548 Epidemiology / Épidémiologie Fumonisin B 1 accumulation and severity of fusarium ear rot and gibberella ear rot in food-grade corn hybrids in Ontario after inoculation according to two methods A.W. Schaafsma, L. Tamburic-Ilincic, and L.M. Reid Abstract: Fusarium verticillioides and Fusarium graminearum are pathogens of corn causing fusarium ear rot and gibberella ear rot, respectively. The mycotoxins fumonisin B 1 (FB 1 ) and deoxynivalenol (DON) are produced by F. verticillioides and F. graminearum, respectively, and commercial millers monitor the levels of these toxins in corn to avoid problems with contamination in finished foods. The objectives of the present study were: (1) to evaluate the susceptibility of corn hybrids to F. graminearum and F. verticillioides, following silk-channel or kernel-wounding inoculations, (2) to evaluate the variability in FB 1 production among food-grade corn hybrids, following silk or wound inoculation with F. verticillioides, and (3) to determine the association between the severity of fusarium ear rot and FB 1 to see if visual ratings of fusarium ear rot are predictive of the tendency of corn hybrids to accumulate FB 1. The experiments were conducted in the field from 1999 to 2002 with different sets of commercial hybrids each year. Mean fusarium ear rot severity and FB 1 accumulation varied among years regardless of the inoculation method. Severity of fusarium ear rot and gibberella ear rot (rating on a scale of 1 7) varied respectively from 1.5 to 2.7 and 1.8 to 3.9, with silk inoculation, and from 2.0 to 2.3 and 2.8 to 4.2, with wound inoculation. For a same inoculation method, hybrids responses differed between F. graminearum and F. verticillioides inoculations, likely influenced by the different environmental conditions favoured by each fungus. There was a high level of variation in FB 1 accumulation among the hybrids within each year; for example, FB 1 accumulation across hybrids grown under the same environmental conditions ranged from not detected to 211.2 ppm in 2001. Severity of fusarium ear rot and FB 1 accumulation after wound inoculation with F. verticillioides were correlated, suggesting that severity symptoms of fusarium ear rot after wound inoculation might be useful to select hybrids that are less likely to accumulate FB 1. Key words: Fusarium graminearum, Fusarium verticillioides, maize, fumonisin, mycotoxin, resistance, visual rating, 557 inoculation methods. Résumé : Le Fusarium verticillioides et le Fusarium graminearum sont des agents pathogènes du maïs respectivement responsables de la fusariose de l épi et du grain et de la fusariose de l épi. Les mycotoxines fumonisine B 1 (FB 1 )et désoxynivalénol (DON) sont respectivement produites par le F. verticillioides et le F. graminearum, et les meuneries commerciales surveillent la concentration de ces toxines dans le maïs afin d éviter les problèmes reliés à la contamination des aliments transformés. Les objectifs de la présente étude étaient (1) d évaluer la sensibilité de maïs hybrides au F. graminearum et au F. verticillioides inoculés par voie des soies et par blessure des grains, (2) d évaluer la variabilité de la production de FB 1 parmi les hybrides de maïs de qualité alimentaire par suite d inoculations des soies ou par blessure avec le F. verticillioides et (3) de déterminer l association entre l intensité de la fusariose de l épi et du grain et la FB 1 de façon à déterminer si la notation visuelle de la fusariose de l épi et du grain permet de prédire la tendance des maïs hybrides à accumuler la FB 1. Des expériences furent menées sur le terrain de 1999 à 2002 avec différentes séries d hybrides commerciaux à chaque année. L intensité moyenne de la fusariose de l épi et du grain et l accumulation de la FB 1 varia selon l année, quelle que soit la méthode d inoculation. Avec l inoculation des soies, l intensité de la fusariose de l épi et du grain et de la fusariose de l épi (cote sur une échelle de 1à7)varia respectivement de 1,5 à 2,7 et de 1,8 à 3,9 et, avec l inoculation par blessure, de 2,0 à 2.3 et 2,8 à 4,2. Pour une même méthode d inoculation, la réponse des hybrides différait entre les inoculations avec le F. graminearum et Accepted 31 October 2006. A.W. Schaafsma 1 and L. Tamburic-Ilincic. University of Guelph, Ridgetown Campus, Ridgetown, ON N0P 2C0, Canada. L.M. Reid. Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada, Ottawa, ON K1A 0C6, Canada. Contribution No. 07-756 from the Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada. 1 Corresponding author (e-mail: aschaafs@ridgetownc.uoguelph.ca). Can. J. Plant Pathol. 28: 548 557 (2006)
Schaafsma et al.: corn / fusarium ear rot / gibberella ear rot / mycotoxins / inoculation methods 549 celles avec le F. verticillioides, vraisemblablement influencée par les différentes conditions environnementales privilégiées par chaque champignon. Il y avait une forte variation dans la teneur en FB 1 parmi les hybrides à l intérieur de chaque année; par exemple, dans les mêmes conditions environnementales, la teneur en FB 1 d un hybride à l autre alla d indétectable à 211,2 ppm en 2001. L intensité de la fusariose de l épi et du grain et la teneur en FB 1 après inoculation par blessure avec le F. verticillioides étaient corrélées, ce qui suggère que les symptômes d intensité de la fusariose de l épi et du grain après inoculation par blessure pourraient être utiles pour la sélection d hybrides moins susceptibles d accumuler de la FB 1. methods Mots clés : Fusarium graminearum, Fusarium verticillioides, maïs, fumonisine, mycotoxine, résistance, cote visuelle, méthodes d inoculation. Schaafsma et al.: corn / fusarium ear rot / gibberella ear rot / mycotoxins / inoculation Introduction Interest in expanding food-grade corn production in Canada to offset imports has grown in recent years (Doidge 1997). One of the main barriers to viable food-grade corn production is the potential for Fusarium toxins to accumulate in the grain. Early-maturing corn hybrids suitable for both the food industry and agriculture in Ontario were identified in a concurrent study (Darrah et al. 2001), which also provided the opportunity to examine these same hybrids for their reaction to Fusarium spp. and their propensity to accumulate mycotoxins. Fusarium graminearum Schwabe (teleomorph Gibberella zeae (Schw.) Petch) and Fusarium verticillioides (Sacc.) Nirenberg (= Fusarium moniliforme Sheldon) (teleomorph Gibberella fujikuroi (Sawada) Wr.) are species of Fusarium frequently isolated from corn ears in Ontario, Canada (Vigier et al. 1997). Fusarium graminearum causes gibberella ear rot and produces the mycotoxin deoxynivalenol (DON) in the grain, while F. verticillioides causes fusarium ear rot and produces the mycotoxin fumonisin B 1 (FB 1 ) (Nelson et al. 1993). Canada has no guidelines for either toxin in foodgrade corn, but both may cause potentially serious animal and human health problems (Miller and Trenholm 1994). Commercial millers monitor the levels of these toxins in corn to avoid problems with contamination in finished foods. Fumonisins and DON are found frequently in corn samples in Ontario. Fumonisins were recovered from naturally infected corn grown in Ontario for the first time in 1993 (Miller et al. 1995). Naturally contaminated corn samples from Eastern Canada have been reported to contain fumonisin concentration averaging 0.31 µg g 1 with a maximum of 1.71 µg g 1 (Campbell et al. 2002). In contrast, corn inoculated with F. verticillioides, using the silk method, contains much more fumonisin, with concentrations ranging from 4.0 µg g 1 to 255.0 µg g 1 (Kleinschmidt et al. 2005). DON has been reported in 73% of the corn samples in Ontario at concentrations 0.2 µg g 1, and approximately 23% of the samples were contaminated with FB 1 at concentrations 1.0 µg g 1 (Hooker and Schaafsma 2005). Fusarium graminearum growth in Ontario is favoured by temperatures around 28 C and wet conditions (Sutton 1982; Miller et al. 1995), whereas F. verticillioides is favoured by temperatures 2 to 3 C higher and dry conditions (Miller et al. 1995; Reid et al. 1999). High incidence of FB 1 (58%) and DON (88%) in corn occurred in 1999, in Ontario, when near-normal rainfall and above-normal temperatures prevailed from mid-june to mid-october (Hooker and Schaafsma 2005). Significant interactions between genotype and environment were reported for fumonisin accumulation in Argentina (de la Campa et al. 2005). In the same study, insect damage significantly increased fumonisin production. However, hybrid-corn genetics had more impact than environment on DON and FB 1 accumulation in Ontario (Hooker and Schaafsma 2005). Selecting for genetic resistance of corn to F. graminearum may help mitigate the risk of mycotoxin contamination. However, Schaafsma et al. (1997) found that only a few hybrids exhibited consistent resistance to F. graminearum across environments, with wound inoculations and silk inoculations giving different results. They also reported that rating hybrids visually for severity of gibberella ear rot, without the high cost of DON analysis, may be appropriate if done over several years. Kleinschmidt et al. (2005) were the first to report on visually evaluating resistance of hybrids to F. verticillioides in relation to their propensity to accumulate fumonisins. They used only the silk-channel method of inoculation and found that the amount of visible fusarium ear rot after inoculation was not highly correlated with the amount of FB 1. Furthermore, these silk inoculations resulted in unnaturally high levels of FB 1 in some of the food-grade corn hybrids tested (Kleinschmidt et al. 2005). Fumonisin has been reported in asymptomatic kernels (Rheeder et al. 1992), and kernel wounding factored prominently in the contamination of grain with this toxin (de la Campa et al. 2005). The objectives of the present study were: (1) to determine if the susceptibilities of food-grade corn hybrids to fusarium ear rot and gibberella ear rot are correlated, following silkchannel or kernel-wounding inoculations, (2) to compare FB 1 concentrations after silk or wound inoculation with F. verticillioides, (3) to determine if fusarium ear rot severity and FB 1 accumulation are correlated, and (4) to determine a suitable method of inoculation as well as a suitable method of rating to screen corn hybrids for their tendency to accumulate FB 1. Materials and methods Experimental design and plant material Two similar experiments were conducted each year in early May in 1999, 2000, 2001, and 2002 at Ridgetown, Ontario; one for F verticillioides without mist irrigation and the other for F. graminearum with mist irrigation. Each experiment was designed as a split plot arranged in a randomized complete block design, with blocks replicated three
550 Can. J. Plant Pathol. Vol. 28, 2006 times. Main plots were hybrids and these were split into two equal subplots with one assigned to the silk-channel inoculation and the other to the kernel-wounding inoculation. Main plots were single rows spaced 0.76 m apart and 4 m long, with 35 seeds planted per plot. Subplots were one half of each main plot or 2 m rows. Different sets of commercial hybrids were planted each year, following the annual list for performance testing of food-grade hybrid-corn candidates (Darrah et al. 2001), and all hybrids were tested to each pathogen. One hybrid (Pioneer 34P93) was common to all 4 years of the study. Corn hybrids were planted in early May and thinned to 25 plants per plot during June of each year. The crop was maintained according to provincial recommendations for weed control and plant nutrition (Baute 2002). Inoculum preparation and inoculation Single-spore cultures of F. verticillioides (isolate DAOM 195167) and F. graminearum (isolate DAOM 180378) were generated and inoculum was produced according to Schaafsma et al. (1993). For the silk-inoculation method, a 2 ml aliquot of a suspension of F. graminearum or F. verticillioides macroconidia at a concentration of 10 5 conidia/ml was injected with a needle into the silk channels of individual ears (Reid et al. 1993). For the wound-inoculation method, the kernels were damaged directly via penetration of the husk with 25 brass pins embedded in a lead square measuring 2.5cm 2.5cm.A2mLaliquot of a suspension with 10 5 conidia/ml was sprayed directly into the wound area (Schaafsma et al. 1993). Ten primary ears of each hybrid were inoculated once per replicate, per inoculation method in each subplot, for each of the two pathogens. Silk inoculations were done at 50% silk browning (Reid et al. 1992), while wound inoculations were done after 600 crop heat units (Brown and Bootsma 1993) accumulated post 50% silking, when kernels were most susceptible to wound infection (Schaafsma et al. 1993; Reid et al. 2002). The single inoculations were spread over 3 weeks to accommodate the range of hybrid maturities. Plots inoculated with F. graminearum were irrigated using overhead misting (Schaafsma et al. 1997), from the first date of inoculation until 3 weeks after the last one for each year. Plots inoculated with F. verticillioides were not irrigated, to encourage dryer conditions more favourable for this fungus (Miller et al. 1995). Evaluation of severity of fusarium ear rot and gibberella ear rot Ears were rated for severity of fusarium ear rot and gibberella ear rot in mid-october of each year. For the silkchannel inoculation method, the percentage of kernels covered with fungal growth was rated according to a 1 7 scale (Reid et al. 1993): 1, 0%; 2, 1% 3%; 3, 4% 10%; 4, 11% 25%; 5, 26% 50%; 6, 51% 75%; 7, >75%. This scale was modified for disease-severity rating under the wound-inoculation method: 1 3, no girdling of the cob observed; 4 7 girdled cobs observed. When plots were inoculated with F. verticillioides, if the mycelium was white to salmon in colour, we assumed that fusarium ear rot was the disease manifested. When plots were inoculated with F. graminearum, if the mycelium showed pink, red, and purple colouration, we assumed that gibberella ear rot was manifested. An average severity rating was calculated per replication for each subplot in both experiments. Determination of FB 1 levels In the experiment with F. verticillioides, 10 ears from each plot were harvested at maturity for FB 1 analysis and threshed by hand in November of each year. Only the hybrids from the F. verticillioides experiment were used for FB 1 analysis. The grain from each plot was pooled and these single-plot samples were ground through a No. 60 grist mill (C.S. Bell Co., Tiffin, Ohio); the resulting meal was thoroughly mixed by hand. A 250 g subsample of meal was then ground finely through a grinding subsampling mill (model 2A, Romer Labs, Inc., Union, Mo.). Extractions were made from a 50 g sample of this flour, using 100 ml of acetonitrile (C 2 H 3 N) and 4% aqueous KCl in a ratio of 9:1 (v/v). Quantification of FB 1 was achieved under thin layer chromatography with a limit of detection of approximately 1.0 µg g 1 (Schaafsma et al. 2002). Statistics Data for minimum and maximum daily temperatures and rainfall were obtained from the Environment Canada automated weather station located at Ridgetown, Ontario, for 30 days after corn inoculation in each year and these were compared with the 30-year averages from the same weather station for the same period. Normality and homogeneity of variance for the data was tested using PROC UNIVARIATE version 8 (SAS Institute Inc. 1999), and the data were transformed using ln(x +1)as required. Analyses of variance with the general linear model (PROC GLM) were conducted by year (separately for the variables gibberella ear rot severity, fusarium ear rot severity, and FB 1 accumulation) because different corn hybrids were used each year. Hybrid response to the variables FB 1 accumulation, fusarium ear rot severity, and gibberella ear rot severity were compared using a Pearson s and Spearman s rank correlation coefficient (SAS Institute Inc. 1999) because the hybrids were tested in two different experiments. The level of significance used for statistical analyses was P= 0.05 unless otherwise stated. Results Environmental conditions varied among the 4 years of the study (Fig. 1). Average temperature and rainfall were near normal in 1999 and 2001 compared with the 30-year averages. In 2000, temperatures were lower while mean rainfall was much higher than the 30-year averages, and in 2002, temperatures were very high and rainfall was low. Gibberella ear rot severity, after silk inoculation with F. graminearum, ranged from 2.7 to 5.5, 2.1 to 5.5, 1.6 to 5.8, and 1.2 to 2.8 in 1999, 2000, 2001, and 2002, respectively. After wound inoculation, gibberella ear rot severity ranged from 2.9 to 5.7, 3.3 to 4.6, 2.1 to 4.7, and 1.8 to 3.7 in 1999, 2000, 2001, and 2002, respectively. Mean severity of gibberella ear rot after either silk or wound inoculation with F. graminearum was similar in 1999, 2000, and 2001, but lower in 2002 (1.8 and 2.8, respectively) (Table 1).
Schaafsma et al.: corn / fusarium ear rot / gibberella ear rot / mycotoxins / inoculation methods 551 Table 1. Gibberella ear rot severity in food-grade corn hybrids after silk or wound inoculation with Fusarium graminearum, and fusarium ear rot severity and fumonisin B1 (FB1) levels in food-grade corn hybrids after silk or wound inoculation with Fusarium verticillioides, at Ridgetown, Ontario, 1999 2002. Disease severity a Gibberella ear rot Fusarium ear rot Concentration of FB 1 (ppm) d Hybrid Silk b Wound c Silk b Wound c Silk Wound 1999 Dekalb DK483 4.3 5.1 1.3 1.8 9.4 50.0 Dekalb DK540 4.6 4.5 1.3 1.2 25.3 36.7 Dekalb DK579 4.0 2.9 1.6 1.1 10.8 7.3 Direct D75 4.2 4.2 1.8 2.2 14.4 73.3 Direct 9979xFG 4.3 4.8 1.2 1.4 22.0 25.0 Enerfeast 2.8 3.1 1.2 2.3 10.0 46.7 Garst 8527W 3.6 4.0 1.2 1.6 10.2 13.7 Maizex dry 1 5.5 4.1 1.9 2.5 46.7 66.7 Maizex dry 2 4.2 4.9 1.6 2.7 22.0 50.0 Maizex wet 1 4.4 4.0 1.3 1.9 25.6 60.0 Maizex wet 2 3.8 4.5 1.6 2.2 23.5 60.0 Mycogen X29565 3.7 3.7 1.9 1.7 8.0 46.7 Novartis G-4030 4.0 3.0 1.0 1.8 2.0 80.0 Novartis MAX 23 2.8 3.8 1.1 3.2 7.3 20.0 Novartis MAX 86 3.1 4.9 1.3 2.5 9.1 50.0 Novartis MAX 357 2.7 4.0 1.4 1.8 32.4 70.0 Novartis N47-H7 3.8 3.9 1.6 1.9 27.2 9.3 Novartis NX4616 3.4 3.9 2.1 2.1 43.3 33.3 Novartis Wilson 1096 3.4 3.7 1.3 2.0 50.7 63.3 Pioneer 34K77 4.6 4.2 1.2 1.6 17.4 46.7 Pioneer 34P93 4.4 3.9 1.2 1.4 3.1 10.0 Pioneer 3443W 5.2 5.7 2.0 2.4 27.4 40.0 Pioneer 3515 3.5 3.7 1.1 2.3 2.5 40.0 Pioneer 3532 4.2 4.3 1.5 1.6 13.9 66.7 Pioneer 36B08 3.9 4.8 1.7 2.4 27.4 110.0 Pioneer 36K50 3.8 3.2 1.5 1.5 6.7 36.7 Pioneer 36Y95 4.3 4.2 1.5 2.6 25.6 90.0 Pioneer 36Y96 4.5 4.4 1.8 3.0 100.0 85.0 Pioneer 3737 3.9 5.1 1.2 1.9 4.1 36.7 Pioneer 3790 3.4 3.3 1.4 2.3 23.6 42.7 Pride K378 3.3 3.8 1.6 2.2 22.9 34.0 Pride K469 3.0 3.8 1.3 2.4 20.0 86.7 Pride L-ZM 548/23 4.2 4.5 1.1 1.6 3.0 24.0 Pride L-ZM 548/28 4.9 4.8 1.6 1.4 9.3 23.3 Renk RK 569 3.5 3.5 1.8 1.9 31.5 66.7 Renk RK 606 3.2 4.0 1.8 2.4 22.1 80.0 Renk RK 864 3.4 4.6 1.4 1.7 20.0 63.3 Mean 3.9 4.2 1.5 2.1 15.8 51.7 Coefficient of variation 25.0 16.6 17.4 24.5 43.0 65.8 2000 Great Lakes 5910 5.5 4.4 2.9 2.5 9.0 10.0 Hyland HL2632 3.3 4.2 4.7 2.8 10.0 9.2 NK E9406 4.0 4.1 1.8 2.0 1.0 10.0 Pioneer 34K77 2.9 3.4 2.1 2.0 4.0 2.8 Pioneer 34P93 4.5 3.8 2.3 2.4 11.0 3.3 Pride K469 2.1 3.3 2.4 2.0 12.0 1.3 Pride X0321 3.8 4.6 2.9 2.1 14.0 4.5 Pride X9324 2.7 3.6 2.3 2.2 6.0 3.8 Mean 3.6 3.9 2.7 2.3 8.0 6.3 Coefficient of variation 40.1 30.4 18.9 12.6 47.0 35.6 2001 Asgrow RX776W 1.6 2.1 1.8 2.2 24.1 5.2
552 Can. J. Plant Pathol. Vol. 28, 2006 Table 1 (concluded). Disease severity a Gibberella ear rot Fusarium ear rot Concentration of FB 1 (ppm) d Hybrid Silk b Wound c Silk b Wound c Silk Wound B73 Mo17 2.0 3.1 2.6 2.5 18.2 69.7 IFSI 95-2 5.8 4.4 1.8 1.7 11.6 5.1 Lfy (FR810 Lfy728w) 3.1 4.0 2.7 2.4 14.2 211.2 Lfy (MBS62W Lfy728W) 2.5 3.1 2.5 2.4 1.5 16.0 Monsanto EXP 162W 3.6 4.3 2.0 1.4 4.8 Nd NC+ RE557W 2.8 3.4 4.2 2.3 28.3 11.9 Pioneer 32H39 2.6 2.8 1.8 2.6 7.2 46.4 Pioneer 32K72 2.8 3.0 1.3 2.3 1.8 5.7 Pioneer d 33T17 3.5 3.6 1.6 2.8 7.4 7.0 Pioneer 3394 2.4 2.6 1.4 2.1 3.3 6.1 Pionner 34P93 3.0 2.7 2.4 3.4 12.7 22.9 Pioneer 3751 2.6 3.6 1.5 2.0 4.8 7.8 Vineyard V420W 4.1 4.2 1.9 1.8 5.7 4.5 Vineyard V431W 3.8 4.6 1.2 3.5 22.4 151.9 Vineyard V433W 2.7 3.1 1.6 2.2 2.0 12.9 Vineyard V445W 3.3 2.9 1.9 2.8 5.9 14.5 Vineyard Vx6122W 2.4 2.5 1.5 1.4 4.2 0.6 Whisnand 50AW 5.8 4.5 2.6 2.3 19.0 11.9 Whisnand 100W 2.9 4.1 2.3 2.0 Nd Nd Zimmerman 1790W 1.9 4.1 1.1 2.0 0.1 1.0 Zimmerman E2010 5.0 4.0 1.7 2.3 2.5 3.0 Zimmerman E8272 4.8 4.7 2.0 2.8 1.5 18.4 Zimmerman Z75W 3.2 3.5 2.0 2.7 2.0 42.8 Mean 3.3 3.5 2.0 2.3 6.3 10.0 Coefficient of variation 21.5 14.1 19.8 37.2 80.9 67.9 2002 Asgrow RX776W 1.4 2.0 1.1 1.7 3.6 17.5 Guelph CGX004W 1.6 2.7 1.4 1.5 5.7 13.2 Guelph CGX010W 2.6 3.0 1.4 2.1 6.0 31.8 Guelph CGX013W 2.3 3.2 1.4 1.6 3.9 7.2 Monsanto EXP 162W 1.4 3.3 2.2 2.8 33.4 48.6 NC+ 4950W 1.2 1.8 1.2 2.2 6.8 15.0 NC+ 5642W 1.5 2.3 1.2 1.7 4.8 11.1 Pioneer 32A85 2.0 2.6 1.5 2.1 19.0 39.5 Pioneer 32K72 1.6 2.4 1.3 1.6 1.8 13.0 Pioneer 33T17 2.5 3.0 1.8 2.3 19.8 62.2 Pioneer 34P93 2.8 3.3 1.4 2.0 8.6 35.1 Pioneer Brand 32W8 2.1 2.8 1.5 1.7 15.8 24.4 Pioneer Brand 3394 2.2 2.7 1.5 1.8 7.4 7.6 Vineyard V433W 1.7 2.7 1.3 1.8 5.5 8.3 Vineyard Vx7120W 1.5 2.5 1.2 1.7 4.5 12.3 Whisnand 50AW 1.5 3.1 1.8 3.5 22.0 85.6 Whisnand 100W 1.2 3.6 1.9 2.4 19.8 42.2 Yellow check B73 Mo17 1.4 3.0 1.7 1.7 7.4 19.0 Zimmerman 1790W 2.1 2.7 1.1 2.2 1.7 39.6 Zimmerman WX7528 1.3 3.7 1.6 2.1 6.9 21.2 Mean 1.8 2.8 1.5 2.0 7.9 25.1 Coefficient of variation 14.0 14.9 23.9 21.4 44.1 24.5 Note: Nd, not detected. a Rated on a scale of 1 to 7, according to the percentage of kernels that were covered with mold: 1, 0%; 2, 1% 3%; 3, 4% 10%; 4, 11% 25%; 5, 26% 50%; 6, 51% 75%; 7, >75%. b A macroconidial suspension (2 ml) of F. graminearum or F. verticillioides at a concentration of 10 5 conidia/ml was injected into the silk channels of individual ears with a syringe. c The kernels were directly damaged via penetration of the husk with 25 brass pins embedded in a lead square measuring 2.5 cm 2.5 cm, previously dipped in a conidial suspension of F. graminearum or F. verticillioides, delivering 2 ml of a suspension with 10 5 conidia/ml into the wound area. d Transformed using ln(x + 1).
Schaafsma et al.: corn / fusarium ear rot / gibberella ear rot / mycotoxins / inoculation methods 553 Fig. 1. Mean temperature and cumulative rainfall for the 30 days after corn inoculation in 1999 2002, and the 30-year averages based on data from June and July in each year, at Ridgetown, Ontario. Fig. 2. Disease severity (on a scale of 1 to 7) + standard error in food-grade corn hybrid Pioneer 34P93 after silk or wound inoculation at Ridgetown, Ontario, 1999 2002. Gibberella ear rot severity was rated after inoculation with Fusarium graminearum, and fusarium ear rot severity was rated after inoculation with Fusarium verticillioides. Fig. 3. Fumonisin B 1 accumulation + standard error in the foodgrade corn hybrid Pioneer 34P93 after silk or wound inoculation with Fusarium verticillioides at Ridgetown, Ontario 1999 2002. Fusarium ear rot severity, after silk inoculation with F. verticillioides, ranged from 1.0 to 2.1, 1.8 to 4.7, 1.1 to 4.2, and 1.1 to 2.2 in 1999, 2000, 2001, and 2002, respectively. After wound inoculation, fusarium ear rot severity ranged from 1.1 to 3.2, 2.0 to 2.8, 1.4 to 3.5, and 1.5 to 3.5 in 1999, 2000, 2001, and 2002, respectively. Fusarium ear rot severity in Pioneer 34P93 was consistently higher across all years after wound inoculation than after silk inoculation (Fig. 2). The highest fusarium ear rot severity (4.7) across all years was found in hybrid Hyland HL2632 in 2000 after silk inoculation (Table 1). FB 1 levels, after silk inoculation with F. verticillioides, ranged from 2.0 to 100.0 ppm, 1.0 to 14.0 ppm, not detected to 28.3 ppm, and 1.7 to 33.4 ppm in 1999, 2000, 2001, and 2002, respectively (Table 1). After wound inoculation, FB 1 levels ranged from 7.3 to 110.0 ppm, 1.3 to 10.0 ppm, not detected to 211.2 ppm, and 7.2 to 85.6 ppm in 1999, 2000, 2001, and 2002, respectively. The highest mean concentration of FB 1 across all years and hybrids, after both methods of inoculation (15.8 and 51.7 ppm, respectively), occurred in 1999 (Table 1). Generally hybrids had higher mean levels of FB 1 after wound inoculation than after silk inoculation in all years, except in 2000 (Table 1). In addition, the FB 1 concentration in hybrid Pioneer 34P93 across the years was also higher after wound inoculation than after silk inoculation, except in 2000 (Fig. 3). In 1999, Pioneer 36Y96 had the highest level of FB 1 after silk inoculation, while Pioneer 36B08 had the highest level of FB 1 after wound inoculation (Table 1). After silk inoculation, Pioneer 36Y96 had low fusarium ear rot severity but high FB 1 level in 1999. In contrast, Dekalb DK579 had low fusarium ear rot severities and FB 1 levels after wound or silk inoculation in 1999 (Table 1).
554 Can. J. Plant Pathol. Vol. 28, 2006 Table 2. Analysis of variance for the effect of the hybrid, the method of inoculation, and their interaction on gibberella ear rot severity in food-grade corn hybrids after silk or wound inoculation with Fusarium graminearum and on fusarium ear rot severity and fumonisin B1 (FB 1 ) levels in food-grade corn hybrids after silk or wound inoculation with Fusarium verticillioides, at Ridgetown, Ontario, 1999 2002. df Mean square F P >F a Severity of gibberella ear rot 1999 Hybrid (H) 36 4.399 4.93 0.0001 Method (M) 1 4.027 8.04 0.0059 H M 36 0.655 1.31 0.1651 2000 Hybrid 7 3.139 1.39 0.2851 Method 1 1.488 3.59 0.0764 H M 7 1.027 2.48 0.0631 2001 Hybrid 23 4.559 12.41 0.0001 Method 1 2.695 7.57 0.0084 H M 23 0.987 2.77 0.0014 2002 Hybrid 19 0.895 6.14 0.0001 Method 1 31.110 113.37 0.0001 H M 19 0.620 2.26 0.0149 Severity of fusarium ear rot 1999 Hybrid 36 0.567 2.32 0.0012 Method 1 14.635 88.56 0.0001 H M 36 0.379 2.30 0.0013 2000 Hybrid 7 2.108 10.02 0.0002 Method 1 2.231 25.46 0.0001 H M 7 0.697 7.95 0.0003 2001 Hybrid 23 1.186 2.03 0.0207 Method 1 3.438 8.87 0.0045 H M 23 1.031 2.66 0.0021 2002 Hybrid 19 0.713 4.30 0.0001 Method 1 8.965 59.44 0.0001 H M 19 0.203 1.34 0.2112 FB 1 level 1999 Hybrid 36 2 792.515 2.16 0.0027 Method 1 22 662.382 27.55 0.0001 H M 36 926.226 1.13 0.3273 2000 Hybrid 7 50.857 1.18 0.3762 Method 1 27.000 0.65 0.4336 H M 7 52.333 1.25 0.3336 2001 Hybrid 23 16 695.207 2.91 0.0001 Method 1 60 382.843 14.09 0.0005 H M 23 10 607.058 2.48 0.0041 2002 Hybrid 19 1 601.289 3.58 0.0004 Method 1 11 154.832 25.17 0.0001 Table 2 (concluded). df Mean square F P >F a H M 19 456.203 1.03 0.4523 Note: The analysis of variance was conducted separately for severity of gibberella ear rot, severity of fusarium ear rot, and FB 1 accumulation. df, degree of freedom. a The level of significance for the statistical analyses was P = 0.05. The lowest mean level of FB 1 across years and hybrids occurred in 2000 for silk and wound inoculations combined (8.0 and 6.3 ppm, respectively) (Table 1). There was a high level of variation in FB 1 accumulation among the hybrids (from not detected to 211.2 ppm after wound inoculation) in 2001. In the same year, hybrid Lfy (FR810 Lfy728w) had the highest detected level (211.2 ppm) of FB 1 across years and hybrids (Table 1). Novartis G-4030 had high FB 1 levels after wound inoculation but not after silk inoculation (80.0 vs. 2.0 ppm) in 1999 (Table 1). There was no FB 1 detected in hybrid Whisnand 100W after both methods of inoculation in 2001 (Table 1). However, in 2002, Whisnand 100W had 19.8 and 42.2 ppm of FB 1 after silk and wound inoculations, respectively (Table 1). A comparison across all years of hybrid performance of Pioneer 34P93 showed that gibberella ear rot severity was higher than fusarium ear rot severity for all growing seasons except for 2001, when fusarium ear rot severity after wound inoculation was higher. Fusarium ear rot severity was more severe after wound inoculation than silk inoculation with the opposite for gibberella ear rot severity, except in 2002 (Fig. 2). In addition, FB 1 accumulation was increased about twofold by wound inoculation compared with silk inoculation, except for the 2000 growing season (Fig. 3). Hybrids and the method of inoculation significantly influenced fusarium ear rot severity in all years and the gibberella ear rot severity and FB 1 accumulation in 1999, 2001, and 2002 (Table 2). There was a significant interaction between hybrids and the method of inoculation for fusarium ear rot severity in all years, with the exception of 2002 (Table 5). This interaction was also significant for FB 1 accumulation in 2001 and for gibberella ear rot severity in 2001 and 2002 (Table 2). Pearson s and Spearman s rank correlations were calculated and found to be similar, therefore, only Spearman s rank correlation coefficient is presented in Table 3. Data on gibberella ear rot severity after silk or wound inoculation were correlated after inoculation with F. graminearum in all years, except in 2002 (Table 3). Data on fusarium ear rot severity after silk or wound inoculation with F. verticillioides were correlated in 2000 and 2002, but not in 1999 and 2001 (Table 3). FB 1 levels after both inoculation methods were significantly correlated within the years, with the exception of 2000 (Table 3). There was a significant correlation between fusarium ear rot severity and FB 1 accumulation after silk inoculation in all years, except in 2001 (Table 3). However, the highest and most consistent correlation among all variables tested was between fusarium ear rot severity and FB 1 accumulation after wound inoculation (r = 0.51, 0.63, 0.73, and 0.78 in 1999, 2000, 2001, and 2002, respectively) (Table 3).
Schaafsma et al.: corn / fusarium ear rot / gibberella ear rot / mycotoxins / inoculation methods 555 Table 3. Spearman s correlation coefficients among gibberella ear rot severity in foodgrade corn hybrids after silk or wound inoculation with Fusarium graminearum, fusarium ear rot severity, and fumonisin B 1 (FB 1 ) levels in food-grade corn hybrids after silk and wound inoculation with Fusarium verticillioides, at Ridgetown, Ontario, 1999 2002. Gibberella ear rot Fusarium ear rot FB 1 Silk Wound Silk Wound Silk 1999 Fumonisin B 1 Wound NS NS 0.32* 0.51** 0.52** Silk NS NS 0.49** 0.36* Fusarium ear rot Wound NS NS NS Silk NS NS Gibberella ear rot Wound 0.48** 2000 Fumonisin B 1 Wound 0.65* 0.81* NS 0.63** NS Silk NS NS 0.66* NS Fusarium ear rot Wound NS NS 0.69* Silk NS NS Gibberella ear rot Wound 0.69* 2001 Fumonisin B 1 Wound NS NS NS 0.73** 0.37* Silk NS NS NS NS Fusarium ear rot Wound NS NS NS Silk NS NS Gibberella ear rot Wound 0.71** 2002 Fumonisin B 1 Wound NS 0.45* 0.56* 0.78** 0.66** Silk NS 0.60* 0.81** 0.62** Fusarium ear rot Wound NS 0.40* 0.47* Silk NS 0.75** Gibberella ear rot Wound NS Note: *, P < 0.05; **, P < 0.0001; NS, not significant. Discussion Disease severities were greater for both fungi in 1999 and 2001, when average temperatures and rainfall were near normal compared with the 30-year averages (Fig. 1). Both F. verticillioides and F. graminearum reach maximum growth around 28 C, however, a decline was observed for F. graminearum at higher temperatures, but not for F. verticillioides (de la Campa et al. 2005). In 2000, growth of Fusarium graminearum was favoured over that of F. verticillioides by lower temperatures and higher rainfall than the 30-year averages (Fig. 1). In 2002, in contrast, F. verticillioides growth and FB 1 accumulation were favoured over those of F. graminearum by higher temperatures and lower rainfall than the 30-year averages. Other studies (Sutton 1982; Miller et al. 1995; Reid et al. 1999; de la Campa et al. 2005) have also found that higher rainfall stimulates F. graminearum growth, while hot and dry conditions stimulate F. verticillioides growth and FB 1 accumulation. This study was not designed to compare hybrids across environments and to study genotype environment interactions. However, ear rot severity and FB 1 accumulation varied among years regardless of inoculation method. In addition, hybrids and the method of inoculation significantly influ-
556 Can. J. Plant Pathol. Vol. 28, 2006 enced fusarium ear rot severity in all years and the gibberella ear rot severity and FB 1 accumulation in 1999, 2001, and 2002. These results emphasize the complexities involved with hybrid screening as affected by the different environments experienced and hybrids used during the 4 years of this study. This emphasizes the need to screen for resistance to gibberella ear rot, fusarium ear rot, and (or) fumonisin accumulation under specific targeted conditions. The range in gibberella ear rot severity produced with inoculation of F. graminearum on food-grade corn hybrids are consistent with those found earlier in commodity corn hybrids (Schaafsma et al. 1993). Moreover, gibberella ear rot severity after silk or wound inoculation with F. graminearum correlated well in all years, except in 2002. We assume that hot and dry weather conditions experienced in 2002 likely stressed and shortened silking, providing unfavourable conditions for F. graminearum propagules to germinate and grow in the silk-channel method. The woundinoculation method is less dependent on environment in that the inoculum is introduced directly into wounded, developing kernels that have high water potential. The hybrids with high gibberella ear rot severity did not necessarily have high fusarium ear rot severity, suggesting that the mechanisms for resistance to each pathogen responsible for these disease are likely different, or the disease may be affected by the different environmental conditions that favour F. graminearum and F. verticillioides growth. Our data suggest that screening for resistance to each type of ear rot ought to be done separately, using the appropriate species of Fusarium under optimized conditions for each species. Canada has no recommended tolerance levels for fumonisins, but the United States Food and Drug Administration recommends 2 to 4 µg g 1 or less for corn and corn products destined for human consumption (Kleinschmidt et al. 2005). FB 1 varied from not detected to 211 ppm in the current study. It was detected in most of the hybrids across all years of the study and its levels were generally higher in our inoculated experiments than those reported from naturally infected corn, which, according to Hooker and Schaafsma (2005), ranged from 0 to 7 ppm with a mean of 1.5 ppm in Ontario from 1993 to 2000. However, the high levels of FB 1 in our inoculated plots were similar to those reported by Kleinschmidt et al. (2005) where they ranged from 4 to 255 µg g 1, after silk inoculation. FB 1 levels for both inoculation methods were correlated across the years of this study, with the exception of 2000, when temperatures were significantly lower while rainfall was much higher than the 30-year averages. The hybrid list tested in 2000 was also smaller than for the other years, which may help to explain why there was no correlation in this year. The correlation between fusarium ear rot severity and FB 1 accumulation after silk inoculation was consistently low throughout all years of this study, supporting the finding of Kleinschmidt et al. (2005) who suggested that disease severity cannot be used to judge the tendency of corn hybrids to accumulate FB 1 when the silk-inoculation technique is applied. For example, we found that the hybrid Pioneer 36Y96 had low fusarium ear rot severity but high FB 1 level after silk inoculation in 1999. Furthermore, high concentrations of FB 1 arising from systemic infections with F. verticillioides can be detected in asymptomatic kernels (Rheeder et al. 1992). Fusarium ear rot severity and FB 1 accumulation after wound inoculation with F. verticillioides were consistently correlated. This relationship was not tested by Kleinschmidt et al. (2005). Therefore, fusarium ear rot symptoms after wound inoculation may be a useful criterion to screen hybrids for their resistance to FB 1 accumulation, perhaps with the goal of keeping the highly susceptible hybrids out of the foodproduction chain. Robertson et al. (2006) also recommended, on the basis of genetic correlation and the employment of wound inoculations 1 week after silk inoculations, that hybrids be screened visually for ear rot without the high cost of FB 1 analysis. In conclusion, this work emphasizes the importance of screening for resistance to fusarium ear rot and gibberella ear rot separately, because the causal organisms respond quite differently to hybrids and the environment. Gibberella ear rot symptoms may be used as a surrogate predictor for DON accumulation after either silk or wound inoculation, as noted in previous studies. However, this is not the case for fusarium ear rot and FB 1 accumulation, where only ear rot severity after wound inoculation is predictive of FB 1. Finally, there is a measurable variability in susceptibility to fumonisin accumulation among food-grade corn hybrids, which is a promising avenue, and that breeding for resistance constitutes a viable path to take. Acknowledgements The authors thank two anonymous reviewers for their useful suggestions. They also want to extend their appreciation to the Ontario Corn Producers Association, Guelph, Ont.; the Ontario Ministry of Agriculture, Food and Rural Affairs; Pioneer Hi-bred Canada Ltd.; and the Canadian Adaptation Council for their financial support. Laboratory and field technical support by Diane Paul and Todd Phibbs of Ridgetown Campus, University of Guelph, Ontario, are gratefully acknowledged. References Baute, T. 2002. Agronomy guide for field crops. Ontario Ministry of Agriculture, Food and Rural Affairs, Publication 811. Queens Printer, Toronto, Ont. Brown, D.M., and Bootsma, A. 1993. Crop heat units for corn and other warm-season crops in Ontario. Ontario Ministry of Agriculture, Food and Rural Affairs, Factsheet No. 93-119, Agdex 111/31. Queens Printer, Toronto, Ont. Campbell, H., Choo, T.M., Vigier, B., and Underhill, L. 2002. Comparison of mycotoxin profiles among cereal samples from eastern Canada. Can. J. Bot. 80: 526 532. 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