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Pathology (1989) 38, 571-576

{Pennisetum americanum)graminicola)

an d S. F. L. BALL*ent of Ag riculture, U niversity o f Reading, Earley Gate, Reading R G 6 2.4T. i 'K

{Pennisetum americanum} were{Sclerospora graminicola). Individual florets

the host tissues were accompanied by both sexual and asexual sporulation of the fungus

Scleros-can result in severe grain lossespearl m illet (Pennisetum americanum). Primary

borne sporangia and zoospores. M ycelium haset al..80), althou gh w hether seed transm ission occu rs

Diseased plants show symptoms only on theleaf, with subsequent

orescence may show partia l or co mp lete phyl-

The evidence for seedling infection by both

g & Webster, 1970; Singh & W illiams, 1980;

colonization have yet to be elucidated. It has beensuggested by Singh & Williams (19S0) and Ball(1983) that the pathogen reaches meristems ofyoung shoots and tillers, colonizes undifTeren-tiated tissues and thereafter causes disease onleaves an d/o r inflorescences Plants infectedbefore panicle initiation produce no inflores-cence. Later infection may affect the tillers only,with the main shoot escaping disease. M alforma-tion and phyllody of the organs within theinflorescence is well-known ('green-ear' disease),but information on direct infection of the inflor-escence is lacking. Generally it has been assumedthat pan icle malformation is the result of s\ stemicinfection of seedlings and tillers. Some plantsproduce panicles that are only partially mal-formed with some normal grain set. In theseinstances we noticed that the malformationsoccurred only on the gynoecia that becamegrossly elongated green structures, although stillclearly bifurcated at the style-tips. Sometimesthese malformed stigmas occurred at the base ofthe panicle, sometimes towards the tips or ran-domly scattered over the panicle.

This paper presents evidence of panicle infec-tion by zoospores of S. graminicola in which


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572 S. T. Semisi and S. F. L. Ballcontribution of infection by zoospores to epi-demic progress and yield losses could be signifi-cant because late infection gives no opportunityfor yield compensation by surrounding plants(Williams, 1984).M A T E R I A L S A N D M E T H O D SHostsG rowth and maintenanceSeeds of pearl millet P. americanum cultivar 7042(susceptible to downy mildew) were supplied bythe Internation al C rops R esearch Institute for theSemi-Arid Tropics (ICRISAT), Hyderabad,India. To minimize variation, the same seed batchwas used throughout this investigation. Seedswere surface-sterilized for 10 min in 1 g/l m ercuricchloride followed byfivewashes in sterile distilledwater (5 min each). Excess moisture w as drainedoff and the seeds were dried at 40 C for 48 h andstored in sterile petri dishes at 5 C until required.Only samples with germination of >80% wereused. Seeds were sown into 15-cm diameterplastic pots containing peat-based compost andplaced on a greenhouse bench. Night/day tem-peratures were maintained at an average of 24

30C, respectively, with 14-h daylength. Supplementary feeding with macron utrients (NP K) wasupplied at sowing time and every 10 days untcompletion of the experiments.

Florat initiationWhen seedlings reached the 3-4 leaf stage thewere thinned out, leaving two seedlings per poFloral initiation was induced by short days (On& Everard, 1979; Coaldrake & Pearson, 1986) bcovering the plan ts w ith black polyethylene sheetbetween 20.30 and 08.30 hours for 14 consecutivdays. Th e test pla nts were grown un til the desirefloral developm ent stages were reached (Table 1

Pathogen maintenance and productionAn accession of 5. graminicola from BengouNiger was supplied as oospores in dried, fiineground, pearl millet material. Seeds of pearmillet cultivar 7042 were soaked in oosporsuspensions (4-5 x lO^ml) for 1 h. Five seeds wesown in each pot in which 1 ml of oosporsuspension had been added to each sowing hole

Table 1. Effect of inoculation with Sclerospora graminicola zoospores at defined floral development stages oPennisetum americanum cv. 7042

Treatmenttit :t jUtst6

t7

U

Number of daysfrom sowing172227293132343641

Num ber of leaveson mainshoot6-78-9

10-1111-1212 1413-1414-1514 1514-15

Inflorescence developmentstage at inoculationPanicle initiationBranch primordiaSpikelet primordiaSpikelet differentiation(empty glume)Spikelet differentiation(stamens)Spikelet differentiation(pistil)Spikelet differentiation(ovule/carpel)Boot (undifferentiatedgynoecia; fused styllodia)Panicle emerged from flag

Disease symptomComplete phyllodyComplete phyllodyComplete phyllodyComplete phyllodyPartial phyllodyPartial phyllodyPartial phyllodyPartial phyllodyNormal seed


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Floral infection by zoospores o f p earl millet do wny mildew 573

+ 2C, and with summer daylight, whichly exceeded 50000 lux. After approx imate lydays, diseased leaves could be seen on oosp ore-Zoospore inoculum was prepared in humidows. Petri dishes and their lids (9

sterilized by wip-with paper towels soaked in 70% alcohol an d

1 h

ed in glass beakers. The zoospore concentra-

with sterile tap water.

ated section of the glasshouse to avoid anybility of either cross-infection or c onta mina-

R E S U L T SEffect of the pathogen on inflorescencedevelopmentResults are summarized in Table 1. Completemalformation of the inflorescence occurred wheninoculation w as carried out during panicle initia-tion, branch primordia initiation, spikelet pri-mordia initiation and differentiation into theempty glume (t|-t4. Fig, la). The malformedtissues supported asexual sporulation underfavourable environmental conditions and occa-sionally produced oospores. Infection duringpanicle initiation (ti) caused proliferation ofvegetative tissues (phyllody) without furtherfloral differentiation. Inoculation at branch pri-mordia initiation, spikelet primordia initiationand differentiation into the empty glume (t;-t4)resulted in growth of abnormal appendages inspikelets within 5-7 days of inoculation. No seeddeveloped on plants inoculated during thesestages of floral development. Infection by zoo-spores was by direct penetration of host tissuesfollowed by formation of vesicles that producedintracellular m ycelium within 12 h. In tercellularmycelium with finger-like ha ust od a was observedwithin 48 h. with systemic colonization of hosttissues both by intercellular and intracellularhyphae. Externally both the flag leaf and theadjacent leaf consistently showed basal chlorosisand asexual sporulation.

Inoculation during spikelet differentiation intothe stamen primordia. pistil primordia andgynoecial development stages (ts-tj) resulted inpartial m alformation of the panicle with some ofthe seed setting normally (Fig. lb). Unaffectedareas of inoculated spikelets completed seeddevelopment either as distinct areas of the panicleor scattered among the diseased spikelets. Nofurther differentiation of floral organs occurredon infected spikelets. which appeared malformedat emergence of the panicle. With inoculations atstage t; (spikelet differentiation into the ovulescarpels), malformations were confined to thesefloral organs, the rest having already differen-tiated normally (Fig, Ic), Stigmas, althoughmalformed, were still recognizably bifurcate atpanicle emergence. The anthers that had differen-tiated very early in inflorescence ontogeny werefound to be normal.


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574 S. T. Sentisi and S. F. L. Ball


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Floral infection by zoospores o f pearl millet downy mildew 575more elongated ovaries and styllodia, and stig-mas which always emerged 2 4-48 h before thosein normal spikelets. No seed developed fromdiseased spikelets. In addition, inoculation at thelate boo t stage caused sterile florets to form at thetip of the panicle (Fig. Id). Norm al seed develop-ment frequently occurred on the basal half of thepanicles due to the fact that the inoculum hadf"ailed to reach these areas. When the pathogenwas specifically in troduce d to the b asal part of thepanicles and not to the tips, malformationsoccurred on the Horets in this area and not on theupper half of the panicles, which produced nor-mal seeds. Occasionally, where the inoculum hadrun down to the base of the panicle during theboot stage, malformed and/or sterile floretsoccurred along the inoculum pathway. Micro-scopic observations showed that, 48 h afterinoculation, mycelium was present in the ovarywalls with hypertrophied cell development.Within 7-10 days after inoculation, the ovariesbecame completely deformed and supportedasexual reproduction by the fungus. However,the vascular tissues in the pedicel remained intactand uncolonized.

No malformation of the floral organs occurredon plants inoculated during panicle emergencefrom the flag leaf (t9). Developing stigmasenclosed within the glumes were well protectedfrom the pathogen. Although the pathogen wasobserved to penetrate the glumes, no colonizationof other host tissues occurred. These plantsproduced normal seed at maturity.

Inoculation at full-protogyny (tio) did notcause malformation or phyllody. However, littleor no seed-set occurred on these plants. Within12-24 h after inoculation, brown lesions wereobserved on the tips of the stigmas of inoculatedpanicles. The whole stigmas became completelybrown and withered within 48-72 h of inocula-tion. At this stage, uninfected spikelets in co ntrolinflorescences showed normal, turgid, white stig-mas. Anthers emerged within the next 24-48 hand although pollen was available no seedsdeveloped from infected spikelets. The ovariesdisintegrated and eventually dehisced andbecame necrotic.

Florets that escaped infection and colonizationeveloped norm al seeds. Th roug hou t this investi-ation con trol plan ts for each floral development

zoospores of S. graminicola being able to infectthe floral organs of P. americanum. It has beensuggested that systemic infection of P. america-num seedlings by S. graminicota (from oosporesor zoospores) follows invasion of the apicalmeristem and colonization of undifferentiatedtissues with characteristic sym ptoms on the leavesand panicle phyllody (Williams. 1984), The workrecorded here shows that infection and diseasecould be localized to individual florets, 5, grami-nicola zoospores can germinate and penetrate theinflorescence at any developmental stage beforematurity. However, the extent of the resultingmalformation of host tissues was dependent uponthe developmental stage at infection, maturity ofthe host tissues and escape from infection clearlybeing two major factors in this context. Onlywhen all the tissues were fully differentiated didthe zoospores fail to colonize, as was shownduring full protogyny when there was a hyper-sensitive host reaction to pathogen invasion. Thishost reaction is the first recorded instance of S.graminicola inoculum causing direct damage tofully differentiated tissue of pearl millet.

Infection of individual spikelets on the pearlmillet panicle always resulted in phyllody. mal-formation and/or sterility, Sclerospo ra g ramini-cola colonized the tissue, which prevented furtherdifferentiation and caused malfonnation of dif-ferentiating organs or abortion of differentiatedtissues. These results suggest that transmission ofthis disease from the interior of the seed is veryunlikely. Host tissues were either colonized, inwhich case phyllody occurred with n o seed set. orlate infection of mature stigmas resulted innecrosis, destroying them as receptive organs forpollination. In the field, partially malformedpanicles are a common sight, with abnormalstigmas protruding from diseased florets inter-spersed with spikelets which have normal seed.This has been found even in parts of the S outhe rnSahel in Africa, where conditions are usually hotand dry and not particularly conducive to spreadof downy mildew. Clearly further field laboratoryinvestigations will be necessary to resolve howsuch symptoms are caused. Lack of full under-standing of the infection processes by this fungusis hamp ering the development of rational controlstrategies in spite of the great importance of thiscrop in the semi-arid regions The present workused only one susceptible cultivar but the same


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576 S. T. Semisi and S. F . L. BallA C K N O W L E D G E M E N T SSeed and pathogen were supplied by staff and co-operators from ICRISAT Center, Hyderabad,India and the Sahelian Center, Niamey, Niger.This study was carried out under MAFF licenceNo. PHF 149 117 (87) (S2(66), issued under theImport and Export (Plant Health) (Great Britain)Order 1980 and the Plant Pests (Great Britain)Order 1980. The work was completed by the firstauthor as part of his PhD programme funded bythe G TZ (SGC PP), and M inistry of Agriculture,Western Samoa. Thanks are extended to Dr J.Barnett for help and advice with the electronmicroscopy and to B. Thurley for her technicalassistance.R E F E R E N C E SBall S.L. (1983 ) Path ogenic variability of downy mildew

{Sclerospora graminicola) on pearl millet. I. H ostcultivar reactions to infection by different pathogenisolates. A nnals o f A pp l ied Bio logy 102, 257-264.

Coaldrake P.D. & Pearson C.J. (1986) Environmentalinfluences on panicle differentiation and spikeletn u m b e r of Pennisetum americanum. Journal of Exper-imental Botany 37, 865 875.

Francis S.M. & Williams R.J. (1983) Scterosporagrami-

nicola. CMI Descriptions of Pathogen ic Fun gi andBacteria No. 770 .

Kenneth R .G. (1981). Dow ny m ildews of graminaceouscrops. In The Downy Mildews (Ed. by D. M.Spencer), pp. 367-394. Academic Press, London.

King A.B. & Webster O.J. (1970) Downy mildew ofsorghum in Nigeria. Indian Phytopathology 23, 342-349.

Ong C.K. & Everard E. (1979) Short day induction offlowering in pearl millet (Pennisetum typhoides) an dits effect on p lant mo rpho logy. Experimental Agricul-ture 1 5 , 4 0 1 ^ 1 0 .

Safeeulla K. M. (1976) Biology and Con trol of the DownyMildews of Pearl Millet, Sorghum and Finger Millet.Wesley Press, Mysore, India.

Shetty, H .S. , M athur S.B. & Neergaard P. (1980)Sclerospora graminicola in pearl millet and itstransmission. Transactions of the British MycologicatSociety 74. 127-134.Singh S .D. & W ilhams R.J. (1980) The role of sporangiain the epidemiology of pearl millet downy mildew.Phytopatho logy 70, 1187-1190.

Suryanarayana D. (1962) Occurrence of an unknownfungal mycelium inside the sound grains produced onpartly formed green ears of bajra plants. Science andCulture 28, 536.

Williams, R.J. (1984). Downy mildews of tropicalcereals. Advances in Plant Pathology 2. 1-103.


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