Integrated Control of Allium White Rot With Trichoderma

Plant Pathology

(2006)

55

375ndash386 Doi 101111j1365-3059200601389x

copy 2006 The Authors

375

Journal compilation copy 2006 BSPP

Blackwell Publishing Ltd

Integrated control of

Allium

white rot with

Trichoderma viride

tebuconazole and composted onion waste

J P Clarkson A Scruby A Mead C Wright B Smith and J M Whipps

Warwick HRI University of Warwick Wellesbourne Warwick CV35 9EF UK

Two isolates of

Trichoderma viride

(L4 and S17A) were assessed for biological control of

Allium

white rot (AWR) withdifferent onion accessions and cultivars alone and in combination with a tebuconazole-based seed treatment orcomposted onion waste In glasshouse tests 23 new bulb-onion accessions from previous work to detect resistanceto

Sclerotium cepivorum

showed no differences in susceptibility to AWR but when combined with S17A disease wasreduced by up to two-thirds over all accessions

Trichoderma viride

L4 and S17A also reduced the proportion of infectedplants for five commercial bulb-onion cultivars and one advanced breeding line by at least one-third Further glasshousetests using a salad-onion cultivar showed that L4 S17A tebuconazole or composted onion waste controlled AWR andat least halved the proportion of diseased plants Combination treatments of

T viride

with either tebuconazole or com-post enhanced control and in some treatments disease was almost eliminated In field trials control of AWR by S17Awas significant for 17 out of 18 individual or mixed bulb-onion accessions with disease reduced overall by more thanhalf In another field experiment S17A failed significantly to reduce AWR for two out of three commercial bulb-onioncultivars while tebuconazole reduced the final proportion of AWR-infected plants over all cultivars from 0middot47 to 0middot09Combining S17A and tebuconazole resulted in a similar level of AWR to using tebuconazole alone The use of

T viride

in an integrated strategy with other treatments to enhance control of

S cepivorum

is discussed

Keywords

bulb-onion conditioning fungicide sclerotia


soilborne plant pathogen

Introduction

Allium

white rot (AWR) caused by the sclerotium-formingfungus


is a major disease of

Allium

crops worldwide In the UK losses of 10 caused by AWRcost pound4middot3 million (Defra Horticultural Statistics 2004)Tebuconazole which has good efficacy against

S cepivo-rum

(Melero-Vara

et al

2000 Dennis 2001) is the onlyapproved fungicide for AWR control in the UK underthe lsquooff-labelrsquo scheme This allows the use of pesticideson some minor crops when manufacturers have soughtapproval only on major crops such as cereals Despiteefforts to find resistance to

S cepivorum

within existingcultivars and other

Allium

species (Utkhede

et al

1982Brix amp Zinkernagel 1992) there are no commercial

Allium

cultivars with sufficient resistance that currently can beexploited for AWR control Other potential control strat-egies for AWR have therefore been investigated includingsoil solarization (Melero-Vara

et al

2000 McLean

et al

2001) and application of diallyl disulphide (DADS) tostimulate sclerotial germination in the absence of a host

(Crowe

et al

1994 Hovius amp McDonald 2002) but theseapproaches are generally unsuitable for UK conditionsMore recently cruciferous residues (Smolinska 2000) andcomposted onion and other vegetable wastes (Coventry

et al

2005) have been shown to eradicate

S cepivorum

sclerotia and control AWR when incorporated intosoil but in the UK this type of approach has yet to be fullydeveloped by researchers and exploited by growers Thecontinuing reliance on tebuconazole for the control ofAWR in the UK and elsewhere and the increasing demandfor reduced fungicide inputs has also led to a search forbiological control agents (BCAs) of

S cepivorum

SuccessfulAWR control has been demonstrated both in pot trials andin the field using

Trichoderma

spp

Coniothyrium minitans

and

Sporidesmium sclerotivorum

(Adams amp Ayers 1981Abd-El-Moity

et al

1982 Kay amp Stewart 1994a Gerlagh

et al

1996 Clarkson

et al

2002 2004) However as sooften observed with biological control results in the fieldwith AWR can be variable (Entwistle 1988) Coley-Smith(1987) suggested that this may be caused by prevailingenvironmental conditions soil type and the origins of boththe BCA and the

S cepivorum

isolateIn previous work it was shown that two isolates of

Trichoderma viride

(L4 and S17A) degraded sclerotia fromfour different

S cepivorum

isolates and in four different

E-mail johnclarksonwarwickacuk

Accepted 5 December 2005

Plant Pathology

(2006)

55

375ndash386

376

J P Clarkson

et al

soil types and controlled AWR in the field (Clarkson

et al

2002 2004) However although

T viride

L4 and S17Acould reduce AWR reproducibly disease levels weregenerally reduced by only

c

50 and the degree ofcontrol was variable both of which would be unacceptableto growers One approach to address this problem is tointegrate the use of

T viride

with other methods of AWRcontrol and determine if these combinations are moreeffective (Entwistle 1988) This is a promising approach asshown by two studies that have investigated this strategyIn the first study the use of DADS was combined with thefungicide tebuconazole which was applied with a limetreatment and drilled with the onion seeds (Dennis 2001)DADS and tebuconazole reduced AWR incidence from76 (untreated) to 44 and 37 respectively whereasthe combined treatment reduced disease further to just11 In the second study an iprodione-tolerant strain of

T harzianum

was combined with the fungicide which wasapplied in-furrow (Abd-El-Moity

et al

1982)

Trichodermaharzianum

and iprodione reduced AWR incidence from86 (untreated) to 13 and 51 respectively while thecombined treatment resulted in 6 disease

The aim of this work was to confirm

T viride

L4 andS17A as BCAs of AWR when applied alone and also toevaluate potential improved efficacy when combined witha tebuconazole-based fungicide seed treatment or com-posted onion waste In addition new bulb-onion accessionsderived from plants from previous experiments to detectresistance to

S cepivorum

(Entwistle 1990) were alsoassessed for effects on AWR and compared with a com-mercial cultivar Combinations of

T viride

with these newbulb accessions and current commercial cultivars werealso tested The effect of combining

T viride

with theseother control treatments for an integrated approach tocombat AWR has not been assessed previously

Materials and methods


isolates and production of sclerotia

The isolate of

S cepivorum

used in this study (code Kirton)was obtained as sclerotia from an infected onion bulb froma white rot-infested field site near Kirton Lincolnshire UK

Cultures of

S cepivorum

were obtained after first surface-sterilizing sclerotia [washing in sodium hypochlorite solution(15 available chlorine Hays Chemicals) for 90 s followedby washing three times in sterile distilled water (SDW)] andthen squashing onto potato dextrose agar (PDA Oxoid) in9-cm Petri dishes Cultures were incubated at 20

deg

CTo obtain large numbers of sclerotia for experiments

2-week-old PDA cultures of

S cepivorum

were cut into

c

3-mm

2

pieces and used to inoculate mushroom spawnbags (Biscof amp Klein one Petri dish culture per bag) con-taining a mixture of washed sand (1950 g lt2 mm particlesize Hepworth Minerals and Chemicals) ground maizemeal (80 g Midland Shires Farmers Ltd) and water (350mL) which had been sterilized by autoclaving at 0middot1 MPaand 121

deg

C for 15 min The bags were sealed and incubated

at 18

deg

C for 6 weeks after which sclerotia had formedSclerotia were harvested by flotation in water retrievalin a sieve (212

micro

m mesh diameter) and then dried in anairflow cabinet for 12 h before storage at 5

deg

C The sclerotiaused in onion seedling bioassays were further subjected tolsquoconditioningrsquo to overcome constitutive dormancy (Coley-Smith

et al

1987) This consisted of burial in mesh bagsfor at least 12 weeks in a quarantine field at WarwickHRI After this period sclerotia were washed sieved anddried as before then gently rubbed on a sieve (212

micro

mmesh diameter) to remove any that were soft and degraded

Trichoderma

isolates

The two isolates of

T viride

(IMI 386638 code S17A IMI386639 code L4) used in this study had previously beenselected as BCAs of

S cepivorum

based on their ability todegrade sclerotia in soil and control AWR in glasshouseonion seedling bioassays (Clarkson

et al

2002 2004)

Trichoderma viride

L4 and S17A were also shown to reduceAWR in the field (Clarkson

et al

2002) The

T viride

isolateswere originally isolated from parasitized

S cepivorum

sclerotia cultured on PDA at 20

deg

C under fluorescentlighting with a 12-h daylength and stored in liquid nitrogenSpore suspensions were obtained by adding 20 mL SDWto 3-week-old PDA cultures and scraping gently with aspatula

Wheat bran cultures of

T viride

for use in glasshouseexperiments were obtained by inoculating 250-mL flaskscontaining wheat bran (12 g) and water (30 mL) sterilizedby autoclaving at 0middot1 MPa and 121

deg

C for 30 min withthe spore suspensions (5 mL) and incubating for 3 daysat 20

deg

C This formulation was shown to stimulateproliferation of

Trichoderma

spp and was used effectivelywith BCAs of

S cepivorum

(Kay amp Stewart 1994aClarkson

et al

2002 2004)Alginate pellets of

T viride

S17A were produced foruse in field experiments following the method of Lewis ampPapavizas (1985) This dry-pellet formulation also allows

Trichoderma

spp to proliferate in soil and has been usedsuccessfully to deliver BCAs in several pathosystems(Lewis amp Papavizas 1987 Lewis

et al

1996 Wu ampHsiang 1998)

Trichoderma viride

biomass was producedby inoculating a yeast molasses medium [15 g molasses(United Molasses Ltd) 2middot5 g brewers yeast (Holland ampBarrett Ltd) 500 mL water] with 1 mL spore suspension(

c

1

times

10

6

spores mL

minus

1

) and incubating for 9 days at20

deg

C and 110 rpm Biomass (30 g) was then combinedwith 750 mL sodium alginate (Fisher Scientific) solution(26middot6 g L

minus

1

water) and 250 mL ground wheat bran suspen-sion (200 g L

minus

1

water) and the mixture pumped dropwiseinto a calcium chloride solution (5 g L

minus

1

water) Theresulting alginate pellets were dried under an airflow atroom temperature and stored at 5

deg

C until use

Glasshouse onion seedling bioassays

The effects on AWR of onion accessions and commercialcultivars a tebuconazole-based seed treatment and

Plant Pathology

(2006)

55

375ndash386


Allium

white rot

377

composted onion waste all with or without the additionof

T viride

were assessed in glasshouse onion seedlingbioassays as described previously (Clarkson

et al

20022004) The methodology was adapted according to thetest treatments Coarse loam soil (Dunnington HeathSeries Mackney

et al

1983) used in all the bioassays wassieved (4-mm mesh) and amended with medium-gradevermiculite (4 1 vv soil vermiculite William SinclairHorticulture Ltd) to improve structure and drainageConditioned sclerotia of

S cepivorum

were mixedthoroughly with the amended soil (one sclerotium g

minus

1

) andwhere used

T

viride

was added as 3-day-old wheat brancultures (2 g in 100 g soil) The soil mix was then addedto 7-cm pots and onion seeds were planted one in eachpot Experiments always included an inoculated controltreatment consisting of soil and sclerotia only Pots wereplaced in saucers so that all watering was from below ina cooled glasshouse at 15

deg

C Emerging onion plants wereassessed weekly for symptoms of white rot (yellowingand wilting plants) until a maximum level of disease wasreached in the inoculated control treatments after whichno further plants became infected In all the seedlingbioassays the individual treatments tested were alwaysapplied to groups of 10 pots which are referred to asplots or subplots when describing the design of eachexperiment

Effect of

T viride

with different bulb-onion accessions and commercial onion cultivars on AWR

A wide range of long-day (mainly Rijnsburger type) onionaccessions from the Warwick HRI (WHRI) GeneticResources Unit were screened in 1988ndash90 for theirresistancesusceptibility to AWR (Entwistle 1990)Surviving plants from these accessions which had beenranked as least susceptible to AWR were then half-sibmated in 1992 to produce eight new families (codedAC92006 AC92010 AC92016 AC92026 AC92038AC92059 AC92061 and AC92064) These families wereinitially tested for resistance to AWR in a preliminaryonion seedling bioassay in 2001 but problems with dete-rioration in seed quality and subsequent seedling vigourdictated a further round of seed multiplication Survivingseedlings from these eight accessions were thereforegrown to flowering in 2002 and again half-sib mated toproduce new progeny lines listed in Table 1 Onion seed-ling bioassays were then carried out to assess the responseto AWR of eight of these new accessions (AC02010AC02015 AC02023 AC02035 AC02046 AC02053AC02055 AC02061) and five combinations of accessions(AC02002 + AC02006 AC02009 + AC02011 AC02024+ AC02031 AC02033 + AC02037 AC02066 + AC02068)with and without the addition of

T viride

S17A in com-parison with the commercial bulb-onion cv Hystar Com-binations of accessions (both from the same parent) wereused where only small amounts of seed were producedThe limited seed stock and a requirement to test the sameaccessions in a field experiment also precluded testingthem with

T viride

L4 Three repeat experiments werecarried out each with two replicates A replicate was

divided into four blocks of five plots (total 20 plots) eachcontaining two subplots The 20 plots within each replicatecomprised one plot for each of the 13 accessions or groupsof accessions and seven plots for the cv Hystar controlwith subplots being either with or without

T viride

S17AThe allocation of accessions to blocks across the threerepeat experiments followed an alpha design with theallocation of the Hystar control constrained so that withineach block there were no more than two plots for thistreatment and ensuring that pairs of treatments occurredtogether within blocks as equally often as possible

Five commercial onion cultivars [White Lisbon (saladonion) Hysam Red Baron Summit and Renate (bulbonions)] and one advanced bulb-onion breeding line (codeSS1 developed to produce low-pungency lsquosweet onionsrsquo)were also tested for control of AWR in seedling bioassayseither alone or with the addition of

T viride

S17A or L4Two repeat experiments were carried out each compris-ing five replicate blocks of the 18 treatment plots arrangedin a randomized complete block design

Effect of

T viride and tebuconazole on AWRTrichoderma viride L4 and S17A were tested for controlof AWR in onion seedling bioassays both with andwithout a tebuconazole-based film-coated seed treatment(tebuconazole at 5 g kgminus1 thiram at 2 g kgminus1 carbendazimat 1 g kgminus1 and metalaxyl at 0middot7 g kgminus1 Elsoms Seeds) usingthe commercial salad onion cv White Lisbon White Lisbonwas used as it was a commercial standard in previous tests(Clarkson et al 2002 2004) and unlike daylength-sensitivebulb-onion cultivars allows tests to be carried out at any

Table 1 New bulb-onion accessions and their associated parent accessions tested for effects on Allium white rot

New accession Parent accession

AC02001 AC92006AC02002 AC92006AC02006 AC92006AC02008 AC92006AC02009 AC92010AC02010 AC92010AC02011 AC92010AC02015 AC92010AC02023 AC92038AC02024 AC92038AC02028 AC92038AC02031 AC92038AC02033 AC92059AC02037 AC92059AC02035 AC92059AC02039 AC92059AC02046 AC92061AC02053 AC92061AC02055 AC92061AC02061 AC92064AC02063 AC92064AC02066 AC92064AC02068 AC92064

Plant Pathology (2006) 55 375ndash386

378 J P Clarkson et al

time of year For comparison both tebuconazole-treatedand untreated onion seeds without the addition of T viridewere included as controls Two timings of T viride applica-tion were tested with wheat bran cultures being appliedeither 6 weeks before sowing or at sowing Inclusion ofthe presowing treatments was to allow for the possibilitythat the tebuconazole seed treatment might inhibit T virideand to determine if an earlier application of the BCAs mightimprove AWR control Before sowing pots containingsoil infested with S cepivorum sclerotia were amendedwith T viride L4 or S17A and inoculated control treat-ments (soil and sclerotia only) were also set up in a cooledglasshouse at 15degC These pots were watered from belowas required to keep the soil moist during the 6 weeks andat the end of this period untreated or tebuconazole-treatedonion seeds were sown At sowing further pots were setup with soil infested with the same batch of S cepivorumsclerotia (stored at room temperature in the laboratoryfor the 6 weeks) and amended with T viride L4 or S17AAgain an inoculated control treatment was included (soiland sclerotia only) and both treated and untreated seedswere sown for these and the T viride-amended pots Tworepeat experiments were carried out each comprisingfive replicate blocks of the 12 treatment plots arrangedfollowing a randomized complete block design

Effect of T viride and composted onion waste on AWRTrichoderma viride L4 and S17A were tested for controlof AWR both with and without composted onion waste inonion seedling bioassays using the commercial salad onioncv White Lisbon Small-scale composting of onion waste(peelings and chopped whole bulbs) was carried out afteraddition of urea (4 g kgminus1 waste) for 7 days in aerated2-L flasks immersed in thermostatically controlled waterbaths at 50degC as described by Coventry et al (2005)Onion compost must be incorporated in soil infested withsclerotia for at least 12 weeks before it is effective againstS cepivorum (Coventry et al 2005) Therefore beforethe seedling bioassays were set up in the pots the compostwas added to silty clay soil infested with S cepivorumsclerotia [one sclerotium gminus1 soil adjusted to 15 moisturecontent (minus0middot12 kPa)] at a rate of 50 50 vv in polythenebags (1middot1 kg soil per bag) and thoroughly mixed beforeincubation for 12 weeks at c 15degC in a cooled glasshouseAt the same time bags of soil (2middot2 kg per bag) and sclerotiaonly were also set up Trichoderma viride L4 or S17A wasadded to the infested soilcompost or infested soil onlyas wheat bran cultures (22 g per bag) either at the sametime as the composted onion waste or after 12 weeksThis was to test whether an earlier application of T virideat the same time as the compost would enhance controlcompared with a later application at planting Controltreatments were also included consisting of onion com-post and infested soil (no T viride) and infested soil only(no T viride no compost) After the 12-week incubationperiods the soilcompostT viride treatments from bagswere dispensed into pots and onion plants (cv WhiteLisbon c 20 cm tall) were transplanted (one plant perpot) Transplants were used rather than seeds as it was

shown that composted onion waste can be phytotoxic togerminating seedlings (Coventry et al 2005) Two repeatexperiments were carried out each comprising five replicateblocks of the 10-treatment plots arranged following arandomized complete block design

Field experiments

Field experiments were carried out in 2003 and 2004in a quarantine field at Warwick HRI Wellesbourne UKIn March of each year the beds in the experimental areawere infested with conditioned sclerotia of S cepivorumfrom four different isolates (Kirton 5675 15575 and16675) raked into the top 5 cm of the seedbed at a rateof c 50 000 mminus2 before sowing of bulb-onions (Clarksonet al 2002) In all experiments onion plants were assessedeach week for typical AWR symptoms of wilting or leafyellowing until the foliage naturally senesced just beforethe time when bulbs would normally be harvested

Effect of T viride with different bulb-onion cultivars on AWRIn 2003 a field experiment was carried out to test T virideL4 and S17A for control of AWR with four commercialbulb-onion cultivars Hystar Red Baron Renate and SS1Trichoderma viride was applied as 3-day-old wheat brancultures suspended in a guar gum (J A amp P E Wright) gel(240 g wheat bran culture 50 g guar gum 1 L water)directly into the furrow with the onion seeds via tubes ineach coulter by peristaltic pumps driven by the landwheels of a modified Stanhay drill This fluid drill systemwas previously used successfully to apply Trichodermain field experiments (Clarkson et al 2002) The systemdelivered 7ndash10 mL BCA suspension mminus1 of row of bulb-onion seeds which were drilled in four rows 35 cm apartin beds 1middot83 m wide The tebuconazole-based seedtreatment was included for comparison with the T viridetreatments only for cv Hystar while untreated controltreatments (no T viride no fungicide) were included forall the onion cultivars Fungicide-treated and untreatedcontrol seeds were both drilled with guar gum in wateronly Each treatment was replicated four times and thetrial was effectively arranged as a split-plot design withcultivars allocated to plots and the T viride and controltreatments applied to subplots within these plots Theallocation of cultivars to plots followed a Latin squaredesign Each subplot consisted of a 6-m length of a four-row bed and every week all onion plants in the middletwo rows (c 60 plants per row) were assessed for AWRsymptoms of wilting and leaf yellowing Infection byS cepivorum was confirmed by examining the roots andstem base for mycelium and sclerotia

Effect of T viride with different bulb-onion accessions and cultivars or tebuconazole on AWRIn 2004 two field experiments were carried out in thequarantine field to test the effect on AWR of T virideS17A combined with different onion accessions andcultivars or the tebuconazole seed treatment A cone drill


Integrated control of Allium white rot 379

was used for sowing as it did not require large amountsof seed to operate (unlike the Stanhay drill used in 2003)and delivered all the added seed to a specific length of rowThis was necessary because of the limited seed stockavailable for the bulb-onion accessions In order to applyT viride S17A with the onion seed using the cone drilla dry-pellet formulation was required rather than the guargumwheat bran formulation used before Alginate pelletsof T viride were therefore produced as described earlierand applied in-furrow at a rate of 2 g mminus1 row In bothfield experiments onion seeds were sown c 10 cm apart

The first field experiment assessed AWR developmenton 18 bulb-onion accessions or combinations of accessionsfrom the WHRI Genetic Resources Unit both with andwithout T viride S17A These accessions wereAC02001 AC02008 AC02010 AC02015 AC02023AC02028 AC02035 AC02039 AC02046 AC02053AC02055 AC02061 AC02063 AC02002 + AC02006AC02009 + AC02011 AC02024 + AC02031 AC02033 +AC02037 and AC02066 + AC02068 The commercial cvHystar was also included (again with and without S17A)as a control treatment for comparison The trial was arrangedin three replicates with each replicate divided into twoblocks one with T viride S17A and one without and witheach block divided into six plots Plots contained fourrows 3middot5 m long 35 cm apart in beds 1middot83 m wide witheach row drilled with a different accession group ofaccessions or the cv Hystar control Treatments were allo-cated to plots following an alpha design with each blockcontaining one row for each of the 18 accessions orgroups of accessions and six rows for the Hystar controlThe allocation of the Hystar control rows was constrainedso that each plot contained exactly one of these rowsThe alpha design ensured that pairs of accessions or groupsof accessions occurred together within plots as equallyoften as possible All onion plants in every row (c 35 plantsper row) were assessed for AWR symptoms each week

The second field experiment assessed AWR developmenton the commercial bulb-onion cvs Hystar Red Baronand Renate with T viride S17A the tebuconazole seedtreatment described before or a combination of bothUntreated controls (no T viride no fungicide) were alsoincluded for each cultivar The trial was arranged as anextended Trojan square with five main rows and four maincolumns and with three plots within each main-rowmain-column combination Each main-rowmain-columncombination contained one plot for each cultivar withthe allocation of the four treatment combinations ofplusmnT viride S17A and plusmntebuconazole seed treatment beingsuch that each treatment combination for a particularcultivar occurred with a main-rowmain-column com-bination with each of the four treatment combinationsfor the other two cultivars This design provided the bestcomparison of the 12 cultivarndashtreatment combinationswhile allowing for spatial variability in the levels of AWRinfection Each plot again consisted of four rows 3middot5 m long35 cm apart in beds 1middot83 m wide All onion plants in themiddle two rows of each plot (c 35 plants per row) wereassessed for AWR each week

Statistical analyses

The efficacy of treatments tested in each of the glasshouseonion seedling bioassays and each of the field trialswas assessed by calculating the number of seedlings withwhite rot as a proportion of the number emerged for eachassessment date for each plot These proportions wereanalysed using a generalized linear model (GLM) approachassuming a binomial distribution and logit link functionand allowing for overdispersion For each trial the effectwas estimated of each treatment or treatment combina-tion relative to appropriate controls and the predictedproportion of seedlings with white rot was obtainedfor each treatment or treatment combination in each trialThe accumulated analysis of deviance summarized thesignificance (using an approximate F-test) of differences inthe effect of each treatment tested Estimates on the logitscale for the differences between individual treatmentsand appropriate controls were extracted from eachanalysis together with appropriate standard errors Over-all effects of T viride isolates for treatments where theBCAs were used alone or with other treatments were alsoextracted from combined data sets One-sided t-tests werethen performed on these logit estimates to determine forwhich treatments or treatment combinations significantreductions in the proportions of seedlings with whiterot had occurred compared with the appropriate controlsA more negative logit coefficient indicated a greater treat-ment efficacy Where a treatment resulted in the completeabsence of AWR the GLM approach generally producedboth a large negative estimate of the difference betweenthis treatment and the appropriate control and usuallya larger standard error for this estimate The one-sidedt-test for this effect therefore generally indicated a non-significant probability but assuming that the level ofAWR for the inoculated control (soil and S cepivorumsclerotia only) was substantially greater than zero itwas always concluded that the treatment resulted in apractically significant reduction in AWR levels Althoughanalyses were performed for all assessment dates for bothglasshouse and field experiments only data from thefinal assessments are presented here for clarity Thiswas when the disease levels reached a maximum in theinoculated control treatments and no further plants becameinfected

For the glasshouse seedling bioassays testing onionaccessions initial analyses showed little evidence fordifferent levels of variation between and within plots sothe full design structure was not used in the analysis Eachof the three experiments was analysed taking accountonly of variation between replicates and between blockswithin replicates and the variation caused by accessionswas assessed using the between-plot variability For theother glasshouse bioassays the full design structures wereused in the data analysis

For the 2003 field trial two separate analyses wereperformed for each assessment date The first consideredall four cultivars and the three common treatments(untreated control T viride L4 and S17A) and allowed



for variation between main rows and main columns andthe interaction between these blocking factors with themain effect of cultivar assessed at the between-main-plot stratum (main plot being a main-rowmain-columncombination) and the main effect of treatment and thecultivarndashtreatment interaction assessed at the within-main-plot stratum The second analysis considered onlythe cv Hystar plots and thus assumed a randomizedcomplete block design For the 2004 accession field trialthere was evidence of more variation between the mainplots than between plots within main plots thus theanalyses allowed for variation between replicates blocksand main plots with the overall effect of T viride assessedat the between-block stratum and the main effect ofaccession and the interaction between accessions andT viride assessed at the within-main-plot stratum For the2004 onion cultivarT viridetebuconazole field trialthe analysis allowed for differences between main rowsmain columns and the interaction between these factorswith all treatment effects assessed at the within-main-plotstratum

Results


Disease-progress curves for all treatments followed anasymptotic form in all seedling bioassays and the resultsand analyses presented here correspond to the pointwhere AWR levels were at a maximum for the inoculatedcontrol treatments and no further plants became infectedThis was when treatment effects were also generally ata maximum This time varied between 13 and 22 weeksafter sowing or transplanting

Effect of T viride and different bulb-onion accessions and commercial onion cultivars on AWRThe new bulb-onion accessions showed a wide range ofAWR levels within each experiment (final proportion ofinfected plants for all three experiments was 0ndash0middot9 after15ndash21 weeks data not shown) but there was no consist-ent effect of any of the accessions over all the experimentsThe only accessions or combination of accessions thatresulted in significantly less AWR than the commercial cvHystar used for comparison were AC02053 in the firstexperiment and AC02015 AC02061 and AC02009 +AC02001 in the third experiment (P lt 0middot05) The major-ity of accessions were therefore at least as susceptible toAWR as Hystar When the accessions were planted in soilamended with T viride S17A AWR was reduced consist-ently compared with inoculated control plants (finalproportion of infected plants for all three experiments0ndash0middot4 data not shown) when data from all the accessionswere combined this effect was significant in each of thethree experiments (P lt 0middot01)

In the glasshouse experiments with the commercialonion cultivars AWR reached a maximum level after11 weeks in experiment 1 and 14 weeks in experiment 2In both experiments T viride S17A and L4 significantly

reduced the final proportion of plants with AWR com-pared with inoculated control plants when data for allthe cultivars were combined (P lt 0middot001 Table 2) In thefirst experiment the proportion of diseased plants wasreduced from 0middot74 in inoculated controls to 0middot46 forT viride L4 and 0middot47 for T viride S17A in the secondexperiment the reductions were from 0middot61 to 0middot26 and0middot29 respectively For individual onion cultivars diseasewas reduced significantly compared with the inoculatedcontrol (P lt 0middot05) by T viride L4 and S17A in at least oneof the two experiments carried out Allium white rot wasalso reduced in the few treatments where effects were notsignificant In the absence of T viride L4 or S17A therewere no significant differences detected between AWRlevels for any of the commercial cultivars (P gt 0middot05)

Effect of T viride and tebuconazole on AWRAllium white rot reached a maximum level in the inocu-lated control treatments after 13 weeks in experiment 1and after 22 weeks in experiment 2 In the absence oftebuconazole T viride L4 and S17A significantly reducedthe final proportion of onion plants with AWR comparedwith the inoculated control plants (no T viride no tebu-conazole) in both experiments and irrespective of the timeof T viride application (P lt 0middot001 Table 3) No improve-ment in AWR control was evident for T viride L4 or S17Awhen applied 6 weeks before sowing compared withapplication at sowing Over both experiments for thepresowing T viride treatments the final proportion ofinfected plants was reduced from 0middot7ndash0middot8 (inoculatedcontrols) to 0middot23ndash0middot55 For the T viride treatments appliedat sowing the final proportion of infected plants wasreduced from 0middot53ndash0middot55 (inoculated controls) to 0middot02ndash0middot17 In the absence of T viride the tebuconazole seedtreatment also significantly reduced AWR (P lt 0middot01Table 3) with the proportion of diseased plants reducedto 0middot26ndash0middot39 In both experiments the combinationtreatments of T viride and tebuconazole resulted in evenlower proportions of AWR-diseased plants (0ndash0middot31)compared with inoculated controls (P lt 0middot001 Table 3)and hence were generally better than using either T virideor tebuconazole alone This improved control by com-binations compared with individual treatments wassignificant for those combinations listed in Table 4 A signi-ficant increase in AWR levels was also observed in bothexperiments for the T viride treatments applied 6 weekspresowing compared with the same treatments set up atsowing in the absence of the tebuconazole seed treatment(P = 0middot01) In the presence of the tebuconazole seed treat-ment a significant increase for the T viride treatmentsapplied 6 weeks presowing compared with the sametreatment set up at sowing was observed in the secondexperiment (P = 0middot05) Data analysis for the inoculatedcontrols for these data sets showed that this observedAWR increase was close to significant in experiment 1(P = 0middot057) and significant in experiment 2 (P lt 0middot001) inthe absence of the tebuconazole seed treatment but non-significant (P gt 0middot05) in the presence of the tebuconazoleseed treatment



Table 2 Effect of Trichoderma viride L4 and S17A on Allium white rot for six onion cultivars in glasshouse onion seedling bioassays

Treatmenta

Experiment 1 Experiment 2

Proportion AWRb Logit Cc Proportion AWRb Logit Cc

IC all cultivars 0middot74 0middot61L4 all cultivars 0middot46 minus1middot25 (0middot20) 0middot26 minus1middot50 (0middot19)S17A all cultivars 0middot47 minus1middot22 (0middot19) 0middot29 minus1middot37 (0middot18)

White Lisbon IC 0middot80 0middot61White Lisbon L4 0middot60 NS minus0middot98 (0middot53) 0middot17 minus2middot09 (0middot51)White Lisbon S17A 0middot45 minus1middot60 (0middot54) 0middot31 minus1middot28 (0middot44)

Hysam IC 0middot65 0middot53Hysam L4 0middot47 NS minus0middot78 (0middot44) 0middot30 minus0middot99 (0middot45)Hysam S17A 0middot38 minus1middot14 (0middot46) 0middot30 minus0middot99 (0middot44)

Red Baron IC 0middot80 0middot69Red Baron L4 0middot50 minus1middot40 (0middot49) 0middot32 minus1middot58 (0middot46)Red Baron S17A 0middot48 minus1middot50 (0middot48) 0middot27 minus1middot79 (0middot47)

Summit IC 0middot82 0middot64Summit L4 0middot44 minus1middot76 (0middot49) 0middot25 minus1middot69 (0middot46)Summit S17A 0middot65 NS minus0middot90 (0middot52) 0middot24 minus1middot73 (0middot47)

Renate IC 0middot62 0middot51Renate L4 0middot36 minus1middot07 (0middot45) 0middot22 minus1middot30 (0middot46)Renate S17A 0middot42 NS minus0middot83 (0middot44) 0middot15 minus1middot79 (0middot51)

SS1 IC 0middot76 0middot67SS1 L4 0middot39 minus1middot62 (0middot50) 0middot33 minus1middot43 (0middot45)SS1 S17A 0middot43 minus1middot48 (0middot47) 0middot48 NS minus0middot80 (0middot43)

aCombination of T viride isolate and onion cultivar IC = inoculated control (no T viride) L4 S17A = T viride All cultivars = overall effect of treatments for all the onion cultivarsbFinal proportion of onion seedlings with AWR from the generalized linear model analysis after 11 weeks (experiment 1) and 14 weeks (experiment 2) Significance of reduction compared with inoculated control for each onion cultivar P lt 0middot001 P lt 0middot01 P lt 0middot05 NS = not significantcLogit coefficient A more negative value indicates greater efficacy of T viride isolates compared with controls Standard errors (68 df) in parentheses

Table 3 Effect on Allium white rot of Trichoderma viride (L4 or S17A) and tebuconazole seed treatment alone or in combination in glasshouse onion seedling bioassays

Treatmenta



T viride applied 6 weeks presowingIC 0middot71 0middot84L4 0middot23 minus2middot16 (0middot51) 0middot55 minus1middot48 (0middot50)S17A 0middot37 minus1middot46 (0middot45) 0middot45 minus1middot90 (0middot50)IC + tebuconazole 0middot26 minus1middot95 (0middot47) 0middot39 minus2middot15 (0middot50)L4 + tebuconazole 0middot05 minus3middot94 (0middot80) 0middot16 minus3middot41 (0middot57)S17A + tebuconazole 0middot05 minus3middot97 (0middot80) 0middot31 minus2middot52 (0middot52)

T viride applied at sowingIC 0middot55 0middot53L4 0middot02 minus4middot02 (1middot05) 0middot17 minus1middot76 (0middot49)S17A 0middot09 minus2middot60 (0middot60) 0middot16 minus1middot81 (0middot51)IC + tebuconazole 0middot29 minus1middot12 (0middot43) 0middot26 minus1middot23 (0middot46)L4 + tebuconazole 0middot00d minus10middot20 (13middot40) 0middot02 minus4middot00 (1middot05)S17A + tebuconazole 0middot10 minus2middot42 (0middot56) 0middot06 minus2middot91 (0middot67)

aIC = inoculated control (no T viride) L4 S17A = T viride applied either 6 weeks presowing or at sowingbFinal proportion of onion seedlings with AWR from the generalized linear model analysis after 13 weeks (experiment 1) and 22 weeks (experiment 2) Significance of the reduction compared with inoculated controls (IC at sowing or IC 6 weeks presowing) for different treatments P lt 0middot001 P lt 0middot01 P lt 0middot05 NS = not significantcLogit coefficient A more negative value indicates greater efficacy of T viride isolates andor the tebuconazole seed treatment compared with inoculated controls (IC at sowing or IC 6 weeks presowing) Standard errors (60 df) in parenthesesdTreatment resulted in no AWR for all replicates When the proportion of plants with AWR was zero the GLM analysis resulted in a large negative estimate and a larger standard error suggesting a nonsignificant comparison However this comparison was identified as of high practical significance



Effect of T viride and composted onion waste on AWRAllium white rot reached a maximum level in the inocu-lated control treatments after 16 weeks in experiment 1and 17 weeks in experiment 2 In the absence of compostedonion waste T viride L4 and S17A applied 12 weeks pre-planting to soil in bags or at planting in pots significantlyreduced (P lt 0middot001) the final proportion of onion plantswith AWR from 0middot47 to lt0middot18 in experiment 1 and from0middot94 to lt0middot18 in experiment 2 (Table 5) There was noapparent advantage in applying T viride 12 weeks beforeplanting rather than at planting In the absence of T viridethe composted onion waste also significantly reduced(P lt 0middot001) the final proportion of plants with AWR inboth experiments When T viride and composted onionwaste were combined even less AWR developed (finalproportion of plants infected lt0middot04) and hence diseasereduction was again significant compared with theuntreated control (P lt 0middot001 Table 5) The use ofcomposted onion waste combined with T viride therefore

generally improved AWR control compared with usingT viride alone or composted onion waste alone Howeversignificant additive effects were not detected becauseof the low disease levels in all the treatments The oneexception to this was for T viride S17A applied 12 weekspresowing in experiment 1 where disease levels werereduced significantly when combined with the compostcompared with using T viride S17A alone (P lt 0middot05)

Field experiments

Effect of T viride with different bulb-onion cultivars on AWREnvironmental conditions resulted in very low AWRlevels in the field experiment carried out in 2003 Onionswere sown on 31 March and final proportions of plantsinfected at the end of the experiment on 21 August (c 20weeks after sowing) for the untreated control plants wereonly 0middot21 0middot19 0middot17 and 0middot19 for Hystar Red Baron

Table 4 Trichoderma viride treatments where Allium white rot was significantly reduced when combined with tebuconazole seed treatment compared with using T viride or tebuconazole alone in glasshouse onion seedling bioassays

ExperimentAWR less than using T viride L4 or S17A alone P

AWR less than using tebuconazole alone P

1 L4 at 6 weeks presowing lt0middot05 L4 at 6 weeks presowing lt0middot01S17A at 6 weeks presowing lt0middot001 S17A at 6 weeks presowing lt0middot01

S17A at sowing lt0middot05

2 L4 at 6 weeks presowing lt0middot001 L4 at 6 weeks presowing lt0middot01L4 at sowing lt0middot05 L4 at sowing lt0middot01


Table 5 Effect on Allium white rot of Trichoderma viride (L4 or S17A) and composted onion waste alone or in combination in glasshouse onion seedling bioassays

Treatmenta



IC 0middot47 0middot94IC + compost 0middot04 minus3middot02 (0middot76) 0middot04 minus6middot02 (0middot84)

T viride applied 12 weeks preplantingL4 0middot02 minus3middot85 (1middot03) 0middot16 minus4middot49 (0middot58)S17A 0middot18 minus1middot45 (0middot43) 0middot16 minus4middot49 (0middot58)L4 + compost 0middot02 minus3middot85 (1middot03) 0middot00d minus14middot90 (36middot10)S17A + compost 0middot04 minus3middot03 (0middot76) 0middot00d minus14middot90 (36middot10)

T viride applied at plantingL4 0middot02 minus3middot85 (1middot03) 0middot18 minus4middot34 (0middot57)S17A 0middot06 minus2middot70 (0middot64) 0middot18 minus4middot34 (0middot57)L4 + compost 0middot00d minus10middot90 (21middot90) 0middot00d minus14middot90 (36middot10)S17A + compost 0middot02 minus3middot84 (1middot03) 0middot00d minus14middot90 (36middot10)

aIC = inoculated control (no T viride no compost) L4 S17A = T viride compost = composted onion waste (no T viride) Compost incorporated in infested soil 12 weeks before planting Trichoderma viride applied at compost incorporation (12 weeks preplanting) or at plantingbFinal proportion of onion seedlings with AWR from the generalized linear model analysis after 16 weeks (experiment 1) and 17 weeks (experiment 2) Significance of the reduction for each treatment compared with the inoculated control (IC) P = 0middot001 P = 0middot01 P = 0middot05 NS = not significantcLogit coefficient A more negative value indicates greater efficacy of the treatment compared with IC Standard errors (41 df) in parenthesesdTreatment resulted in no AWR for all replicates When the proportion of plants with AWR was zero the GLM analysis resulted in a large negative estimate and a larger standard error suggesting a nonsignificant comparison However this comparison was identified as of high practical significance



Renate and SS1 respectively Trichoderma viride L4 andS17A reduced AWR in all treatments (final proportion ofinfected plants lt0middot12) except for S17A with Red Baronand L4 with Renate When data for all cultivars werecombined the overall effects of T viride L4 and S17A onreducing AWR compared with untreated plants weresignificant (P lt 0middot05) but for individual cultivars the onlytreatments that reduced disease significantly were L4 andS17A with SS1 (P lt 0middot05 data not shown) The tebucona-zole seed treatment applied to cv Hystar also reducedAWR compared with the untreated control (no T virideno tebuconazole) but again this effect was not significant

Effect of T viride with different bulb-onion accessions cultivars and tebuconazole on AWRIn 2004 onions for both experiments were sown on 31March and final assessments for AWR were made on 9August c 23 weeks after sowing

In the first field experiment testing different bulb-onionaccessions in 2004 the final proportion of plants withAWR varied between 0middot59 and 0middot87 for untreated plantswith no significant difference in disease levels (P gt 0middot05)compared with the commercial cv Hystar Trichodermaviride S17A significantly (P lt 0middot05) reduced the finalproportion of plants with AWR for every accession orcombination of accessions compared with the untreatedcontrol with the exception of accession AC02008(Table 6) Hence the overall effect of T viride S17A wasalso significant (P lt 0middot001) compared with the untreatedcontrol when the data from all accessions or groups ofaccessions were combined

In the second field experiment testing the commercialbulb-onion cultivars in 2004 the final proportion ofinfected plants was 0middot49 0middot57 and 0middot36 for Hystar RedBaron and Renate respectively in the absence of bothT viride S17A and the tebuconazole seed treatmentWhen the data for all cultivars were combined the overallreduction in disease resulting from T viride S17A alonecompared with the untreated control was not significantAllium white rot was however reduced with T virideS17A alone compared with the untreated control forcvs Hystar and Red Baron (Table 7) but this was onlystatistically significant for Red Baron (P lt 0middot05) Therewas no reduction of AWR by T viride S17A with cv

Table 6 Effect on Allium white rot of Trichoderma viride S17A for different onion accessions in the field

Accessioncultivara

Proportion AWR without T viride S17Ab

Proportion AWR with T viride S17Ac Logit Cd

All accessions 0middot73 0middot31 minus1middot98 (0middot35)

Hystar 0middot66 0middot33 minus1middot49 (0middot41)AC02001 0middot66 0middot30 minus1middot62 (0middot63)AC02008 0middot67 0middot54 NS minus0middot61 (0middot61)AC02010 0middot67 0middot36 minus1middot41 (0middot59)AC02015 0middot69 0middot26 minus2middot00 (0middot62)AC02023 0middot73 0middot39 minus1middot60 (0middot59)AC02028 0middot59 0middot29 minus1middot37 (0middot52)AC02035 0middot66 0middot36 minus1middot35 (0middot63)AC02039 0middot68 0middot29 minus1middot82 (0middot60)AC02046 0middot74 0middot43 minus1middot42 (0middot57)AC02053 0middot78 0middot35 minus2middot08 (0middot57)AC02055 0middot78 0middot40 minus1middot85 (0middot62)AC02061 0middot69 0middot23 minus2middot21 (0middot56)AC02063 0middot75 0middot34 minus1middot91 (0middot61)AC02002 + AC02006 0middot73 0middot40 minus1middot55 (0middot59)AC02009 + AC02011 0middot87 0middot39 minus2middot55 (0middot62)AC02024 + AC02031 0middot75 0middot36 minus1middot83 (0middot53)AC02033 + AC02037 0middot71 0middot32 minus1middot81 (0middot53)AC02066 + AC02068 0middot75 0middot24 minus2middot46 (0middot63)

aBulb-onion accessions from Warwick HRI Genetic Resources Unit Hystar = standard commercial bulb-onion cultivar All accessions = overall effect of all the onion accessions with or without T viride S17AbFinal proportion of onion seedlings with AWR from the generalized linear model analysis after 23 weeks for accessions in the absence of T viride S17A (inoculated controls)cFinal proportion of onion seedlings with AWR from the generalized linear model analysis after 23 weeks for accessions when T viride S17A applied Significance of the reduction resulting from T viride compared with the inoculated control for each accession (no T viride S17A) P lt 0middot001 P lt 0middot01 P lt 0middot05 NS = not significantdLogit coefficient A more negative value indicates greater efficacy of T viride S17A compared with the inoculated control for each accession Standard errors (72 df) in parentheses

Table 7 Effect on Allium white rot of Trichoderma viride S17A and tebuconazole seed treatment alone or in combination for different onion cultivars in the field

Treatmenta Proportion AWRb Logit Cc

IC all cultivars 0middot47Tebuconazole all cultivars 0middot09 minus2middot25 (0middot39)S17A all cultivars 0middot37 NS minus0middot46 (0middot31)S17A + tebuconazole all cultivars 0middot15 minus1middot70 (0middot37)

Hystar IC 0middot49Hystar + tebuconazole 0middot07 minus2middot60 (0middot57)Hystar S17A 0middot36 NS minus0middot58 (0middot49)Hystar S17A + tebuconazole 0middot11 minus2middot12 (0middot62)

Red Baron IC 0middot57Red Baron + tebuconazole 0middot11 minus2middot49 (0middot58)Red Baron S17A 0middot36 minus0middot91 (0middot48)Red Baron S17A + tebuconazole 0middot25 minus1middot46 (0middot53)

Renate IC 0middot36Renate + tebuconazole 0middot11 minus1middot59 (0middot54)Renate S17A 0middot38 NS 0middot09 (0middot47)Renate S17A + tebuconazole 0middot10 minus1middot65 (0middot55)

aIC = inoculated control (no T viride) S17A = T viride All cultivars = overall effect of treatments for all the cultivarsbFinal proportion of onion seedlings with AWR from the generalized linear model analysis after 23 weeks Significance of reduction for each treatment compared with the inoculated control (IC) for each cultivar P lt 0middot001 P lt 0middot01 P lt 0middot05 NS = not significantcLogit coefficient A more negative value indicates greater efficacy treatments compared with inoculated control Standard errors (29 df) in parentheses



Renate Combination treatments of T viride S17A andtebuconazole resulted in a significant reduction in AWRfor all cultivars compared with untreated plants as didthe tebuconazole treatments applied alone with the finalproportion of plants infected with AWR being lt0middot25Data analysis showed that the combination treatments forHystar and Renate reduced AWR significantly comparedwith T viride applied alone (P lt 0middot05) but this was notthe case for Red Baron However no additive effects weredetected of T viride in combination with tebuconazolecompared with using tebuconazole alone

Discussion

The potential of T viride L4 and S17A as BCAs of Scepivorum was clearly evident in this study as reductionsin AWR were observed consistently in the absence of anyother treatments in multiple glasshouse and field experi-ments This confirms the results of previous work and thegeneral reproducibility of the biological control activity ofthese isolates against S cepivorum (Clarkson et al 20022004)

New bulb-onion accessions and commercial cultivarstested showed no differences in susceptibility to AWRin glasshouse or field tests in the absence of T virideconfirming the difficulty encountered by other researchersin finding resistance to S cepivorum (Utkhede et al 1982Brix amp Zinkernagel 1992) There was therefore nopotential for enhanced control with T viride L4 or S17Abut the efficacy of the BCAs when combined with theseaccessions and cultivars remained remarkably consistentin the glasshouse bioassays so there was no indication thatplant genotype might affect the performance of T virideL4 or S17A In the field T viride S17A significantlyreduced AWR for 17 out of 18 different bulb-onion acces-sions or accession combinations in 2004 and althoughdisease was reduced for commercial cultivars in 2003and to a lesser extent in 2004 the level of control wasless consistent than in the glasshouse seedling bioassaysThese results highlight the problems encountered withusing BCAs in the field The observed variability is pro-bably caused by environmental heterogeneity or morecomplex influences driven by the demographic interac-tions of the pathogen root and microbial populations(Bailey et al 2004)

When T viride L4 or S17A was combined with atebuconazole-based seed treatment in glasshouse onionseedling bioassays at two different application times AWRcontrol was always improved compared with using eitherT viride or tebuconazole alone and this effect was signi-ficant for a number of treatments This additive effect ofTrichoderma and fungicide confirms the results of Abd-El-Moity et al (1982) where T harzianum and iprodionewere more effective against AWR in combination thanalone The reduced effect of T viride S17A in the field in2004 however meant that although the same com-bination treatments were effective they were comparablewith using tebuconazole alone and AWR control wasenhanced only compared with using T viride S17A alone

Nevertheless these results still suggest that the use ofT viride is potentially compatible with tebuconazole butfurther work on quantifying the effect of the fungicide onT viride L4 and S17A is now required In New ZealandMcLean et al (2001) showed that T harzianum (anothereffective BCA of S cepivorum) although sensitive totebuconazole in vitro was only partially suppressed by thefungicide in soil and populations of the fungus recoveredover time Similarly C minitans was demonstrated tobe compatible with iprodione for control of Sclerotiniasclerotiorum in glasshouse soil despite its sensitivity to thefungicide in agar tests (Budge amp Whipps 2001) In somecases therefore the soil can act as an effective bufferbetween BCAs and fungicides so the selection of fungicide-resistant antagonists of S cepivorum either by spontaneousmutation on selective media (Abd-El-Moity et al 1982)or UV mutation (Kay amp Stewart 1994b) is not necessarilyrequired for successful integration of the two controlmethods

Another approach to avoid potential incompatibilitybetween BCAs and fungicides is to apply them at differenttimes and this has been successful for integrated con-trol of Botrytis cinerea using Trichoderma on grape andtomato (Harman et al 1996 Moyano et al 2003) In thepresent glasshouse experiments T viride L4 and S17Awere applied either 6 weeks before or at the same time asthe tebuconazole-treated seeds were sown The presowingT viride treatments might have been expected to giveenhanced control compared with those at sowing as moretime was available for the BCAs to degrade and destroythe S cepivorum sclerotia and populations would beestablished before tebuconazole-treated seed was plantedin the combination treatments However there was noadvantage to this presowing application and AWR levelswere actually greater in all the T viride treatments appliedat this time and the equivalent inoculated control in bothexperiments In the presowing treatments S cepivorumsclerotia remained in moist soil for 6 weeks at c 15degCand this appears to have increased their infectivity com-pared with dry sclerotia from the same source stored inthe laboratory and used to infest soil for the treatments atsowing One explanation for this is that the sclerotia keptin moist soil in the glasshouse were being lsquoconditionedrsquoAlthough all sclerotia used in onion seedling bioassayswere given a minimum 12-week conditioning period inthe field to overcome constitutive dormancy as suggestedby other researchers (Coley-Smith et al 1987) it could bethat only a proportion of the sclerotia were effectivelyconditioned during this time and that a further period insoil in the glasshouse allowed additional sclerotia to over-come dormancy and cause more AWR This might alsoexplain the variation in maximum AWR level and thetime for this to occur for the inoculated control treatmentsin the onion seedling bioassays where batches of sclerotiaconditioned in the field at different times of year wereused However variability in onion plant growth rates asa result of changing light levels during the year may alsohave affected AWR progress One way to assess the potentialof S cepivorum sclerotia to germinate after periods of



conditioning in the field would be to use germinationstimulants such as onion extracts or DADS in a laboratorytest (Gerbrandy 1989) and hence determine their potentialinfectivity Although time-consuming such tests may helpeliminate variability in maximum AWR levels and times inonion seedling bioassays

The effects of environmental factors on conditioningS cepivorum sclerotia are poorly understood althoughGerbrandy (1989) showed that germination was morerapid at 15degC for sclerotia conditioned in soil at 5 and10degC than for those conditioned at 15ndash25degC This suggeststhat the conditioning of S cepivorum sclerotia buried inthe field may take longer than 12 weeks during warmerperiods of the year Work is currently under way to under-stand factors affecting dormancy in the S cepivorumisolate used in the present study

Composted onion waste was also effective in reducingAWR in onion seedling bioassays confirming the findingsof Coventry et al (2005) They initially thought that activityof the onion compost was related to sulphur-containingvolatiles causing S cepivorum sclerotia to germinate anddie in the absence of a host However their results alsosuggested that other factors such as stimulation of antag-onistic microorganisms might also be involved CombiningT viride L4 or S17A with the composted onion wastealmost completely eliminated AWR in onion seedlingbioassays but because both compost and T viride appliedalone were also extremely effective in these glasshouseexperiments it was not possible to detect any significantadditive effects In the absence of onion compost there wasalso no evidence to suggest that there was any advantagein applying T viride 12 weeks before planting comparedwith application at planting This indicates as for theglasshouse experiments with T viride and tebuconazolethat early application of the BCAs to allow more timefor degradation of S cepivorum is not required for AWRcontrol Combining T viride with composted onion wastetherefore shows potential in an integrated programme forcontrolling AWR Field experiments testing this approachwith composted onion waste and other potentially suppres-sive composts are currently under way Other compostssuch as cruciferous plant residues have also been found toreduce germination of S cepivorum sclerotia (Smolinska2000) and this has been attributed to the production ofisothiocyanates although other toxic sulphur-containingvolatiles are also produced (Gamliel amp Stapleton 1993)These compounds may weaken S cepivorum sclerotia andincrease their susceptibility to invasion by microorganismsif a BCA such as T viride was also present this shouldpotentially enhance the effect

Overall it has been demonstrated that use of theBCA T viride tebuconazole-treated seeds and compostedonion waste are all effective control measures for AWREffects of all these treatments were apparent at the endof long-duration glasshouse and field experimentsindicating that protection of onion plants was achievableover an extended period This was also demonstrated bythe observation that the effects of tebuconazole and theBCAs demonstrated on salad onions in shorter glasshouse

bioassays were also evident for full-season bulb-onion cropsin the field This suggests that these control treatmentswould be effective for use in a variety of different onion-production systems This is the first study to investigatecombining a BCA with tebuconazole or onion composttreatments and results indicate that this approach is aworthwhile strategy for enhancing control and over-coming potential problems with variability in the perform-ance of BCAs Further work on optimizing the timingof treatments testing a combination of all three controlmethods and investigating other potentially suppressivecomposts of S cepivorum should result in an effectiveintegrated strategy for controlling this disease

Acknowledgements

We would like to thank the Department for EnvironmentFood and Rural Affairs through project HH3204for financial support We would also like to thank TimCrowther for producing the bulb-onion accessions andEmma Coventry for providing the onion compost Thetechnical assistance of Clare Grant in the field experimentin 2003 is also acknowledged

References

Abd-El-Moity TH Papavizas GC Shatla MN 1982 Induction of new isolates of Trichoderma harzianum tolerant to fungicides and their experimental use for control of white rot of onion Phytopathology 72 396ndash400

Adams PB Ayers WA 1981 Sporidesmium sclerotivorum distribution and function in natural biological control of sclerotial fungi Phytopathology 91 90ndash3

Bailey DJ Kleczkowski A Gilligan CA 2004 Epidemiological dynamics and the efficiency of biological control of soil-borne disease during consecutive epidemics in a controlled environment New Phytologist 161 569ndash75

Brix HD Zinkernagel V 1992 Screening for resistance of Allium species to Sclerotium cepivorum with special reference to non-stimulatory resistance Plant Pathology 41 308ndash16

Budge SP Whipps JM 2001 Potential for integrated control of Sclerotinia sclerotiorum in glasshouse lettuce using Coniothyrium minitans and reduced fungicide application Phytopathology 91 221ndash7

Clarkson JP Payne T Mead A Whipps JM 2002 Selection of fungal BCAs of Sclerotium cepivorum for control of white rot by sclerotial degradation in a UK soil Plant Pathology 51 735ndash45

Clarkson JP Mead A Payne T Whipps JM 2004 Effect of environmental factors and Sclerotium cepivorum isolate on sclerotial degradation and biological control of white rot by Trichoderma spp Plant Pathology 53 353ndash62

Coley-Smith JR 1987 Alternative methods of controlling white rot disease of Allium In Chet I ed Innovative Approaches to Plant Disease Control New York USA John Wiley 161ndash77

Coley-Smith JR Parfitt D Taylor IM Reese RA 1987 Studies in dormancy of sclerotia of Sclerotium cepivorum Plant Pathology 36 246ndash57

Coventry E Noble R Mead A Whipps JM 2005 Control of Allium white rot (Sclerotium cepivorum) in different soils



using vegetable wastes European Journal of Plant Pathology 111 101ndash12

Crowe FJ Debons J Darnell T Thornton M McGrath D Koepsell P Laborde J Redondo E 1994 Control of Allium white rot with DADS and related products In Entwistle AR Melero-Vara JM eds Proceedings of the 5th International Workshop on Allium White Rot 1994 Cordoba Spain Cordoba SpainWarwick UK Instituto de Agricultura SostenibleHorticulture Research International 7ndash22

Defra Horticultural Statistics 2004 Vegetable Crops httpstatisticsdefragovukesgpublicationsbhs2004summarypdf

Dennis JJ 2001 Progress towards an integrated strategy for onion white rot disease including the use of artificial germination stimulants Acta Horticulturae 555 117ndash21

Entwistle AR 1988 Opportunities for the microbial control of Allium white rot EPPO Bulletin 18 19ndash28

Entwistle AR 1990 Screening accessions of Allium cepa from the Vegetable Gene Bank Wellesbourne for response to Sclerotium cepivorum In Proceedings of the Fourth International Workshop on Allium White Rot Neustadt Weinstrasse Germany Braunschweig Germany Biologische Bundesanstalt Fur Land und Forstwirtschaft 210ndash17

Gamliel A Stapleton JJ 1993 Characterization of antifungal volatile compounds evolved from solarized soil amended with cabbage residues Phytopathology 83 899ndash905

Gerbrandy SJ 1989 The effects of various temperatures during storage in soil on subsequent germination of sclerotia of Sclerotium cepivorum Netherlands Journal of Plant Pathology 95 319ndash26

Gerlagh M Whipps JM Budge SP Goossen van de Geijn HM 1996 Efficiency of isolates of Coniothyrium minitans as mycoparasites of Sclerotinia sclerotiorum Sclerotium cepivorum and Botrytis cinerea on tomato stem pieces European Journal of Plant Pathology 102 787ndash93

Harman GE Latorre B Agosin E San Martin R Riegel DG Nielsen PA Tronsmo A Pearson RC 1996 Biological and integrated control of botrytis bunch rot of grape using Trichoderma spp Biological Control 7 259ndash66

Hovius MHY McDonald MR 2002 Management of Allium white rot (Sclerotium cepivorum) in onions on organic soil with soil-applied diallyl disulfide and di-N-propyl disulfide Canadian Journal of Plant Pathology 24 281ndash6

Kay SJ Stewart A 1994a Evaluation of fungal antagonists for control of onion white rot in soil box trials Plant Pathology 43 371ndash7

Kay SJ Stewart A 1994b The effect of fungicides on fungal antagonists of onion white-rot and selection of dicarboximide-resistant biotypes Plant Pathology 43 863ndash71

Lewis JA Papavizas GC 1985 Characteristics of alginate pellets formulated with Trichoderma and Gliocladium and their effect on the proliferation of the fungi in soil Plant Pathology 34 571ndash7

Lewis JA Papavizas GC 1987 Application of Trichoderma and Gliocladium in alginate pellets for control of rhizoctonia damping-off Plant Pathology 36 438ndash46

Lewis JA Lumsden RD Locke JC 1996 Biocontrol of damping-off diseases caused by Rhizoctonia solani and Pythium ultimum with alginate prills of Gliocladium virens Trichoderma hamatum and various food bases Biocontrol Science and Technology 6 163ndash73

Mackney D Hodgson JM Hollis JM Staines SJ 1983 Legend for the 1250 000 Soil Map of England and Wales Harpenden UK Soil Survey of England and Wales

McLean KL Swaminathan J Stewart A 2001 Increasing soil temperature to reduce sclerotial viability of Sclerotium cepivorum in New Zealand soils Soil Biology and Biochemistry 33 137ndash43

Melero-Vara JM Prados-Ligero AM Basallote-Ureba MJ 2000 Comparison of physical chemical and biological methods of controlling garlic white rot European Journal of Plant Pathology 106 581ndash8

Moyano C Raposo R Gomez V Melgarejo P 2003 Integrated Botrytis cinerea management in southeastern Spanish greenhouses Journal of Phytopathology 151 80ndash5

Smolinska U 2000 Survival of Sclerotium cepivorum sclerotia and Fusarium oxysporum chlamydospores in soil amended with cruciferous residues Journal of Phytopathology 148 343ndash9

Utkhede RSRJ Coley-Smith JR Van der Meer QP Brewer JG Criscola V 1982 Genotypendashenvironment interactions for resistance to onion white rot Canadian Journal of Plant Pathology 4 269ndash71

Wu C Hsiang T 1998 Pathogenicity and formulation of Typhula phacorrhiza a biocontrol agent of gray snow mold Plant Disease 82 1003ndash6

Plant Pathology

(2006)

55

375ndash386

376

J P Clarkson

et al

soil types and controlled AWR in the field (Clarkson

et al

2002 2004) However although

T viride

L4 and S17Acould reduce AWR reproducibly disease levels weregenerally reduced by only

c

50 and the degree ofcontrol was variable both of which would be unacceptableto growers One approach to address this problem is tointegrate the use of

T viride

with other methods of AWRcontrol and determine if these combinations are moreeffective (Entwistle 1988) This is a promising approach asshown by two studies that have investigated this strategyIn the first study the use of DADS was combined with thefungicide tebuconazole which was applied with a limetreatment and drilled with the onion seeds (Dennis 2001)DADS and tebuconazole reduced AWR incidence from76 (untreated) to 44 and 37 respectively whereasthe combined treatment reduced disease further to just11 In the second study an iprodione-tolerant strain of

T harzianum

was combined with the fungicide which wasapplied in-furrow (Abd-El-Moity

et al

1982)

Trichodermaharzianum

and iprodione reduced AWR incidence from86 (untreated) to 13 and 51 respectively while thecombined treatment resulted in 6 disease

The aim of this work was to confirm

T viride

L4 andS17A as BCAs of AWR when applied alone and also toevaluate potential improved efficacy when combined witha tebuconazole-based fungicide seed treatment or com-posted onion waste In addition new bulb-onion accessionsderived from plants from previous experiments to detectresistance to

S cepivorum

(Entwistle 1990) were alsoassessed for effects on AWR and compared with a com-mercial cultivar Combinations of

T viride

with these newbulb accessions and current commercial cultivars werealso tested The effect of combining

T viride

with theseother control treatments for an integrated approach tocombat AWR has not been assessed previously

Materials and methods


isolates and production of sclerotia

The isolate of

S cepivorum

used in this study (code Kirton)was obtained as sclerotia from an infected onion bulb froma white rot-infested field site near Kirton Lincolnshire UK

Cultures of

S cepivorum

were obtained after first surface-sterilizing sclerotia [washing in sodium hypochlorite solution(15 available chlorine Hays Chemicals) for 90 s followedby washing three times in sterile distilled water (SDW)] andthen squashing onto potato dextrose agar (PDA Oxoid) in9-cm Petri dishes Cultures were incubated at 20

deg

CTo obtain large numbers of sclerotia for experiments

2-week-old PDA cultures of

S cepivorum

were cut into

c

3-mm

2

pieces and used to inoculate mushroom spawnbags (Biscof amp Klein one Petri dish culture per bag) con-taining a mixture of washed sand (1950 g lt2 mm particlesize Hepworth Minerals and Chemicals) ground maizemeal (80 g Midland Shires Farmers Ltd) and water (350mL) which had been sterilized by autoclaving at 0middot1 MPaand 121

deg

C for 15 min The bags were sealed and incubated

at 18

deg

C for 6 weeks after which sclerotia had formedSclerotia were harvested by flotation in water retrievalin a sieve (212

micro

m mesh diameter) and then dried in anairflow cabinet for 12 h before storage at 5

deg

C The sclerotiaused in onion seedling bioassays were further subjected tolsquoconditioningrsquo to overcome constitutive dormancy (Coley-Smith

et al

1987) This consisted of burial in mesh bagsfor at least 12 weeks in a quarantine field at WarwickHRI After this period sclerotia were washed sieved anddried as before then gently rubbed on a sieve (212

micro

mmesh diameter) to remove any that were soft and degraded

Trichoderma

isolates

The two isolates of

T viride

(IMI 386638 code S17A IMI386639 code L4) used in this study had previously beenselected as BCAs of

S cepivorum

based on their ability todegrade sclerotia in soil and control AWR in glasshouseonion seedling bioassays (Clarkson

et al

2002 2004)

Trichoderma viride

L4 and S17A were also shown to reduceAWR in the field (Clarkson

et al

2002) The

T viride

isolateswere originally isolated from parasitized

S cepivorum

sclerotia cultured on PDA at 20

deg

C under fluorescentlighting with a 12-h daylength and stored in liquid nitrogenSpore suspensions were obtained by adding 20 mL SDWto 3-week-old PDA cultures and scraping gently with aspatula

Wheat bran cultures of

T viride

for use in glasshouseexperiments were obtained by inoculating 250-mL flaskscontaining wheat bran (12 g) and water (30 mL) sterilizedby autoclaving at 0middot1 MPa and 121

deg

C for 30 min withthe spore suspensions (5 mL) and incubating for 3 daysat 20

deg

C This formulation was shown to stimulateproliferation of

Trichoderma

spp and was used effectivelywith BCAs of

S cepivorum

(Kay amp Stewart 1994aClarkson

et al

2002 2004)Alginate pellets of

T viride

S17A were produced foruse in field experiments following the method of Lewis ampPapavizas (1985) This dry-pellet formulation also allows

Trichoderma

spp to proliferate in soil and has been usedsuccessfully to deliver BCAs in several pathosystems(Lewis amp Papavizas 1987 Lewis

et al

1996 Wu ampHsiang 1998)

Trichoderma viride

biomass was producedby inoculating a yeast molasses medium [15 g molasses(United Molasses Ltd) 2middot5 g brewers yeast (Holland ampBarrett Ltd) 500 mL water] with 1 mL spore suspension(

c

1

times

10

6

spores mL

minus

1

) and incubating for 9 days at20

deg

C and 110 rpm Biomass (30 g) was then combinedwith 750 mL sodium alginate (Fisher Scientific) solution(26middot6 g L

minus

1

water) and 250 mL ground wheat bran suspen-sion (200 g L

minus

1

water) and the mixture pumped dropwiseinto a calcium chloride solution (5 g L

minus

1

water) Theresulting alginate pellets were dried under an airflow atroom temperature and stored at 5

deg

C until use


The effects on AWR of onion accessions and commercialcultivars a tebuconazole-based seed treatment and

Plant Pathology

(2006)

55

375ndash386


Allium

white rot

377


T viride


et al


et al


S cepivorum


minus

1

) andwhere used

T

viride


deg


Effect of

T viride



T viride


T viride



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T viride


Effect of










Field experiments
















Results










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Acknowledgements


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Plant Pathology

(2006)

55

375ndash386


Allium

white rot

377


T viride


et al


et al


S cepivorum


minus

1

) andwhere used

T

viride


deg


Effect of

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Documents

Integrated Control of Allium White Rot With Trichoderma