RESEARCH
Characterization of Nebraska Isolates of Fusarium graminearum Causing Head Blight of Wheat John F. Hernandez Nopsa, Stephen N. Wegulo,* Anita Panthi, Heather E. Hallen-Adams, Steven D. Harris, and P. Stephen Baenziger
ABSTRACT Fusarium head blight (FHB) is a devastating disease of wheat (Triticum aestivum L.) and other small grain cereals. The disease is caused by several species of Fusarium. This study was conducted to identify the major species of Fusarium causing FHB in Nebraska and to characterize isolates of the species for perithecia and deoxynivalenol (DON) production and for aggressiveness (quantified as disease severity and area under the disease progress curve) on spikes of two winter wheat cultivars. Isolates of Fusarium spp. obtained from wheat spikes and grain collected from FHB-affected winter wheat fields and grain elevators, respectively, in 2007 and 2008 were identified as Fusarium graminearum. The isolates varied widely in perithecia and DON production in vitro and in aggressiveness on wheat spikes of soft red winter wheat cultivars Coker 9835 (FHB susceptible) and VA04W-433 (moderately resistant). Fusarium head blight severity for the three most aggressive F. graminearum isolates was higher in Coker 9835 than in VA04W-433 during the first 7 d after inoculation (DAI) but was comparable in both cultivars from 10 to 21 DAI. Deoxynivalenol concentration and aggressiveness were positively and linearly related (R2 ≥ 0.60, P < 0.05). Isolates that produced higher concentrations of DON were more aggressive than those that produced lower concentrations of the toxin regardless of wheat cultivar.
J.F. Hernandez Nopsa, S.N. Wegulo, A. Panthi, and S.D. Harris, Dep. of Plant Pathology, Univ. of Nebraska, Lincoln, NE 68583; H.E. Hallen-Adams, Dep. of Food Science and Technology, Univ. of Nebraska, Lincoln, NE 68583; P.S. Baenziger, Dep. of Agronomy and Horticulture, Univ. of Nebraska, Lincoln, NE 68583. Received 8 Apr. 2013. *Corresponding author (
[email protected]). Abbreviations: AUDPC, area under the disease progress curve; DAI, days after inoculation; ddH 2O, double distilled sterile water; DON, deoxynivalenol; FHB, Fusarium head blight; ITS, internal transcribed spacer; PCR, polymerase chain reaction; PDA, potato dextrose agar.
F
usarium head blight (FHB) is a destructive disease of wheat and other small grain cereals. It is caused by several species of Fusarium. The predominant species associated with FHB worldwide are Fusarium graminearum Schwabe [teleomorph: Gibberella zeae (Schwein.) Petch], Fusarium culmorum (Wm. G. Smith) Sacc., and Fusarium avenaceum (Fr.:Fr.) Sacc. (Dill-Macky, 2010). During wet, warm weather, spores of these fungi are released from crop residue and infect spikelets on wheat spikes mostly during flowering, leading to premature partial or complete bleaching of spikes. Losses to FHB result from yield reduction, presence of Fusariumdamaged kernels, and accumulation of mycotoxins in grain. The most commonly encountered mycotoxin is deoxynivalenol (DON). In the central Great Plains of the United States, FHB epidemics have occurred sporadically due to a variable climate. However, since the early 1990s, FHB outbreaks have become frequent in this region and other wheat growing areas in the United States (Dill-Macky, 2010). In Nebraska, FHB has occurred yearly to varying levels of severity and prevalence since 2007, with the worst epidemics in
Published in Crop Sci. 54:310–317 (2014). doi: 10.2135/cropsci2013.04.0229 © Crop Science Society of America | 5585 Guilford Rd., Madison, WI 53711 USA All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher.
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over 20 yr occurring in 2007 and 2008. It is known that the most important causal agent of FHB in the United States is F. graminearum (Gale, 2003; Zeller et al., 2004). However, the potential exists for emergence of FHBcausing species other than F. graminearum. For example, Gale et al. (2011) reported the prevalence of Fusarium asiaticum in two Louisiana parishes. Therefore, it is necessary to confirm the species of Fusarium responsible for the recent FHB epidemics in Nebraska. Traditional diagnostic methods for identification of F. graminearum are based on morphological characteristics, but this procedure can be time consuming and may not be accurate in distinguishing between similar species (de Biazio et al., 2008). Species identification using molecular and morphological techniques simultaneously can give more confidence than identification using either method alone. Niessen and Vogel (1997) described a duplex polymerase chain reaction (PCR) method for identification of F. graminearum using a set of primers designed to detect a galactose oxidase-producing G. zeae strain. A fragment of 900 bp was amplified using this method. Based on this technique, Knoll et al. (2002) described an identification method using DNA detection with test strips. However, the authors of this set of primers reported that this method failed to identify one F. graminearum strain that was a galactose oxidase producer (de Biazio et al., 2008). A PCR method for detection of F. culmorum, F. graminearum, and F. avenaceum was published by Schilling et al. (1996). This method used internal transcribed spacer (ITS) regions of nuclear ribosomal DNA but they were sufficiently polymorphic to make a clear distinction among the species analyzed. Today we have increased understanding of the limitations of using ITS for species identification. A new protocol for F. graminearum identification was developed using the internal 3 ¢ coding region of the gaoA gene (de Biazio et al., 2008). This test showed specificity for F. graminearum with concentrations as low as 4.0 ng of DNA. Waalwijk et al. (2003) used a set of 18 primers to identify F. avenaceum, F. culmorum, F. graminearum, Fusarium poae, Fusarium proliferatum, Microdochium nivale var. nivale, and Microdochium nivale var. majus. Once the species of Fusarium causing FHB in a region or state is or are identified, it is important to know if isolates of the species differ in characteristics such as pathogenicity (ability to cause disease), aggressiveness, and perithecia and DON production. Aggressiveness refers to quantitative traits related to pathogenicity (Andrivon, 1993; Pariaud et al., 2009; Van der Plank, 1963). These traits include infection efficiency, latent period, rate of spore production, infectious period, lesion size, and rate of disease progression. For FHB, pathogen aggressiveness can be quantified as disease severity, defined as the percentage of diseased spikelets on a spike (Paul et al., 2005; Wilcoxson et al., 1992). Summation of disease severity assessed multiple times during the course of an experiment results in crop science, vol. 54, january– february 2014
area under the disease progress curve (AUDPC) (Shaner and Finney, 1977), which is commonly used to quantify disease intensity. Due to the recent FHB epidemics in Nebraska and the need to develop management strategies for the disease, laboratory and greenhouse experiments were conducted in 2009 through 2012 to (i) use PCR and morphological characteristics to identify the major Nebraska species of Fusarium causing FHB and (ii) quantify perithecia production, DON production, and aggressiveness of selected isolates of the species of Fusarium identified in objective (i).
MATERIALS AND METHODS Isolation of Fusarium Isolates
Samples of wheat kernels were collected from grain elevators and wheat spikes in FHB-affected winter wheat fields in south central and southeastern Nebraska during the growing season in 2007 and 2008. Fusarium-damaged kernels were disinfested using 1% NaClO (sodium hypochlorite) for 1 min, rinsed with double distilled sterile water (ddH2O), and disinfested again with 70% CH3CH2OH (ethanol) for 1 min followed by a second rinse with ddH2O for 1 min. After disinfestation, two Fusariumdamaged kernels per 9-cm-diameter petri plate were incubated at 25°C in 12 h light and 12 h dark on Nash-Snyder (peptone pentachloronitrobenzene) medium (Leslie and Summerell, 2006) for 5 to 7 d in a low temperature illuminated incubator, model 818 (Thermo Electron Corporation). Ten-millimeterdiameter mycelial plugs from the actively growing edges of the cultures were transferred to potato dextrose agar (PDA) plates. After 4 d, approximately 1 cm 2 of a mycelial plug was placed into an Eppendorf tube containing 1 mL of ddH2O. The mycelia were disrupted with a sterile needle and homogenized with a vortex machine. To increase the probability of obtaining single spores, 100 mL of this suspension was used to make serial dilutions (1:10, 1:100, and 1:1000). Three hundred microliters from each dilution were spread and incubated on 2% water agar plates for 12 to 48 h under the same incubation conditions described above. A single conidium was isolated from each of these plates and placed on Nash-Snyder plates. After 72 h of incubation, transfer to PDA plates was done as described above. Mycelia and spores from the PDA plates were kept in vials at –80°C in a 15% glycerol suspension until needed for experiments. A total of 41 pure culture, single conidium isolates from infected kernels were obtained. Seventeen isolates were from samples collected in 2007 (NE90 to NE110), and 24 isolates were from samples collected in 2008 (NE111 to NE165).
Identification of Fusarium Isolates Protocols for morphological (Leslie and Summerell, 2006) and molecular (de Biazio et al., 2008) characterization for F. graminearum were used. Isolates were grown on carnation leaf agar and PDA for morphological identification. A DNA extraction of each isolate was done using mycelia grown in 25 mL of potato dextrose broth in a 125 mL glass flask on a rotation shaker (New Brunswick Scientific Co. Inc.) at 100 revolutions per minute for 48 to 72 h at 25°C in a 12 h light/dark cycle. Approximately 300 mL of mycelial suspension were used to isolate DNA using the
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MP Biomedicals Geneclean Spin Kit according to the manufacturer’s instructions, with slight modifications. The DNA obtained from all isolates was tested for the suitability of PCR amplification using primers ITS4 and ITS5 targeting the ITS 5.8S ribosomal RNA region (White et al., 1990). A specific PCR amplification was conducted to identify F. graminearum using primers GOFW (5¢-ACCTCTGTTGTTCTTCCAGACGG-3¢) and GORV (5¢- CTGGTCAGTATTAACCGTGTGTG-3¢) (de Biazio et al., 2008). These primers will yield a 434 bp product from F. graminearum while no product is obtained from other Fusarium species or other genera. The amplification was performed in a DNA Engine Peltier thermal cycler, single block model, 60 V a unit (BioRad). The reaction was made in PCR tubes according to de Biazio et al. (2008): 50 mL of final reaction volume, PCR buffer (Invitrogen) 1x, 1.5 mM MgCl 2, 0.2 mM deoxyribonucleotide triphosphates mix (Invitrogen), 25 pmol GOFW, 25 pmol GORV, 20 to 400 ng of DNA, and 1.5 U of platinum Taq DNA polymerase (Invitrogen). The PCR reaction for GOFW and GORV consisted of 25 cycles of 1 min and 30 s at 94°C, 1 min and 30 s at 55°C, and 2 min at 72°C. Amplification with ITS4 and ITS5 primers differed from amplification using GOFW and GORV in the annealing temperature (50°C). For both reactions, an initial heating was done at 94°C for 5 min and a final extension time of 72°C for 10 min was applied. A positive control (DNA from F. graminearum, strain PH-1) was used for amplification and a negative control (no DNA) was also used. Ten microliters of PCR product were analyzed in 1% agarose gel containing 0.25 mg mL-1 of ethidium bromide (3,8-diamino-5-ethyl-6-phenylphenanthridinium bromide) in 1x Tris-borate-ethylenediaminetetraacetic acid buffer at 80 V. Molecular weight markers (100 bp DNA ladder; Invitrogen) were used to determine the weight of the PCR products. Polymerase chain reaction products were visualized and photographed using a Molecular Imager Chemi-doc (BioRad).
Perithecia Production Seventeen Nebraska isolates of F. graminearum collected in 2007 were tested for perithecia production in vitro. Additionally, PH-1 (the sequenced strain, known for its readiness to produce perithecia synchronously in culture) was used as a control. Carrot agar was the medium used for this fertility study (Leslie and Summerell, 2006). The protocol used was a modification of the protocol of Pasquali and Kistler (2006). In a 9-cm-diameter petri plate, 20 mL of carrot agar was poured. A 1-cm-diameter PDA plug from the actively growing edge of the culture of each isolate was transferred onto a carrot agar plate. Each isolate was incubated at 25°C in 12 h light and 12 h dark. After 96 h of incubation, 1 mL of 2.5% Tween 60 was applied to each plate and mycelia were homogenized for 30 s with an L-shaped cell spreader (Fisher Scientific). Plates were incubated as previously described. After 10 d, perithecial units were counted and the percentage of perithecia-covered area in each petri plate was estimated visually. A perithecial unit consisted of a single perithecium or a cluster of perithecia. A randomized complete block design with four replications was used and the experiment was conducted twice.
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Deoxynivalenol Production and Quantification Seven F. graminearum isolates (NE90, NE91, NE97, NE98, NE103, NE110, and NE119) were selected for in vitro DON production and quantification. The isolates were selected based on cultural and morphological characteristics on PDA. The characteristics included sparse or abundant production of mycelia and perithecia, the color of pigments (pink or dark purple) produced in PDA, mycelial color (purple or white), and mycelial growth habit (fluffy or smooth). Production of DON was induced in liquid culture following a two-stage protocol (McCormick et al., 2004). The seven isolates were grown on PDA in 9-cm-diameter petri plates for 7 to 10 d, arranged randomly in a low temperature illuminated incubator set at 25°C and a 12/12 h light/dark cycle. A 1 cm 2 mycelial plug was cut from the outer, actively growing edge of the culture of each isolate and used to inoculate 50 mL of first stage medium (3 g NH4CL, 2 g MgSO4·7 H 2O, 0.2 g of FeSO4·7H 2O, 2 g KH 2PO4, 2 g peptone, 2 g yeast extract, 2 g malt extract, and 20 g glucose in 1 L of distilled water) in 125 mL Erlenmeyer flasks. The cultures were grown at 28°C on a rotary shaker at 220 revolutions per minute in the dark for 3 d. The mycelial culture with a small amount of liquid medium was ground using a mortar and pestle. A 3.5 mL concentrated suspension of ground mycelia was used to inoculate 50 mL of second stage medium consisting of 1 g (NH4)2HPO4, 3 g KH 2PO4, 0.2 g MgSO4·7H 20, 5 g NaCl, 40 g sucrose, and 10 g glycerol in 1 L of distilled water in 125 mL Erlenmeyer flasks. The cultures were grown under the same conditions as the first stage medium for 8 d, harvested by filtration through a 1 mm Whatman filter paper using a Buchner funnel, and lyophilized for 2 d. The dried mycelium was ground using a mortar and pestle. The finely ground powder of fungal mycelium was used for DON quantification. Quantification of DON was as follows: the finely ground fungal mycelium from each isolate was weighed, suspended in distilled water at the rate of 1 mL to 100 mg, and vortexed for 5 min. Each sample was then filtered using a 1 mm Whatman qualitative filter paper. The filtrate was used for DON quantification using a competitive direct enzyme linked immunosorbent assay for DON (Veratox DON 5/5, catalog number 8331; Neogen Corporation) following the manufacturer’s instructions. The entire process was conducted three times sequentially, and each run was considered a replication.
Aggressiveness on Wheat Spikes A greenhouse experiment was conducted twice to quantify the aggressiveness of the seven selected isolates of F. graminearum on the spikes of two soft winter wheat cultivars, Coker 9835 and VA04W-433. Based on field evaluations, Coker 9835 is FHB susceptible and VA04W-433 is moderately resistant to FHB (Carl Griffey, personal communication, 2009; Chen et al., 2012). Seed of the two cultivars was planted in 15-cm-diameter pots. The soil mix consisted of clay loam soil:Canadian sphagnum peat moss:sand:vermiculite in a 2:2:1:1 ratio. Seed was planted at a rate of one seed per pot after 7 wk of vernalization at 4°C. The pots were placed on a greenhouse bench and fertilized daily. Fertilizer consisted of 20:20:20 N:P2O5:K 2O injected daily at a rate of 250 mg L-1 during regular watering.
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Temperature ranged from 20°C (night) to 26°C (day) and lighting was set to a 17/7 h light/dark cycle. The experimental design was a split plot in randomized complete blocks with three replications. Cultivar was the main plot and isolate was the subplot. At mid anthesis (Zadoks growth stage 65; Zadoks et al., 1974) two spikes per pot were each inoculated with 0.5 mL of a spore suspension of each of the seven isolates of F. graminearum at 1 × 105 spores mL-1 using a hand-held bottle sprayer. Following inoculation, each spike was covered with a transparent plastic bag for 3 d to maintain high humidity, which is favorable for infection. The plastic bags were then removed and the plants were incubated on a greenhouse bench for 18 more days under the conditions described above. The spore suspension was obtained from isolates grown on PDA for up to 3 wk in 9-cm-diameter petri plates in a low temperature illuminated incubator set at 25°C and a 12 h light/dark cycle. Five milliliters of ddH 2O was poured onto each plate and conidia were dislodged from the surface of the agar with a plastic L-shaped cell spreader and filtered through two layers of sterile cheesecloth. Spores were quantified using a hemocytometer, adjusted to the final concentration, and kept in a 50 mL Falcon tube at 4°C until needed for inoculation. Inoculation was done within 6 h of inoculum preparation. Disease severity was visually assessed as the percentage of bleached spikelets on each spike 5, 7, 10, 14, and 21 d after inoculation (DAI) and used in trapezoidal integration to calculate AUDPC (Shaner and Finney, 1977).
Table 1. Number of perithecial units and perithecia-covered area of 17 Nebraska isolates of Fusarium graminearum collected from wheat fields and grain elevators in Nebraska in 2007. Exp. 1
Isolate
Exp. 2
PeritheciaPeritheciaPerithecial covered area Perithecial covered area (%) (%)† units units
NE90 NE91 NE92 NE93 NE96 NE97 NE98 NE99 NE100 NE101 NE102 NE103 NE105
11.3 efghi‡ 5.8 fghi 53.5 a 21.8 bcde 26.5 bc 8.3 fghi 0.0 i 21.0 bcde 0.8 hi 16.5 cdef 11.0 efghi 12.8 defgh 13.8 defg
8.8 cde 1.6 ef 18.0 ab 20.0 ab 19.8 ab 4.0 ef 0.0 f 8.5 cde 0.4 f 13.4 bcd 6.1 def 7.0 ddf 13.0 bcd
18.3 b 81.3 ab 129.5 a 26.0 b 11.5 b 13.0 b 0.5 b 5.5 b 0.3 b 13.8 b 6.5 b 18.5 b 16.8 b
11.5 cdefg 14.3 bcdef 28.8 a 15.8 abcde 6.5 defg 6.0 defg 0.1 g 1.1 fg 0.4 g 4.8 efg 6.3 defg 2.0 fg 11.3 cdefg
NE107 NE108 NE109 NE110 PH-1
24.0 bcd 0.0 i 4.0 ghi 32.8 b 14.3 defg
15.8 abc 0.0 f 1.9 ef 23.3 a 4.9 ef
24.3 b 0.5 b 25.3 b 47.8 ab 132.8 a
19.8 abc 0.1 g 8.5 cdefg 27.0 ab 18.0 abcd
†
Percent of the surface area of a 9-cm-diameter petri plate covered by perithecia.
‡
Means within a column followed by the same letter are not significantly different according to the least significant difference test at P = 0.05.
Data Analysis Data were subjected to analysis of variance using the GLM (in vitro experiments) and GLIMMIX (greenhouse experiment) procedures of SAS version 9.2 (SAS Institute, 2008). Data from replicate experiments were analyzed separately (perithecia experiment) or subjected to a combined analysis (aggressiveness experiment) based on heterogeneous and homogeneous error variances, respectively, between the replicate experiments (Gomez and Gomez, 1984). The F-values for treatment effects were considered significant at P ≤ 0.05. The least significant difference test at P = 0.05 (Gomez and Gomez, 1984) was used to compare pairs of treatment means. Linear regression analysis was used to model the relationships between DON concentration measured in vitro and disease severity and AUDPC measured on wheat spikes in the greenhouse.
RESULTS
Fusarium Isolates Polymerase chain reaction analysis using primers ITS4 and ITS5 was tested to verify the quality of amplification of DNA from 41 Nebraska isolates of F. graminearum. A DNA fragment of approximately 550 bp was obtained, as reported by White et al. (1990), indicating that the DNA obtained had optimal conditions for amplification. Amplification with the specific set of primers GOFW and GORV produced a product between 400 and 500 bp for 40 of the 41 isolates, showing that these were F. graminearum. All 41 isolates produced perithecia and macroconidia characteristic of F. graminearum, including crop science, vol. 54, january– february 2014
isolate NE145 whose fragment did not amplify with F. graminearum-specific primers. Sporulation (production of macroconidia and ascospores) was abundant in all isolates except NE160 and NE161.
Perithecia Production Fifteen of the 17 isolates of F. graminearum collected in 2007 produced perithecia on carrot agar. In Exp. 1, the F-value for number of perithecial units (one unit is a single perithecium or a cluster of perithecia) among isolates was highly significant (P < 0.01), indicating that isolates differed in the number of perithecial units they produced (Table 1). In Exp. 2, the F-value for number of perithecial units was nonsignificant at the 5% level. The F-value for the percentage of the petri plate surface covered with perithecial units was highly significant in both experiments (P < 0.01). Isolates NE98 and NE108 produced no or very few perithecia (Table 1). Isolate NE92 produced the largest number of perithecial units, which, along with those of isolate NE110, also covered the largest area on the petri plate (Table 1). Isolates differed in the size of perithecial units they produced. However, this difference was not consistent for some isolates, which produced both small (1- to 3-mm-diameter) and large (4- to 10-mm-diameter) perithecial units.
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Relationship between Deoxynivalenol Concentration in vitro and Aggressiveness on Wheat Spikes Regression analysis showed that DON concentration measured in vitro for seven selected F. graminearum isolates was positively and linearly related to disease severity and AUDPC on wheat spikes (R 2 ≥ 0.60, P < 0.05). This relationship was significant for all 12 regressions at P < 0.05 (Table 3). The results indicated that variation in DON concentration explained 60 to 73% of the variation in FHB severity and AUDPC. Isolates that produced higher concentrations of DON were more aggressive than those that produced lower concentrations of the mycotoxin (Table 3). Figure 1. Deoxynivalenol concentrations determined in vitro for seven selected Nebraska isolates of Fusarium graminearum. Error bars represent standard errors of the mean.
Deoxynivalenol Production and Quantification All seven selected isolates produced DON in vitro. Differences in DON concentration among the isolates were highly significant (P < 0.01). Isolates NE90, NE91, and NE97 produced DON in low concentrations (F
0.67 0.66 0.65 0.63 0.61 0.64
0.0248 0.0264 0.0279 0.0336 0.0378 0.0300
2.5 10.1 19.6 28.0 28.1 379.1
1.5 2.2 2.8 3.2 3.7 49.7
0.67 0.68 0.68 0.60 0.73 0.67
0.0238 0.0226 0.0224 0.0410 0.0146 0.0251
R
The seven F. graminearum isolates were inoculated onto wheat spikes of two soft red winter wheat cultivars, Coker 9835 and VA04W-433, in the greenhouse and Fusarium head blight severity was measured at 5, 7, 10, 14, and 21 d after inoculation (DAI). The same seven isolates were grown on potato dextrose agar in the laboratory and DON concentration was determined in mycelia using enzyme-linked immunosorbent assay.
†
population in Nebraska. Therefore, the level of FHB severity in a given field may depend on the level of aggressiveness of the predominant F. graminearum isolate in that field or localized region. Knopf and Miedaner (2008) spray inoculated spring wheat plots with binary F. graminearum mixtures and single isolates. As in this study, they found that aggressiveness of the isolates varied significantly. Similarly, Malbrán et al. (2012) found 112 isolates of F. graminearum to induce significantly different levels of disease severity under field conditions, indicating variation in aggressiveness. The observation of a wide variation in DON production in vitro among F. graminearum isolates in this study is supported by results reported by other investigators. Gilbert et al. (2002) found DON production in rice (Oryza sativa L.) culture to vary between 0.2 and 249 mg g-1 among 15 Canadian isolates of F. graminearum. In Germany, Ludewig et al. (2005) similarly found that DON production on autoclaved rice varied from 0.1 to 812 mg g-1 among 31 isolates of F. graminearum. crop science, vol. 54, january– february 2014
In this study, regression of FHB severity and AUDPC on DON concentration measured in vitro showed that for the seven selected F. graminearum isolates, variation in DON concentration explained 60 to 73% of the variation in FHB severity and AUDPC (0.60 ≤ R 2 ≤ 0.73). This strong, positive linear relationship between DON concentration in vitro and aggressiveness on wheat spikes suggested that isolates that produced higher concentrations of DON were more aggressive than those that produced lower concentrations of the toxin. The results are consistent with Mesterházy et al. (1999) and Mesterházy (2002) who found significant positive relationships between aggressiveness of F. graminearum and F. culmorum isolates on wheat spikes and their DON production in infected grain. For the three most aggressive isolates (NE103, NE110, and NE119), FHB severity assessed 10, 14, and 21 DAI and AUDPC did not differ between the two wheat cultivars used in this study. Under field conditions, Coker 9835 is susceptible to FHB whereas VA04W-433 is moderately
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resistant (Carl Griffey, personal communication, 2009; Chen et al., 2012). This lack of differences in FHB severity and AUDPC between the two cultivars beyond 7 DAI may have been due to high disease intensity in the greenhouse compared to field conditions. The results suggest that under the conditions in which the experiment was conducted, differences in resistance to FHB between the two cultivars were most apparent before 10 DAI, implying that disease severity assessments beyond 7 DAI were unnecessary. The fact that FHB severity increased over time in VA04W-433 indicates that resistance in this cultivar was expressed, at least in part, as Type II resistance (resistance to pathogen spread in plant tissue) (Schroeder and Christensen, 1963; Mesterházy, 1995) or that Type I resistance (resistance to initial infection) was overcome due to a high inoculum concentration and a favorable environment for infection and disease development. Susceptibility of Coker 9835 and resistance of VA04W-433 were observed during the first 7 d following inoculation and were detected only with the three most aggressive isolates (NE103, NE110, and NE119), indicating that isolates with low aggressiveness may not be suitable for detection of differences in resistance to FHB among cultivars. Fusarium head blight severity during this period was higher in Coker 9835 compared to VA04W-433 regardless of isolate. This finding has implications in the management of FHB in these two cultivars. If this observation holds true under field conditions, the timing of fungicide application to suppress FHB would be more critical for Coker 9835 than for VA04W-433. There was a statistically significant interaction between cultivar and isolate for disease severity assessed at 5 and 7 DAI. Such interaction is sometimes considered to indicate race specificity (Van der Plank, 1968). Races of a pathogen that interact differentially with cultivars are said to vary in virulence (Van der Plank, 1968). In this study, however, the numbers of cultivars used (two) and experiments conducted (two) were insufficient to determine if the interaction was due to race specificity. The observed cultivar × isolate interaction was due to the fact that three isolates with high aggressiveness differentiated cultivars as resistant and susceptible whereas the rest of the isolates did not.
Conclusions This study identified the species of Fusarium causing head blight of wheat in Nebraska as F. graminearum. Results from the study suggest that in Nebraska, there are populations of F. graminearum that differ in perithecia production, DON production, and aggressiveness. The results further indicate that F. graminearum isolates that produce high concentrations of DON are more aggressive than those that produce low concentrations of the mycotoxin. Because F. graminearum also causes ear and stalk rots in corn (Zea mays L.), and wheat is often grown in rotation with corn in Nebraska, this confirmation reinforces the need for growers to adopt 316
crop rotation schemes that avoid planting wheat following corn, especially where a reduced or no-till system is practiced to conserve soil and moisture. Further research is needed to determine the geographical distribution of these populations and hence devise FHB management strategies suited to specific regions in the State. Acknowledgments and Disclaimer This material is based on work supported by the U.S. Department of Agriculture, under Agreement No. 59-0790-7-080. This is a cooperative project with the U.S. Wheat and Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture. We thank Carl Griffey for providing seed for Coker 9835 and VA04W-433.
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