Medentsev A.G. & V.K. Akimenko. 1998.

Naphthoquinone metabolites of the fungi.

Phytochemistry 47:935-959

Scandalias J. G. 1993. Oxygen stress and superoxide

dismutase. Plant Physiol. 101:7-12.

Smirnoff N. 2000. Ascorbic acid: metabolism and func-

tions of a multi-facetted molecule. Current Opinion

in Plant Biology 3:229-235.

Sutherland M.W. 1991. The generation of oxygen radi-

cals during host plant responses to infection.

Physiological and Molecular Plant Pathology

398:79-93.

XXIV PROMUSA INFOMUSA — Vol 11, N° 1

The following publications are available fromheadquarters:INIBAP 2002. Networking bananas and plantains: Annual Report 2001.INIBAP 2002. The Global Musa Genomics Consortium. Strategy for the Global Musa

Genomics Consortium: Report of a meeting held in Arlington, USA, 17-20 July 2001.INIBAP/CTA/CIRAD 2001. J. Daniells, C. Jenny, D. Karamura & K. Tomekpe. Musalogue: a

catalogue of Musa germplasm. Diversity in the genus Musa (E. Arnaud & S. Sharrock,compil.).

INIBAP/CTA 2001. B. Panis & N.T. Thinh. Cryopreservation of Musa germplasm (J.V. Escalant et S. Sharrock, eds). INIBAP Technical Guidelines 5.

INIBAP 2001. Networking bananas and plantains: Annual Report 2000.CIRAD/INIBAP 2000. Bananas.INIBAP 2000. G. Orjeda (compil.). Evaluating bananas: a global partnership. Results of

IMTP Phase II. INIBAP/EARTH/IDRC 1999. F.E. Rosales, S.C. Tripon & J. Cerna (eds). Organic/environ-

mentally friendly banana production. Proceedings of a workshop held at EARTH,Guácimo, Costa Rica, 27-29 July 1998.

INIBAP/CRBP/CTA/CF 1999. C. Picq, E. Fouré & E.A. Frison (eds). Bananas and foodsecurity/Les productions bananières: un enjeu économique majeur pour la sécurité ali-mentaire. Proceedings of an International Symposium held in Douala, Cameroon, 10-14 November 1998.

INIBAP/FHIA 1999. F.E. Rosales, E. Arnaud & J. Coto (eds). A tribute to the work of PaulH. Allen: a catalogue of wild and cultivated bananas.

INIBAP/RF/SDC 1999. E.A. Frison, C.S. Gold, E.B. Karamura & R.A. Sikora (eds). MobilizingIPM for sustainable banana production in Africa. Proceedings of a workshop on bananaIPM held in Nelspruit, South Africa, 23-28 November 1998.

INIBAP 1999. E. Akyeampong (ed.). Musa Network for West and Central Africa. Report ofthe second Steering Committee meeting held at Douala, Cameroon, 15-16 November1998.

INIBAP 1999. K. Shepherd. Cytogenetics of the genus Musa.INIBAP 1998. E. Akyeampong (ed.). Musa Network for West and Central Africa. Report of

the first Steering Committee meeting held at Douala, Cameroon, 8-10 December 1997.INIBAP 1998. E.A. Frison & S.L. Sharrock (eds). Banana streak virus: a unique virus-Musa

interaction? Proceedings of a workshop of the PROMUSAvirology working group heldin Montpellier, France, 19-21 January 1998.

INIBAP 1998. C. Picq (ed.). Segundo seminario/taller de la Red regional de información sobrebanano y plátano de America Latina y el Caribe. San José, Costa Rica, 10-11 July 1997.

INIBAP/CTA/FHIA/NRI/DFID 1998. B.K. Dadzie. Post-harvest characteristics of blackSigatoka resistant banana, cooking banana and plants hybrids. INIBAP TechnicalGuidelines 4.

INIBAP/CTA 1998. G. Orjeda in collaboration with the PROMUSA working groups on Sigatoka and Fusarium. Evaluation of Musa germplasm for resistance to Sigatokadiseases and Fusarium wilt. INIBAP Technical Guidelines 3.

CIRAD/INIBAP 1998. Les bananes.

INIBAP/ACIAR 1997. E. Arnaud & J.P. Horry (eds). Musalogue, a catalogue of Musagermplasm: Papua New Guinea collecting missions 1988-1989.

INIBAP/CTA/FHIA/NRI/ODA 1997. B.K. Dadzie & J.E. Orchard. Post-harvest RoutineScreening of Banana and Plantain Hybrids: Criteria and Methods. INIBAP TechnicalGuidelines 2.

INIBAP/CTA 1997. P.R. Speijer & D. De Waele. Screening of Musa Germplasm forResistance and Tolerance to Nematodes. INIBAP Technical Guidelines 1.

INIBAP/The World Bank 1997. E.A. Frison, G. Orjeda & S. Sharrock (eds). PROMUSA: A Global Programme for Musa Improvement. Proceedings of a meeting held in Gosier,Guadeloupe, March 5 and 9, 1997.

INIBAP-IPGRI/CIRAD. 1996. Descriptors for Banana (Musa spp.).

The following publications are available from Asia and the Pacific office:INIBAP-ASPNET 2001. A.B. Molina, V.N. Roa & M.A.G. Maghuyop (eds). Advancing banana

and plantain R & D in Asia and the Pacific Vol. 10. Proceeding of the 10th INIBAP-ASP-NET Regional Advisory Committee (RAC) meeting held at Bangkok, Thailand, 10-11November 2000.

INIBAP-ASPNET/MARDI 2001. A.B. Molina, N.H. Nik Masdek & K.W. Liew (eds). BananaFusarium wilt management: towards sustainable cultivation. Proceedings of the inter-national workshop on the management of Fusarium wilt disease held in Genting,Malaysia, 18-20 October 1999.

INIBAP-ASPNET 2000. A.B. Molina & V.N. Roa (eds). Advancing banana and plantain R & D in Asia and the Pacific. Proceedings of the 9th INIBAP-ASPNET RegionalAdvisory Committee meeting held at South China Agricultural University, Guangzhou,China, 2-5 November 1999.

INIBAP-ASPNET/FFTC 2000. A.B. Molina, V.N. Roa, J. Bay-Petersen, A.T. Carpio & J.E.A.Joven (eds). Managing banana and citrus diseases. Proceedings of a regional workshopon disease management of banana and citrus through the use of disease-free plantingmaterials held in Davao City, Philippines, 14-16 October 1998.

INIBAP-ASPNET 2000. R.V. Valmayor, S.H. Jamaluddin, B. Silayoi, S. Kusumo, L.D. Danh,O.C. Pascua & R.R.C. Espino. Banana cultivar names and synonyms in Southeast Asia.

INIBAP-ASPNET 1999. V.N. Roa & A.B. Molina (eds). Minutes: Eighth meeting of INIBAP-ASPNET Regional Advisory Committee (RAC) hosted by the Queensland HorticultureInstitute (DPI) in Brisbane, Australia, 21-23 October 1998.

INIBAP-ASPNET 1998. Minutes: Seventh meeting of INIBAP/ASPNET Regional AdvisoryCommittee (RAC) hosted by the Vietnam Agricultural Science Institute (VASI) inHanoi, Vietnam, 21-23 October 1997.

INIBAP-ASPNET 1997. V. N. Roa & R. V. Valmayor (eds). Minutes: Sixth meeting of INIBAP-ASPNET Regional Advisory Committee (RAC) hosted by National ResearchCenter on Banana (ICAR) in Tiruchirapalli, India, 26-28 September 1996.

INIBAP-ASPNET 1996. R. V. Valmayor, V. N. Roa & V. F. Cabangbang (eds). RegionalInformation System for Banana and Plantain - Asia and the Pacific (RISBAP):Proceedings of a consultation/workshop held at Los Baños, Philippines, 1-3 April 1996.(ASPNET Book Series No. 6).

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The International Magazine on Banana and Plantain · 2018. 3. 28. · Evaluation of the agronomic characteristics of plantain hybrids Options for in vitro propagation of the banana