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Published article online:30 Jul 2007Issue online:10 Sep 2007

Received January 18, 2007;accepted March 18, 2007

To cite this article: E. M. DEL PONTE,J. M. C.FERNANDES,G. C. BERGSTROM(2007) lnfluence of GrowthStage on Fusarium Head Blight and DeoxynivalenolProduction in WheatJournal of Pllytopathology 155 (10) ,577-581doi: 10.1111/j.1439-0434.2007 .01281.x

Influence of Growth 5tage on Fusarium HeadBlight and Deoxynivalenol Production in WheatE. M. OEL PONTE!, J. M. C. FERNANDES'Z and G. C. BERGSTROM]

Authors' addresses: lDepartment of Fitossanidade, Federal University

of Rio Grande do Sul, 91540000, Porto Alegre, RS, Brasil; 2Embrapa

Trigo, Rodovia BR 285, km 174, Passo Fundo, RS, Brasil; 3Departmentof Plant Pathology, Cornell University, Ithaca, NY 14853-5904, USA(correspondence to E. M. Dei Ponte. E-mail:er-r1I.'I"·on. dlél!~onLc'(iluf ru.:,.lll)

Fusarium Ilead blight is a major concern for wheat productionworldwide. The fungi that cause tlle disease may infect headtissues from flowering to late stages of kernel development, but abetter understanding of the influence of the time of infection ongrain weight reduction and mycotoxin accumulation resulting fromthe infection process is needed. We investigated the influence ofwheat reproductive stage at the time of inoculation on disease andgrain quality parameters, especially production of deoxynivalenol(DON) in mature grains. Heads of Norm wheat were sprayinoculated with a macroconidial suspension of a DON-producingisolate of Fusarium graminearum at each of six reproductive stagesfrom flowering to hard dough. Plants were incubated in a mistchamber for 48 h and then moved to the greenhouse untilmaturity. Norm wheat was susceptible at ali stages inoculated butthe highest grain weight reduction and DON accumulation occurredin plants inoculated past flowering to late milk stages. However,high incidences of kernel infection and significant levels of DONaccumulation resulted from inoculations as late as the hard doughstage, even though there was no corresponding reduction in grainweight compared to non-inoculated plants. The occurrence ofcommercially significant levels of DON in plump, high-yieldingwheat may result from infections that occur during favourableenvironments well after the flowering stages. Late infection and

J. Phytopalhology, 155, 577-581 (2007)Journal cOll1pilation © 2007 Blackwell Verlag, BerlinNo c1aill1 10 original US government works

Influence of Growth Stage on Fusariulll Head Blight alld DeoxynivalenolProduction in Wheat

E. M. DEL PONTE I, J. M. C. FERNANDES2 and G. C. BERGSTROM3

Authors' addresses: IDepartmenl or Fitossanidade, Federal Universily or Rio Grande do Sul, 91540000, Porto Alegre, RS,Brasil; 2EIllbrapa Trigo, Rodovia BR 285, kll1 174, Passo Fundo, RS, Brasil; 3Department or Plant Pathology, CorneUUniversily, llhaca, NY 14853-5904, USA (correspondence to E. M. Dei Ponte. E-mail: emerson.delponte@ufrgs.br)

Received January 18, 2007; accepted March 18, 2007

AbstractFusarium head blight is a major concern for wheatproduction worldwide. The fungi that cause the diseasemay infecl head tissues from flowering 10 Iate slages ofkernel development, but a better understanding of theinfluence of the time of infection on grain weighl reduc-lion and mycotoxin accumulation resulling fram lheinrection process is needed. We investigated lhe influ-ence of wheat reproductive stage at the time of inocula-lion on disease and grain quality parameters, especiaIlypraduction 01'deoxynivalenol (DON) in mature grains.Heads of Norm wheal were spray inoculaled with amacroconidial suspension of a DON-producing isolaleof FusariwII graminearum at each of six reproductivestages from flowering to hard dough. Plants were incu-bated in a mist chamber for 48 h and then moved tothe greenhouse unlil maturity. Norm wheat was sus-cepti ble aI aIl stages inoculaled bul the highesl grainweight reduction and DON accumulalion occurred inplants inoculaled past fioweringo lale milk stages.However, high incidences of kernel infection and signi-ficant levels of DON accumulation resulled from inocu-lations as late as the hard dough stage, even lhoughthere was no corresponding reduction in grain weightcompared to non-inoculated pJants. The occurrence ofcommerciaIly significant levels of DON in plump, high-yielding wheat may result from infections that occurduring favourable environments weIl after the floweringstages. Late infection and DON production shouldtherefore be a future research focus for wheat breedingand integrated management of FHB and an importantconsideration for grading systems that employ the pres-ence of visibly damaged kernels as a means of estima-ting DON content of wheat.

IntroductionFusarium head blight (FHB), also known as scab, is adestructive disease of cereais that has reached epidemic

proportions in North America and several other partsof the world in the past decades (McMullen et al.,1997). The major causal organism of this diseaseworldwide is Gibberella zeae (Schwein) Petch (ana-morph: Fusarium gra11linearu/1/ Schwabe) (Schmale andBergstrom, 2003). The disease alfects wheat, barley,and other small grains both in tempera te and semi-tropical areas causing reduction in grain yield andquality. Costs to farmers include increased manage-rnent costs, reduced income from outright food or feed10sses, and lower selling prices for commodities con-taminated with mycotoxin (Jones and Miracha, 1999).The occurrence of DON is of concern for human andanimal health, as this toxin is known to cause foodrefusal, vomiting, and depressed immune function(World Health Organization, 200 I). Although progressis being made in disease reduction through chemicalcontrol, cultural controI, and the development ofresistant cultivars, satisfactory levels of control haveyet to be attained (Bai and Shaner, 1994; Cromeyet al., 2001). The fungus overwinters as a saprophytein debris of infected cereais and grasses, including cornand wheat (Dill-Macky and Jones, 2000). Ascosporesand macroconidia af the fungus are produced on over-wintered residues (Khonga and Sutton, 1988) and bothspore types are able to infect wheat any time fromf!owering through kernel filling stages if conditions areconducive (Shaner, 2003). However, there is lack ofconsellSUS about the exact size of the phellological Will-dow of vulnerability 10 illfection, and especially for theaccumulatioll of DON in the kernels. Some authorsdemonstrated that illfection occurred principally atanthesis with the anthers as the maill illfection site(Atanasolf, 1920; Pearce et al., 1976). However, othershave observed an extended window of infectioll with apeak after anthesis (Andersen, 1948; Schroeder andChristensen, 1963).

DON accumulation ill the kernels is illfluenced bymallY factors including host resistance, chemotype,

aggressiveness of the prevalent fungal species, andenvironment (mainly temperature and moisture) duringdisease development (Hart et a!., 1984; Mesterházyet a!., 1991). The dynamics of toxin praduction in thefield seem to be of a complex nature. Miller andYoung (1985) reported that DON levels in headsinoculated at flowering increased for six weeks, thendeelined. This study was conducted to determine theinfluence of growth stage on disease and grain qualityparameters. A brief, preliminary report of a portion ofthese findings was published (DeI Ponte et aI., 2003).

Materiais and Methodslnoculum preparationCultures of a single-conidium isolate of G. zeae(GZOI4NYI998), with documented virulence and abil-ity to produce DON in wheat, were grawn in Petridishes containing 1/4 strength polato-dextrose-agar(PDA) at a constant 25°C and a photoperiod of 12 hfluorescent light for 7 to 10 days. Macroconidia wereharvested by washing the culture surface with sterile,distilled water. The resulting inoculum suspensionswere adjusted with water to a concentration of 105

conidia/ml. Inoculul11 was used within 3 h aflerpreparation.

Experimental designTen seeds of hard red spnng wheat cv. Norm weresown in elay pots (2.5 I) containing a soil substrateand grown in the greenhouse at temperatures rangingfrom 20-23°C. By the beginning of anthesis, groups ofsix pots (each pot was one replica te) of homogeneousphenology were established. In each pot, 7 to 10 maintilIers were maintained and secondary tilIers were con-sistently eliminated during lhe experiment. Each treat-ment consisled of single-time spray-inoculations at onedistincl growth slage, as follows, according to Zadoksscale (Zadoks et aI., 1974): 65 - Anthesis half-com-plete; 71 - kernel watery ripe; 73 - early milk; 77 - lalemilk; 83 - earIy dough; and 87 - harel dough. Controllrealment consisted of a group of w'heal heads sprayedwith sterile water. A hand-sprayer was used lo applywater and the spore suspension; wheat heaels weresprayed to saturation. After applying lhe treatments,plants were moved into a mist chamber for 48 h unelercontinuous moisture anel temperatures ranging fram21 to 23°C. Thereafter, p\ants were moved back to thegreenhouse and grown until maturity. The experimentwas repeated once with six replications (a potteelplant with 7 to 10 main tillers) of trealments in eachexperiment.

Disease and grain quality parameters evaluatedWheat heads were inspecled at the sixth day followinginoculation and the percentage of spikelets which weresymptomatic (RDS - relative disease severity) wasrecorded. A spikelet was 'recorded as visually infectedwhen showing premature bleaching anel discolouration.At maturity, alI kernels of each replicate were hanel-harvested, hand-threshed anel dried lo a uniform mois-

ture content of 13% by weight. Five sub-samples of 50kernels were taken from each treatment to determinethe mean percentage of visually scabby kernels (VSK),e.g. discoloureel, shriveleel or pinkish white kernels.Mean percentage of kernel infection (KI) was estima-ted by plating 10 sub-samples of 100 kernels in 10plates with a selective medium - SNA W (Mandanharand Cunfer, 1991). The fungal isolale inoculated pre-viously was idenlified based on cultural and morpholo-gical characteristics. Three sub-samples of 100 kernelswere weighed and the average used to estimate 1000-kernel weight (KW). DON levels were determined bydirect competitive enzyme immunoassay RIDA-SCREEN® DON (Digen Ltel., Oxford, UK) accordingto manufacturer's instructions. For each sample, 5-ggrain samples were ground in a coffee mill for 12-15 s,and then stored in sealed plastic containers before ana-Iysing. One hunelreel millimetres of extraction bufferwas added to the 5-g graunel sample. Samples wereshaken for 3 minutes and filtered into a collecliontube. When toxin levels exceeded the test range, thetesl was repeated after the extract was diluled withbuffer. The OON concentration was calculateel accord-ingly.

Data analysisData fram the two eXI)eriments were pooled elue tolack of a significant difference between them (data notshown), and statistical analysis was based on ali 12combined replicates. DON analysis was an exceptionin that there were three replicates per experiment (totalof six replicates for both experiments). Summary sta-tistics and Pearson correlations were maele in R soft-ware (R Foundation for Statistical Computing,Vienna, Austria). For the correlations of DON x KWand DON x VSK, averages for two pairs of the 12replicates were useel, in order to for111six pair valuesof KW and VSK to correlate with six DON replicatevalues and lo make lhe graphs.

ResultsNorm wheat was susceptible to infection from flower-ing through ali stages of kernel development. Diseaseseverity could not be evaluated for heads inoculateel athard dough stage, due to natural senescence. The timefram inoculalion to appearance of symptomsdecreased from flowering until earIy dough stage inoc-ulations. Typical elisease symptoms were first observed3 to 4 days following inoculation, except in headsinoculated at flowering, in which the incubation periodranged from 7 to 10 days. Hence, RDS value at 6 daysfor inoculations at anthesis was recorded as zero,although it increased dramatically afterwards (data notshown). Mean values. for disease and grain qualityparameters evaluated are presented in Table i. Theincidence of visualIy scabby kernels and the degree ofelamage was highest for lhe earliest inoculations andleast for inoculations at hard dough, which did notaffect grain weight compared to the contraI. A higherincidence of visual symptoms was observed in kernels

Table IErrect of FlIS"rÚIIII gralllilleartllll inóculated at six growth stages on disease and grain quality parameters of wheat

Mean (standard error) for grainMean (standard error) for disease parameters quality paramelers

Zadoks stage at inoculation" RDS" (%) VSK" (%) KI"(%) KW" (g) DONf (mg/kg)

65 0.0 94.0 (1.2) 52.0 (2.3) 2304 (0.1) 24.7 (5.7)71 31.3 (304) 97.3 (0.7) 72.2 (2.5) liA (0.1) 98.0 (3.2)73 40.6 (5.1) 95.2 (1.6) 940 (IA) 17.8 (0.1) 89.7 (5.5)77 67.0 (6.5) 996 (0.2) 99.3 (0.5) 26.9 (0.0) 49.0 (3.0)83 8204 (3.6) 72.3 (3.7) 96.2 (1.3) 37.8(0.1) 133 (1.5)87 na 2304 (1.8) 71.7 (3.6) 429(0.1) 1.2 (0.2)Non-inoculated control 0.0 0.0 0.0 41.0 (0.1) 0.0

"65 - Anthesis half-complete; 71 - Kernel watery ripe; 73 - Early milk; 77 - Late milk; 83 - Early dough; 87 - Hard dough (Zadoks et aI.,1974)."Relative disease severity - % of diseased spikelets per heael at 6 úays afler inoculation (n = 12).'Visllally scabby kernels (li = 12)."Kernel infection by FlIsarilllll gralllinearwlI on cullure meelia (n = 12).e 1000-kernel weight.IDeoxynivalenol concentration (11 = 6).

inoclllated at early dough (72%) but the impacl ongrain weight was not so evident. Nearly one hundredpercent of kernels from inoculalions at early milk loearly dough slages were colonized by the fungus.

OON levels ranged from 1.2 to 98 mg/kg with thehighest toxin levels detected in kernel~ from inocllla-tions at watery ripe or early milk stages. These valueswere around three and two times higher than toxinlevels in kernels from earlier (anthesis) or later (latemilk) inoculatíons, respectively. A high amount(13.3 mg/kg) of DON was detected in kernels fromheads inoculated at early dough, and lowest levels(1.2 mg/kg) were delecled in kernels inoculated al harddough (Table I). Kernel weight was greally reduced(two to fom times lower than the control) by inocula-tíons at anlhesis up to late milk stages. lnoculations atdough stages resulled in 1000-kernel weights similar tothe non-inoclllated control. Both positive and negativecorrelations among the paramelers were observed byanalysing combined data from alI six replicales in eachof lhe six inoculation times (11 = 36). A significanl neg-alive correlation (P < 0.001) was observed betweenkernel weight and OON (r = -0.88) and kernel weightand VSK (r = -0.77). A significant positive correlalion(P < 0.001) was observed belween DON and VSK(r = 0.64). Correlation between Kl and DON was notsignificanl (r = 0.09). Rela tionships for DON andVSK and between DON and KW are presenled ínFig. I. DON conlent in the kernels ranged framaround I lo 100 mg/kg. Deoxynivalenol decreased line-arly with increasing kernel weight from inoculationspcrformed at laler stages of kernel development, yel asignificant amount of loxin was observed in some ker-nels lhat were nearly as plump as kernels from non-inoculated plants.

Discussionln the present sludy we demonstrated that Norm, acultivar already known as highly susceplible to FHB,has a wide window of vulnerability lo infection by

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Fig. I Relalionships belween visllally scabby kernel and e1eoxynival-enol concentralion (lop) anel between 1000-kernel weight anel e1co·xynivalenol concentration (bottom) reslllting frol11 single-til11einocllialions at six dislinct stages of kernel e1evelopl11cnt (11 = 36).Mean kernel weight in the non-inoclllateel control was 41 g

F. gral11inearul11. A high incidence af fungai coloniza-tion of kernels was observed following antl1esisthrough the dough slages af kernel development. Thisis ín agreement with Andersen (1948) who reported

that wheat heads were most susceptible at postanthesisstages. Strange et aI. (1974) suggested that anthers playan important role in infection based on the presence inanthers of the compounds choline and betaine that sti-mulated rungal growth. Some have suggested that sus-ceptibility increases arter flowering precisely becausesenescent anthers adhere to glumes (Takegami, 1957).However, Engle et aI. (2002) found no effect of cholineand betaine on germination 01' conidia and ascospores,suggesting that endogenous compounds 01' flowers donot promote colonization by F. grolllillearUlII. In ourstudy, a significant praportion 01' kernels became infec-ted arter inoculation at sort and hard dough stages,although visual symptoms on kernels and toxin con-tent were lower than in kernels inoculated earlier indevelopment. Susceptibility of wheat inoculated at dif-ferent growth stages was previously e10cumented in afield study by Fernando et aI. (1997) who exposedwheat heads to field inoculum (ascospores) at specifictimes during kernel development. In that study, themost susceptible period was anthesis (70% 01' kernelswere symptomatic) to early milk; whereas only 20% ofkernels sYl11ptomatic were sYl11ptomatic in headsexposed at late milk stage and 5% of kernels sympto-matic in heads exposed at hard dough stage. In ourcontralled inoculation study, a high incipence 01'kernelinfection was recorded at all reproductive stages,although DON levels eleclineel steaelily between kernelwatery ripe anel hard elough. Early elough stage inocu-lations resulteel in a slightly lower percentage 01' visu-ally scabby kernels than did earlier inoculations. It ispossible that the consistently high levei of kernel inrec-tions we observed at the late stages, in contrast withresults by Fernando et aI. (1997), might be due to dif-ferences in the methodology, i.e. inoculum type anddosage and wetness duration, since our study was con-ducted with high inoculum and a highly ravourableenvironment. The lack 01'a relationship between kernelinfection incidence anel DON was reported by Argyriset aI. (2003) who collected wheat, kernels at 5-dayintervals during kernel filling. They observed thatalthough kernel infection increased in later stages,DON was present at significant levels as early as10 days arter anthesis and it remained within a narrowrange through seed e1evelopment. Those results, how-ever, conflict with a previous paper (Trigo-Stockliet aI., 1995) that reported significant correlalionbetween kernel infection and DON, when analysingmature kernels and not kernels harvested e1uring fillingstages. Mesterházy et aI. (2005) rounel strong correla-tions belween FDK anel DON contamination resulting1'1'0111inoculations at flowering, suggesting that scabby01' tombstone kernels are the most important carriersof the toxins, anel selections for low FDK at a reason-ably high infection pressure would leael to lower toxinlevels in the grain. In our study, we round a significantpercentage 01' kernels (23%) with visually lighter dam-age in kernels inoculated at hard dough stages that didnot impact kernel weight yet l11ayhave contributed tothe DON levels observed.

Our findings also agree with a previous study byHart et aI. (1984) who observeel that production ofDON in wheat kernels varied according to the dur-ation of wetness, regarelless of the stage of kerneldevelopment, arter they were filleel. Neverlheless, wehave observed a very high accul11ulation (49 to 98 mg/kg) 01' mean DON in kernels inoculateel rrom wateryripe through milk stages and coml11ercially significantmean toxin levels (1-13 mg/kg) in kernels inoculatedat dough stages. Infections during flowering directlyaffect kernel development resulting in floret abortionor the development 01' small, shriveled and lightweightkernels that l11ay be lost during harvest and cleaningoperations (Schaafsma et aI., 200 I). ln the presentstudy, toxin-contaminated kernels resulting from inoc-ulations at dough stages were similar in weight to non-inoculated kernels, suggesting that late infected kernelsmay also contribute to lower grain quality, i.e., myco-toxin contal11ination, even ir they have a negligibleeffect on grain yield. Depending on the country andinteneled grain use, wheat may be substantially e1is-counteel or even refused by buyers at DON levels aslow as I mg/kg, the 'mean levei 01' contaminationround in this study for kernels inoculated at latedough stage. Empirical evidence exists for the potentialrole of late infections in commercially significant toxinaccumulation in mature grains. ln several recent yearsin New York State, USA, an apparent uncoupling ofDON contamination in soft winter wheat from theoccurrence of FHB symptoms and grain weight reduc-tion has been observed. That is, plump and high-yield-ing wheat has been contaminated with DON above2 mg/kg (Bergstrom, unpublished). Similarly, Cowgerand Sutton (2005) reported that severity 01' FHB in aportion 01' the south-eastern USA in 2003 was bettercorrelated with weather parameters after wheat flQ\.ver-ing than with those before or at the time of f1owering,thus suggesting an important role for postfloweringinfections. Moreover, accuracy of preeliction modelsfor disease severity and DON increased when theyincludeel weather variables rrom periods past flowering(Schaafsma and Hooker, 2003; Dei Ponte et aI., 2005).A possible explanation suggesteel by the current find-ings as well as the, previous findings by Hart et aI.(1984) is that DON ÓII1 be praduced from infectionslate in kernel development without proelucing dramaticreductions in grain weight, especially if multiple infec-tion events occur during grain filling. This biologicalfact complicates the alj'eady challenging task of timelyprediction and contrai of FHB and toxin contal11in-ation. If, in fact, the results of this study and othersrurther confirm an extended phenological window ofwheat vulnerability to FHB, especially for DON accu-l11ulation, then (i) wheat breeelers would neeel to consi-der screening methods for resistance to DONproduction resulting from late infections; (ii) farmerswould need to consider management strategies aimedto protect wheat heads from infection for severa Iweeks rather than several days around flowering; and(iii) practices which use damaged kernels as a predictor

of DüN may be less reJiable in years when conclitionsaJIow for infection late in the growing season.

AcknowledgernentsThis research was conducted at Cornell University and was suppor-ted by Cornell I-Iatch Project NYCI53433 and a fellowship framCNPq-Brazil lhat allowed Emerson Dei Ponte, a former graelualestudent at lhe Federal University 01' Pelotas, to conduct research aICornell. We gralefully acknowledge John Cianchelti and StanleyKawamoto 01' Cornell for technical assistance; and Linda Geiger anelFrancois Lachance 01' Star 01' the West Milling Company in Church-ville, NY for conducting the DON analyses reported in this paper.

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