Timing of inoculation and Fusarium species affect the severity of Fusarium head blight on oat

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1 Timing of inoculation and Fusarium species affect the severity of Fusarium head blight on oat Allen G. Xue 1, Yuanhong Chen 1, Geneviève Marchand 1, Wei Guo 1,2, Changzhong Ren 1,3, Marc Savard 1, and Arthur R. B. McElroy 4 1 Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa, Ontario, Canada K1A 0C6 ( allen.xue@agr.gc.ca); 2 Heilongjiang Bayi Agricultural University, 5 Xinfeng Road, Daqing, Heilongjiang , China; 3 Baicheng Academy of Agricultural Sciences, 17 Sanhe Road, Baicheng, Jilin , China; and 4 PhytoGene Resources Inc., 1855 Rue des Arbres, Orleans, Ontario, Canada K1E 2T7. Received 15 August 2014, accepted 18 December Published on the web 19 December Xue, A. G., Chen, Y., Marchand, G., Guo, W., Ren, C., Savard, M. and McElroy, A. R. B Timing of inoculation and Fusarium species affect the severity of Fusarium head blight on oat. Can. J. Plant Sci. 95: The influence of timing of inoculation and pathogenicity of four Fusarium spp. (F. culmorum, F. graminearum, F. sporotrichioides, and F. avenaceum) causing Fusarium head blight (FHB) were examined on 12 oat genotypes under controlled environmental conditions in two separate sets of experiments. In the first set, early inoculations with F. graminearum at or before the complete emergence of ears resulted in little or no visible FHB symptoms but deoxynivalenol (DON) contents ranging from 0.4 to 2.6 ppm were detected in the harvested grain. Severe levels of FHB were observed on these genotypes with infected spikelets ranging from 30 to 74% and DON concentrations, from 6.6 to 10.0 ppm, when plants were inoculated at or after the 50% anthesis stage. Inoculation at 50% anthesis was considered the most appropriate as it allowed sufficient time for disease development and assessment prior to the physiological maturity of the plants. In the second set of experiments, F. culmorum and F. graminearum were equally highly pathogenic, having areas under the disease progress curve of 45.3 and 47.3, respectively. Fusarium sporotrichioides was significantly less pathogenic than the two highly pathogenic species. The pathogenicity of F. avenaceum was intermediate and was not significantly different from those of either the highly pathogenic or the weakly pathogenic Fusarium species. Key words: Fusarium head blight, oat, Avena sativa L., deoxynivalenol, timing of inoculation Xue, A. G., Chen, Y., Marchand, G., Guo, W., Ren, C., Savard, M. et McElroy, A. R. B Le moment de l inoculation ainsi que l espe` ce de Fusarium influencent le de veloppement de la bruˆlure fusarienne de l e pi de l avoine. Can. J. Plant Sci. 95: Les effets du moment de l inoculation ainsi que du pouvoir pathogène de quatre espe` ces de Fusarium (F. culmorum, F. graminearum, F. sporotrichioides, and F. avenaceum) causant la bruˆlure fusarienne de l e pi ont e té e value s en deux séries d expe rience respectives sur 12 ge notypes d avoine sous conditions environnementales controˆle es. Lors de la premie` re série d expe riences, l inoculation haˆtive avec l espe` ce F. graminearum, avant ou au moment de l e mergence comple` te de l e pi, a produit peu ou pas de symptoˆmes observables, mais des concentrations de de oxynivale nol (DON) variant entre 0.4 et 2.6 ppm ont été mesure es dans les grains apre` s la récolte. Des taux d infection e leve s ont e té observés chez ces ge notypes, avec les taux d e pillets infecte s variant entre 30 et 74% et les concentrations de DON, entre 6.6 et 10.0 ppm lorsque les plants ont e té inocule s avant ou au moment ou` 50% des anthe` res étaient sorties. L inoculation à ce moment a permis d e valuer le de veloppement de la maladie avant la maturite physiologique des plants, et a e té choisi comme e tant le plus approprié pour la suite des expériences. Lors de la deuxième série d expériences, F. culmorum et F. graminearum se sont révélées avoir un pouvoir pathoge` ne e leve et e quivalent, re sultant en des aire sous la courbe de progression de la maladie de 45.3 et 47.3, respectivement. Le pouvoir pathogène de F. sporotrichioides e tait significativement moins éleve que celui des deux espe` ces au pouvoir pathoge` ne élevé. Celui de F. avenaceum était interme diaire et ne diffe rait pas statistiquement de celui des espe` ces de Fusarium au pouvoir pathogène éleve ou faible. Mots clés: Bruˆlure de l e pi cause e par la fusariose, avoine, Avena sativa L., de oxynivale nol, moment de l inoculation Oat (Avena sativa L.) is a common crop grown for both feed and food in Canada. In 2012, Canada produced over 2.6 million tonnes of oats, making it the third largest producer of oats next to the European Union and Russia (Statistics Canada 2013). Oat contains high amounts of valuable nutrients such as b-glucans, unsaturated fatty acids antioxidants, proteins, dietary fiber, vitamins, and minerals (Wood 1990; Behall et al. 1997; Tsopmo et al. 2010; Sobotka et al. 2012). Due to their beneficial effects on human health, the use of oat and oat products for human food has been increasing in recent years (Peterson 1992; North American Millers Association and the National Oat Improvement Committee 2008). In Canada, the beneficial effects of oats consumption on Abbreviations: AUDPC, area under the disease progress curve; DON, deoxynivalenol; FHB, Fusarium head blight; IS, infected spikelets; ZGS, Zadoks growth stage Can. J. Plant Sci. (2015) 95: doi: /cjps

2 518 CANADIAN JOURNAL OF PLANT SCIENCE reduction of blood LDL-cholesterol and total cholesterol levels have been recognized by Health Canada (Health Canada 2010). During the past 20 yr, there has been an increase in the incidence of Fusarium head blight (FHB) on oats grown in the two main production regions, the Canadian prairies (McCallum et al. 1999; Tekauz et al. 2004, 2008, 2011) and the eastern provinces of Ontario and Que bec (Couture and Le vesque 1995; Couture et al. 1996; Tamburic-Ilincic 2010; Xue and Chen 2010, 2014). As a result, seeds harvested from these regions may contain a considerable portion of Fusarium-infected kernels resulting from infection by several Fusarium spp. (Clear et al. 1996, 2000; Tekauz et al. 2008; Tamburic-Ilincic 2010, 2011). A number of authors have reported that FHB lowers grain yield and quality; the fungi also produce deoxynivalenol (DON), zearalenone and HT-2 mycotoxins in kernels, which are harmful to livestock and pose a safety concern in human food (Placinta et al. 1999; Clear et al. 2000; Bottalico and Perrone 2002; Parikka et al. 2008; Tamburic-Ilincic 2010). Consequently, there has been an increase in disease awareness and the development of management strategies to mitigate the adverse effects of FHB on oats (Gavrilova et al. 2008; Mitchell Fetch et al. 2008; Tekauz et al. 2008; Yan et al. 2008, 2010). Several Fusarium species including F. acuminatum Ellis and Everhart, F. avenaceum (Corda: Fr.) Sacc., F. culmorum (W.G. Smith) Sacc., F. equiseti (Corda) Sacc., F. graminearum Schwabe (teleomorph Gibberella zeae (Schwein.) Petch.), F. poae (Peck) Wollenw., and F. sporotrichioides Sherb. have been frequently isolated from Fusarium-infected kernels (Clear et al. 1996; Tekauz et al. 2008, 2011; Tamburic- Ilincic 2010; Xue and Chen 2010, 2014). Among these species, F. graminearum is the principal causal agent of FHB in Canada (Clear et al. 1996, 2000; Tekauz et al. 2008, 2011). Studies on the pathogenicity of these Fusarium species to wheat and barley revealed that only F. graminearum and F. culmorum were highly pathogenic, and the other species were intermediate or weakly pathogenic (Wong et al. 1995; Stack et al. 1997; Xue et al. 2004, 2006). There has been no study on the comparative pathogenicity of Fusarium species causing head blight on oat. Although FHB severity can be reduced or controlled by foliar fungicide applications, the use of genetic resistance is considered as one of the most practical and environmental measures for disease management (Tekauz et al. 2004; Mitchell Fetch et al. 2008; Tekauz et al. 2008; Yan et al. 2008, 2010). High levels of resistance to FHB have been identified in oat germplasm and considerable breeding has been conducted to develop resistant cultivars and lines (Gavrilova et al. 2008; Mitchell Fetch et al. 2008; Tekauz et al. 2008; Yan et al. 2008). The reactions of oat varieties to FHB have commonly been evaluated by field screening, which depends on the natural occurrence of Fusarium spp. or artificial inoculation in a FHB nursery (Mitchell Fetch et al. 2008; Yan et al. 2010). These methods do not account for the effect of the timing of inoculation on oat reactions to FHB, differences in pathogenicity of Fusarium spp., and any possible Fusarium species by oat genotype interactions. As a result, breeders have frequently encountered inconsistent disease reactions in their breeding lines, leading to a significant loss of time and resources (Yan et al. 2010). The objectives of this research were to determine the effect of timing of inoculation on reaction of oat variety to FHB and to compare the pathogenicity of four commonly observed Fusarium spp. in causing FHB under controlled environmental conditions. MATERIALS AND METHODS Plant Materials and Growth Conditions Twelve oat cultivars and lines originating from diverse sources and which had shown different levels of resistance to FHB based on field evaluations (A. McElroy, personal communication) were used (Table 1) for both timing of inoculation and effect of Fusarium species experiments. Seeds were planted in 15-cm-diameter pots containing a mixture of loam soil, sand and composted cow manure (1:1:1, vol/vol/vol), and were maintained at 23258C during the day and at 18208C during the night in a greenhouse. Supplemental light was provided by 300-W metal halide lamps to ensure a 16-h photoperiod and a minimum intensity of 360 mol m 2 s 1. Plants were thinned to three plants per pot (there were three to five tillers from each plant, which yielded at least 10 panicles per pot at the time of inoculation). Five weeks after planting, the plants were supplied with a 1% solution of (NPK) fertilizer. Due to genotypic Table 1. Origins of 12 oat genotypes used in this study Genotype AC Morgan CDC Dancer LAO LAO ND OA Prescott (OA1021-1) SO00013 (OT399) Spurs (IL951241) W00058 W00276 WI-X Origin LRC z, AAFC y, Lacombe, Alberta, Canada CDC, U of S x, Saskatoon, Saskatchewan, Canada LRC, AAFC, Lacombe, Alberta, Canada LRC, AAFC, Lacombe, Alberta, Canada NDStU w, Fargo, North Dakota, USA ECORC v, AAFC, Ottawa, Ontario, Canada ECORC, AAFC, Ottawa, Ontario, Canada CDC, U of S, Saskatoon, Saskatchewan, Canada IAES u, U of I, Urbana, Illinois, USA CRC t, AAFC, Winnipeg, Manitoba, Canada CRC, AAFC, Winnipeg, Manitoba, Canada U of W s, Milwaukee, Wisconsin, USA z LRC, Lacombe Research Centre. y AAFC, Agriculture and Agri-Food Canada. x CDC, U of S, Crop Development Centre, University of Saskatchewan. w NDStU, North Dakota State University. v ECORC, Eastern Cereal and Oilseed Research Centre. u IAES, U of I, Illinois Agricultural Experiment Station, University of Illinois. t CRC, Cereal Research Centre. s U of W, University of Wisconsin.

3 XUE ET AL. * FUSARIUM SPECIES EFFECTS ON FHB OF OAT 519 differences in maturity, seeds were planted over a 2-wk period, so that all genotypes were at the same growth stage for inoculation. Pathogen Species and Inoculum Production To determine the inoculation timing effect on FHB reactions of 12 oat genotypes, a mixture of inoculum from three isolates of F. graminearum, DAOM , DAOM , and DAOM , was used. These isolates were recovered from wheat, and selected based on their pathogenicity on barley and wheat (Xue et al. 2004, 2006) and had been used previously for inoculation in the FHB nursery in Ottawa to screen oat lines for resistance to FHB (Yan et al. 2010). Four Fusarium spp. including F. avenaceum, F. culmorum, F. graminearum, and F. sporotrichioides were used to examine the species effect on the varietal reactions to FHB in the 12 oat genotypes. These Fusarium spp. are either reported to cause FHB on oat or frequently isolated from Fusariuminfected kernels in Canada (Clear et al. 1996, 2000; Tekauz et al. 2008, 2011; Tamburic-Ilincic 2010; Xue and Chen 2010, 2014). One isolate from each of the four Fusarium spp. was used: DAOM , DAOM , DAOM , and DAOM , representing F. avenaceum, F. culmorum, F. graminearum, and F. sporotrichioides, respectively. These four isolates were selected as they were originally isolated from oats and have demonstrated pathogenicity on wheat and barley (Xue et al. 2004, 2006). All Fusarium isolates used in the present study were obtained from the Canadian Collection of Fungal Cultures at Agriculture and Agri-Food Canada s Eastern Cereal and Oilseed Research Centre (ECORC-AAFC), Ottawa, Canada. DAOM stands for Department of Agriculture, Ottawa, Mycology, and is an internationally recognized acronym for the national mycological herbarium of Canada. All isolates were cultured on a modified potato dextrose agar (PDA, with a lowered dextrose content of 10 g L 1, amended with 34 mmole L 1 streptomycin sulfate) and incubated at 22258C under mixed longwave UV and fluorescent lighting on a 12 h light:12 h dark cycle for 14 d. The modified PDA medium was used to reduce mycelium growth, possible mutation and poor vigour, and to increase spore production by the pathogen (Xue et al. 2004). To prepare the inoculum, 0.5 ml of a concentrated macroconidial suspension (approx spores ml 1 ) obtained from the above was spread over the surface of the modified PDA in 9-cm petri dishes and incubated as previously described for 48 h. Ten milliliters of sterile distilled water containing 0.01% Tween 20 (polyoxyethylene sorbitan monolaurate) was then added to each dish, and the surface was scraped gently with a sterile microscope slide to dislodge spores. The resulting conidial suspension was filtered through two layers of cheesecloth and adjusted to spores ml 1 using a haemocytometer. Separate conidial suspensions were prepared for each isolate. For the timing of inoculation experiments, the final suspension used consisted of 1:1:1 (vol/vol/vol) mixture of each of the three F. graminearum isolates. Inoculation To determine the inoculation timing effect on variety reactions of oat to F. graminearum, plants were inoculated at six different stages of development, including the first spikelet just visible [ZGS (Zadoks growth stage) 51], half of ear emerged (ZGS 55), emergence of ear completed (ZGS 59), beginning of flowering (ZGS 61), flowering halfway completed (50% anthesis) (ZGS 65), and flowering completed and some kernels at the early milk stage (ZGS 6973), as classified by 9ks et al. (1974). To determine the effect of the Fusarium species on the varietal reactions to FHB, all plants were inoculated at 50% anthesis stage (ZGS 65). Each panicle was spray-inoculated with 0.2 ml of spore suspension at a concentration of spores ml 1. The inoculum was applied using a DeVilbiss model 15 atomizer (The DeVilbiss Co., Somerset, PA), and the plants were dried for 30 min after inoculation. They were then transferred to a polyethylene humidity chamber in a growth chamber for 48 h, and subsequently returned to the greenhouse bench. The growth chamber was operated at 258C with 12-h photoperiod at a light intensity of 250 mmol m 2 s 1. The chamber was maintained at or near 100% relative humidity by the continuous operation of two ultrasonic humidifiers. Air temperature and humidity in the chamber were monitored with a portable datalogger (Model 21XL Micrologger, Campbell Scientific Canada Corp., Edmonton, AB). Due to limited space in the humidity chamber, and to the inherent difficulty in synchronizing growth stages between oat genotypes for the inoculation timing experiments, the inoculation timing effect was evaluated in six experiments and the Fusarium species effect in four experiments, with two and three oat genotypes in each experiment, respectively. For each inoculation timing or Fusarium species and oat genotype combination, four replicate pots per genotype were used. Pots were arranged in a randomized complete block design in both greenhouse and the humidity chamber. Each experiment included four additional pots of FHB-susceptible oat cultivar Spurs sprayed with sterile distilled water plus the surfactant at the 50% anthesis stage and four pots sprayed with a mixture of spore suspensions of three aggressive F. graminearum isolates (DAOM , DAOM , and DAOM ). These were included as checks against any possible extraneous airborne inoculum in the growth room and to ensure suitability of the inoculum and the environment for infection, respectively. The data from these checks were not included in the statistical analysis. FHB and DON Assessment For the inoculation timing effect experiments, symptoms of FHB were rated as percentage of infected

4 520 CANADIAN JOURNAL OF PLANT SCIENCE spikelets (IS) for all inoculated panicles at the early dough to soft dough stage (ZGS 8385), 2023 d after the 50% anthesis, when panicles were green but infected spikelets showed bleached, ashen grey or pink discoloration. For the Fusarium species effect experiments, the percentage of IS was assessed at four time points (6, 10, 13, and 21 d) after inoculation, to compare the disease development caused by each Fusarium sp. over time. The severity of FHB over time was summarized as area under the disease progress curve (AUDPC) for each pot, using the formula described by Wilcoxson et al. (1975). Plants were hand-harvested at maturity, air-dried, and threshed in the greenhouse using an ALMACO LPT-Stationary Type Plot Thresher (Allan Machine Co., Nevada, IA) to ensure the capture of all small, shrivelled kernels. DON contents in the harvested grains were determined on two FHB susceptible oat genotypes, ND and Spurs, for the experiments to examine the effect of timing of inoculation, and on all 12 oat genotypes for the experiments on Fusarium species effect on the varietal reaction of oat to FHB. The analyses to determine DON contents were done at the Mycotoxin Research Laboratory of ECORC-AAFC, using a 5-g seed sample from each pot. Samples were ground to a fine powder in a Retsch Ultra Centrifugal Mill Type ZM1 (Brinkman Instruments, Inc., Rexdale, ON) and filtered with a 0.75-mm wire mesh. From each ground sample, a 1-g sub-sample was used for DON analysis. The concentration of DON was determined by the competitive direct enzyme-linked immunosorbent assay procedure using monoclonal antibodies as described by Sinha et al. (1995). Statistical Analyses The IS and DON data for each of the six inoculation timing effect experiments and for each of the four Fusarium species effect experiments were checked for homogeneity of variance using Bartlett s test (Steel et al. 1997). The error terms were approximately equal in each experiment of these studies, and hence data of the six inoculation timing effect experiments and those of the four Fusarium species effect experiments were pooled based on the homogeneity of error variance. The data were also checked for normality within each experiment. An angular transformation of the percentage of IS and logarithmic transformation of DON values were used in the analysis of variance to stabilize variances (Snedecor and Cochran 1989). The AUDPC was subjected to analysis of variance without transformation. Treatment means were separated by Fisher s protected least significant difference (LSD) test at a probability level of P 50.05, when treatment effects were significant. Analyses were performed using SAS/STAT (SAS Institute Inc., Cary, NC). RESULTS Inoculation Timing Effect Symptoms of FHB were not visible on any of the 12 oat genotypes inoculated at or before half of the ear emerged (ZGS 5155) and were observed on two of the 12 oat genotypes with IS less than 7% when plants were inoculated at the complete emergence of ears (ZGS 59) (Table 2). Low to moderate levels of FHB, with IS ranging from 0.5 to 32%, developed when the 12 oat genotypes were inoculated at the beginning of flowering (ZGS 61), and severe levels of FHB were observed on all the oat genotypes, with IS ranging from 30 to 74%, when inoculated at or after the 50% anthesis stage (ZGS 6569) (Fig. 1). DON contamination was detected in two selected genotypes (ND and Spurs) at all six time points after inoculation (Table 2). DON concentration ranged from 0.4 to 2.6 ppm when plants were inoculated at or before the complete emergence of ears (ZGS 5159), from 2.7 to 6.9 ppm at the beginning of flowering (ZGS 61), and from 6.6 to 10.0 ppm at or after the 50% anthesis stage (ZGS 6569). Analysis of variance indicated highly significant differences (P50.01) in IS percentage and DON content among the oat genotypes and among the six time points of inoculation (Table 3). The genotype inoculation timing interaction was also highly significant for both IS Table 2. Quantitative differences in percentage of infected spikelets (IS) and deoxynivalenol (DON) content of oat genotypes inoculated with a mixture of three Fusarium graminearum isolates (DAOM , DAOM , and DAOM ) at six time points of plant development under controlled environmental conditions Zadoks growth stage (ZGS) AC Morgan CDC Dancer LAO LAO ND IS (%) OA Prescott SO00013 W00058 Spurs W00276 WI- X DON (ppm) ND Spurs ZGS b 0.0d 0.0b 0.0d 0.0b 0.0c 0.0d 0.0c 0.0d 0.0c 0.0c 0.0d 1.3d 0.4e ZGS b 0.0d 0.0b 0.0d 0.0b 0.0c 0.0d 0.0c 0.0d 0.0c 0.0c 0.0d 0.8e 1.5d ZGS b 0.0d 0.0b 1.0d 0.0b 0.0c 0.0d 0.0c 0.0d 0.0c 0.0c 7.0c 0.6f 2.6c ZGS ab 6.0c 0.5b 21.0c 0.5b 0.5c 22.0c 12.0b 22.0c 12.0b 2.0c 32.0b 2.7c 6.9b ZGS a 40.8b 66.0a 54.0b 70.0a 44.0b 74.0a 51.6a 74.0a 51.6a 30.0b 60.0a 6.9b 10.0a ZGS b 47.6a 67.2a 63.0a 50.4a 61.3a 63.0b 60.2a 63.0b 60.2a 49.0a 63.0a 9.6a 6.6b Mean 15.7fg 15.7efg 22.2def 23.2bcd 20.1efg 17.6fg 26.5ab 20.6cde 26.5ab 20.6cde 13.5g 27.0a 3.6b 4.7a ag Means followed by the same letter within a column or within the row of the treatment mean are not significantly different at P50.05 (LSD).

5 XUE ET AL. * FUSARIUM SPECIES EFFECTS ON FHB OF OAT 521 the experiments aiming to elucidate the effects of the Fusarium species as it allows sufficient time for disease development and assessment prior to the physiological maturity of the plants. Fig. 1. Effect of timing of inoculation with a mixture of three Fusarium graminearum isolates (DAOM , DAOM , and DAOM ) on infected spikelets (IS) and deoxynivalenol (DON) content of oats under controlled environmental conditions. Values are mean across 12 oat genotypes (AC Morgan, CDC Dancer, LAO , LAO , ND990118, OA1019-4, Prescott, SO00013, Spurs, W00058, W00276, and WI-X8177-1) and four replicates for IS and two oat genotypes (ND and Spurs) and four replicates for DON content. Inoculation time points with the same letter above bars are not significantly different at P50.05 (LSD). and DON, indicating that the inoculation timing had different effects on different oat genotypes. On average of 12 oat genotypes for IS and two genotypes for DON, there were no statistical differences in either IS percentage or DON content among the three time points for inoculation before flowering (ZGS 51, ZGS 55, ZGS 59) and between inoculation at 50% anthesis (ZGS 65) and at the flowering completed to early milk stages (ZGS 69) (Fig. 1). However, the IS developing from inoculation at the flowering completed to early milk stages (ZGS 69) were often concurrent with the natural senescence of some early maturing oat genotypes, making the rating of FHB disease severity more difficult. As a result, the 50% anthesis stage (ZGS 65) was chosen for inoculation for Table 3. Analysis of variance for percentage of infected spikelets (IS) and deoxynivalenol (DON) content of oat genotypes inoculated with a mixture of three Fusarium graminearum isolates (DAOM , DAOM , and DAOM ) at six time points of plant development under controlled environmental conditions Mean square Source DF IS DON Replicate 3(3) z Genotype 11(1) 328.8** 1.16** Error A 33(3) Timing 5(5) ** 9.31** GenotypeTiming 55(5) 202.2** 1.85** Error B 180(30) z Values in parentheses are degrees of freedom for DON content. **Significant at P Fusarium spp. Effect Infection with each of four Fusarium species resulted in different rates of FHB symptom development on the 12 oat genotypes (Fig. 2). Symptoms appeared on four genotypes six days after inoculation with F. avenaceum or F. sporotrichioides, but were visible at that time on all 12 genotypes inoculated with F. culmorum or F. graminearum (data not shown). In the early stages (0 to 12 d after inoculation), F. avenaceum usually resulted in a low disease severity, similar to that of F. sporotrichioides, but showed a more rapid disease progression starting 13 d after inoculation (Fig. 2). Twenty-one days after inoculation, F. avenaceum infected 30.6% of spikelets, which was not significantly different from F. culmorum (29.7%) and F. graminearum (30.1%), averaged across the 12 oat genotypes. The disease reached maximum severity 24 d after inoculation, when early maturing genotypes were at or near physiological maturity (data not shown). Significant differences (P 50.05) were observed in AUDPC and DON content in harvested grains among Fusarium spp. and oat genotypes (Table 4). There were also highly significant oat genotype Fusarium spp. interactions for AUDPC and DON content; however, the interaction effects contributed to less than 12.2 and 0.1% of the total variance for AUDPC and DON, respectively, and were considered relatively low compared with the Fusarium spp. and oat genotype effects. DON contents ranged from 1.7 to 74.2 ppm in the harvested grain from plants inoculated with F. culmorum and F. graminearum Fig. 2. Fusarium head blight progress curves of oats inoculated with Fusarium avenaceum, F. culmorum, F. graminearum, or F. sporotrichioides under controlled environmental conditions, as measured by the percentage of infected spikelets (IS). Values are mean across 12 oat genotypes (AC Morgan, CDC Dancer, LAO , LAO , ND990118, OA1019-4, Prescott, SO00013, Spurs, W00058, W00276, and WI-X8177-1) and four replicates.

6 522 CANADIAN JOURNAL OF PLANT SCIENCE Table 4. Analysis of variance for area under the disease progress curve (AUDPC) and deoxynivalenol (DON) content of 12 oat genotypes inoculated with four Fusarium spp. Mean square Source of variation DF AUDPC DON Replicate Fusarium spp * ** Genotype ** 1.4** GenotypeFusarium spp ** 1.1** Error *,**Significant at P50.05 and P50.01, respectively. (Table 5), but were not detectable from those inoculated with F. avenaceum or F. sporotrichioides. Among the four Fusarium species, F. culmorum and F. graminearum were equally highly pathogenic, resulting in AUDPC of 45.3 and 47.3, and DON concentrations of 10.4 and 14.3 ppm, respectively, averaged over 12 oat genotypes (Table 5). There was no significant difference between these two species in either AUDPC or DON content. F. sporotrichioides resulted in the lowest mean value of AUDPC (31.2) and was significantly less pathogenic than the two highly pathogenic species (Table 5). Fusarium avenaceum resulted in a mean value of AUDPC of 36.7, which was not significantly different from those of either the highly pathogenic or the weakly pathogenic species. Among 12 oat genotypes, AC Morgan, CDC Dancer, LAO , OA1019-4, and Prescott had AUDPC ranging from 19.2 to 22.8 averaged across the four Fusarium spp. and were considered moderately resistant to FHB; ND990118, Spurs and W00276 had AUDPC ranging from 28.3 to 47.0 and were considered susceptible; and the remaining genotypes had AUDPC higher than 60 and were considered highly susceptible (Table 5). DISCUSSION This study demonstrated that timing of inoculation had a significant effect on FHB severity and DON concentrations in harvested grains of oat. Severe levels of FHB and high DON contents in harvested grains were observed in plants inoculated at or after the 50% anthesis stage (Fig. 1), suggesting that the inoculation at the 50% anthesis stage is the most appropriate timing as it allowed sufficient time for disease development and assessment prior to physiological maturity of the plant. The results of this study were in agreement with Tekle et al. (2012) who conducted a similar experiment in Norway and reported that oats were more susceptible to F. graminearum at the anthesis stage. The 50% anthesis stage has also been considered the most appropriate timing of inoculation for FHB development on wheat and barley (Xue et al. 2006; Del Ponte et al. 2007). Subsequent to anthesis, dead anthers often stay attached to the ear, and their tissues can serve as favorable growth substrate and infection sites for the Fusarium pathogens (Skinnes et al. 2010; Tekle et al. 2012). Current research on FHB management in oat in Canada has been focussing mainly on the control of F. graminearum and DON, recognized as the primary causal species of FHB and source of mycotoxin contamination, respectively (Clear et al. 1996, 2000; Tekauz et al. 2004; Yan et al. 2010). Little effort has been dedicated to monitoring the presence of other Fusarium spp. and mycotoxins. Results of the present research indicate that F. avenaceum and F. sporotrichioides were also capable of causing moderate to severe development of FHB in oats (Fig. 2). Both species produce no detectable levels of DON using the available ELISA method (Sinha et al. 1995; Desjardins 2006). However, these species, as a group, are important producers of other mycotoxins including beauvericin, diacetoxyscirpenol, enniatins, moniliformin, neosolaniol, HT-2 and T-2-toxins, which Table 5. Quantitative differences in area under the disease progress curve (AUDPC) and deoxynivalenol (DON) content of 12 oat genotypes inoculated with a single isolate of either Fusarium avenaceum (Fa), F. culmorum (Fc), F. graminearum (Fg), or F. sporotrichioides (Fs) AUDPC DON (ppm) Genotype Fa Fc Fg Fs Mean Fc Fg Mean AC Morgan 14.3e 35.0bc 16.9fg 12.4cd 19.6d 10.7abc 4.2fg 7.5cde CDC Dancer 6.9e 28.4bc 41.9cdef 13.9cd 22.8d 4.5c 5.0efg 4.8f Spurs (IL951241) 27.4de 30.0bc 83.8a 46.8abc 47.0bc 7.9abc 10.9bcd 9.4bcd LAO e 16.1c 22.3efg 25.0cd 19.2d 6.7abc 7.8bcdef 7.3cdef LAO a 99.3a 50.3bcd 14.1cd 67.2a 18.3ab 9.5bcde 13.9bc ND de 34.4bc 47.4bcde 13.8cd 28.3cd 17.4ab 6.2defg 11.8bcd OA de 23.6bc 11.6g 22.6cd 22.5d 10.3abc 1.7g 6.0ef Prescott (OA1021-1) 4.4e 33.0bc 37.8def 7.8d 20.7d 5.9bc 10.2bcde 8.1cde SO00013 (OT399) 83.0ab 50.0abc 88.1a 31.3bcd 63.1ab 19.6a 15.5bc 17.6ab W cd 82.5ab 66.3abc 81.3a 68.5a 9.9abc 74.2a 42.1a W de 50.6abc 31.8defg 41.3bcd 38.0cd 5.7bc 7.5cdef 6.6def WI-X bc 60.6abc 69.4ab 64.0ab 64.5ab 8.4abc 18.3b 13.4bc Mean 36.7ab 45.3a 47.3a 31.2b a 14.3a 12.3 ag Means followed by the same letter within a column or within the row of the treatment mean are not significantly different at P50.05 (LSD).

7 XUE ET AL. * FUSARIUM SPECIES EFFECTS ON FHB OF OAT 523 are equally as or more toxic than DON (Thrane et al. 2004; Desjardins 2006; Kokkonen et al. 2010). The presence of different mycotoxins, though at low levels, could result in chronic adverse health effects to humans and livestock fed contaminated oat grain or products. However, only DON levels were determined in this study and the possible presence of other Fusarium mycotoxins and their concentrations in the grains were not examined. This was due to the availability of a method developed at ECORC for quantifying DON levels in grains (Sinha et al. 1995). A corresponding method and expertise were not available for the other mycotoxins mentioned above. The potential role of these moderately pathogenic species, F. avenaceum and F. sporotrichioides, as competitors of F. graminearum on the oat panicle and their prevalence as affected by the environment and oat genotypes may be worthy of future study. Additional research is also needed to define the geographic distribution and importance of F. avenaceum and F. sporotrichioides as contaminants of oat grain in Canada and the possible negative effects if multiple mycotoxins contaminated grains. Oat cultivar resistance to FHB has recently been identified (Yan et al. 2010) in Canada. Of the 12 oat genotypes used in the present study, AC Morgan, CDC Dancer, LAO , OA1019-4, and Prescott were significantly more resistant than other genotypes except for ND990118, Spurs, and W00276, which showed intermediate reactions, on the average of four Fusarium spp. (Table 5). The cultivar reactions were in agreement with previous field observations. Yan et al. (2010) reported that AC Morgan and CDC Dancer were moderately resistant to FHB under field conditions in Ontario and Quebec, and represented the highest level of resistance commercially available in Canadian oat cultivars. 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