Paul B. Schwarz

Fusarium Species Pathogenic to Barley and Their Associated Mycotoxins

Epidemics of Fusarium head blight (FHB) occurred on barley in Minnesota, North Dakota, and South Dakota from 1993 to 1998. The Red River Valley region was most severely impacted by the disease based on assessments of FHB severity in grain samples harvested from commercial fields. Fusarium graminearum was the primary pathogen causing these FHB epidemics. It comprised from 62 to 64% of all Fusarium species isolated from infected kernels from 1994 to 1996. Fusarium poae (range of isolation 13 to 20%),F. sporotrichioides (10 to 17%), and F. avenaceum (6 to 10%) also were isolated from barley kernels and were likely involved in causing some FHB infection, but to a very limited extent. All four Fusarium species were pathogenic on barley in inoculation tests conducted in both the greenhouse and the field. Mycotoxin screens were performed on barley spikes inoculated with the respective species in the greenhouse. Spikes infected with F. graminearum contained deoxynivalenol and 15-acetyldeoxyni-valenol; those infected with F. sporotrichioides contained T-2 toxin, HT-2 toxin, and T-2 tetraol; and those infected with F. poae contained nivalenol. Some isolates of F. poae also produced 15-acetoxyscirpenol and scirpentriol. Although F. graminearum and DON are recognized as the primary FHB pathogen and mycotoxin, respectively, in barley, the possible presence of other Fusarium species and mycotoxins should not be overlooked.

Identification of QTLs associated with Fusarium head blight resistance in Zhedar 2 barley

Fusarium head blight (FHB) in barley and wheat, caused by Fusarium graminearum, is a continual problem worldwide. Primarily, FHB reduces yield and quality, and results in the production of the toxin deoxynivalenol (DON), which can affect food safety. Identification of QTLs for FHB severity, DON level and related traits heading-date (HD) and plant-height (HT) with consistent effects across a set of environments, would provide the basis for marker-assisted selection (MAS) and potentially increase the efficiency of selection for resistance. A segregating population of 75 double-haploid lines, developed from the three-way cross Zhedar 2/ND9712//Foster, was used for genome mapping and FHB severity evaluation. A linkage map of 214 RFLP, SSR and AFLP markers was constructed. Phenotypic data were collected in replicated field trials from five environments in two growing seasons. The data were analyzed using MQTL software to detect quantitative trait locus (QTL) × environment (E) interactions. Because of the presence of QTL × E, the MQM procedure in MAPQTL was applied to identify QTLs in single environments. We identified nine QTLs for FHB severity and five for low DON. Many of the disease-related QTLs identified were coincident with FHB QTLs identified in previous studies. Only two of the QTLs identified in this study were consistent across all five environments, and both were Zhedar 2 specific. Five of the FHB QTLs were associated with HD, and two were associated with HT. Regions that appear to be promising candidates for MAS and further genetic analysis include the two FHB QTLs on chromosome 2H and one on 6H, which were also associated with low DON and later heading-date in multiple environments. This study provides a starting point for manipulating Zhedar 2-derived resistance by MAS in barley to develop cultivars that will show effective resistance under disease pressure.

Evaluation of gaseous ozone and hydrogen peroxide treatments for reducing Fusarium survival in malting barley.

The use of Fusarium-infected barley for malting can lead to mycotoxin production and decreased malt quality. Methods for treatment of Fusarium-infected barley might prevent these safety and quality defects and allow use of otherwise good-quality barley. Gaseous ozone and hydrogen peroxide (HP) were evaluated for effectiveness in reducing Fusarium survival while maintaining germinative energy (GE) in barley. Gaseous ozone treatments (GOT) included concentrations of 11 and 26 mg/g for 0, 15, 30, and 60 min. HP treatments included 0, 5, 10, and 15% concentrations with exposure times of 0, 5, 10, 15, 20, and 30 min. For GOT, in naturally Fusarium-infected barley, a statistically significant (P < 0.05) decrease (24 to 36%) of Fusarium survival occurred within 15 min of exposure at either concentration. GE was significantly (P < 0.05) affected by 30 min at both concentrations in naturally Fusarium-infected barley, but not in sound barley. GOT did not cause any significant (P > 0.05) effect on GE in sound barley at either concentration over the full 30-min exposure time. For HP, Fusarium survival was significantly decreased (50 to 98%) within 5 min of exposure. With the exception of two treatments (10 and 15% HP agitated for 20 min), GE was not statistically significantly different from the control in naturally Fusarium-infected barley. In sound barley, HP had no significant (P > 0.05) effect on GE. The results suggest that GOT and HP might have potential for treatment of Fusarium-infected malting barley.

Mycotoxins and fermentation--beer production.

Along with food safety issues due to mycotoxins, the effects of Fusarium infections on malt and beer quality can be disastrous. While some of the Fusarium head blight mycotoxins, such as DON, present in infected barley may be lost during steeping, the Fusarium mold is still capable of growth and mycotoxin production during steeping, germination and kilning. Therefore, detoxification of grain before malting may not be practical unless further growth of the mold is also prevented. Methods for reducing the amount of mold growth during malting are needed. Physical, chemical and biological methods exist for inhibiting mold growth in grain. Irradiation is a promising means for preventing Fusarium growth during malting, but its effects on malt quality and mycotoxin production in surviving mold need to be evaluated in more detail. Chemical treatments such as ozonation, which do not leave chemical residues in beer, also appear to be promising. Although biological control methods may be desirable, the effects of these inhibitors on malt and beer quality require further investigation. In addition, storage studies are needed to determine the effect of biological control on Fusarium viability and malt quality. It may also be possible to incorporate detoxifying genes into fermentation yeasts, which would result in detoxification of mycotoxins present in wort. Development of these types of technological interventions should help improve the safety of products, such as beer, made from Fusarium infected grain.

Activity of lipoxygenase isoenzymes during malting and mashing

The objective of this study was to evaluate the development and stability of the lipoxygenase (LOX) isoenzymes LOX-1 and LOX-2 during malting and mashing. LOX-2 was detected only in germinating bar...

The effect of grain storage conditions on the viability of Fusarium and deoxynivalenol production in infested malting barley.

A continuing outbreak of Fusarium head blight occurred on barley in the upper Midwest from 1993 to 1995. This resulted in barley with levels of the mycotoxin deoxynivalenol (DON) reaching levels of concern for maltsters and brewers. This study evaluated the effect of 7 months of storage under different conditions (ambient outdoor temperature from October to April), -20 degrees C, 4 degrees C, 24 degrees C with quiescent air, and 24 degrees C with forced air) on the viability of Fusarium and Alternaria infesting stored grain. Additionally, the ability of Fusarium to produce DON after storage and during malting was evaluated. Initial levels of infestation of barley by Fusarium and Alternaria were 85 and 75%, respectively. All storage condition reduced the viability of both molds slightly and significantly for Fusarium. Forced air ventilation at 24 degrees C was the type of storage most effective in reducing the viability of Fusarium, dropping the percentage of infected kernels to 66%. DON levels did not change after 7 months with respect to storage conditions. However, DON levels were lower in malt produced from barley stored at 24 degrees C with or without aeration. On-farm storage of infected barley at elevated temperatures may provide a means to reduce the level of DON in finished malts.

Evaluation of Hot Water and Electron Beam Irradiation for Reducing Fusarium Infection in Malting Barley

The use of Fusarium-infected barley for malting may lead to mycotoxin production and decreased product quality. Physical methods for the treatment of Fusarium-infected barley may prevent these safety and quality defects and allow the use of otherwise good quality barley. Hot water and electron beam irradiation were evaluated for their effectiveness in reducing Fusarium infection while maintaining germinative energy in barley samples. Hot-water treatments involved temperatures of 45, 50. 55, and 60 degrees C and treatment times of 0, 1, 5, 10, and 15 min. Electron beam irradiation involved doses ranging from 0 to 11.4 kGy. Treatment with water at 45 degrees C for 15 min resulted in a reduction in Fusarium infection from 32 to 1% after 15 min, with only a very slight reduction in germination. Treatment with water at 50 degrees C for 1 min resulted in a reduction in Fusarium infection from 32 to 2%, and no effect on germination was observed for up to 5 min of treatment. At higher water temperatures. Fusarium infection was essentially eliminated, but germination was also severely reduced. Electron beam irradiation of Fusarium-infected barley reduced Fusarium infection at doses of >4 kGy, and a slight increase in germination for dry samples was observed with doses of 6 to 8 kGy. Doses of >10 kGy significantly decreased germination. Physical methods may have potential for the treatment of Fusarium-infected malting barley.

Trichothecene Profiling and Population Genetic Analysis of Gibberella zeae from Barley in North Dakota and Minnesota

Gibberella zeae, the principal cause of Fusarium head blight (FHB) of barley, contaminates grains with several mycotoxins, which creates a serious problem for the malting barley industry in the United States, China, and Europe. However, limited studies have been conducted on the trichothecene profiles and population genetic structure of G. zeae isolates collected from barley in the United States. Trichothecene biosynthesis gene (TRI)-based polymerase chain reaction (PCR) assays and 10 variable number tandem repeat (VNTR) markers were used to determine the genetic diversity and compare the trichothecene profiles of an older population (n = 115 isolates) of G. zeae collected in 1997 to 2000 with a newer population (n = 147 isolates) collected in 2008. Samples were from across the major barley-growing regions in North Dakota and Minnesota. The results of TRI-based PCR assays were further validated using a subset of 32 and 28 isolates of G. zeae by sequence analysis and gas chromatography, respectively. TRI-based PCR assays revealed that all the G. zeae isolates in both populations had markers for deoxynivalenol (DON), and the frequencies of isolates with a 3-acetyldeoxynivalenol (3-ADON) marker in the newer population were ≈11-fold higher than those among isolates in the older population. G. zeae populations from barley in the Midwest of the United States showed no spatial structure, and all the isolates were solidly in clade 7 of G. zeae, which is quite different from other barley-growing areas of world, where multiple species of G. zeae are commonly found in close proximity and display spatial structure. VNTR analysis showed high gene diversity (H = 0.82 to 0.83) and genotypic diversity but low linkage disequilibrium (LD = 0.02 to 0.07) in both populations. Low genetic differentiation (F(ST) = 0.013) and high gene flow (Nm = 36.84) was observed between the two populations and among subpopulations within the same population (Nm = 12.77 to 29.97), suggesting that temporal and spatial variations had little influence on population differentiation in the Upper Midwest. Similarly, low F(ST) (0.02) was observed between 3-ADON and 15-acetyldeoxynivalenol populations, indicating minor influence of the chemotype of G. zeae isolates on population subdivision, although there was a rapid increase in the frequencies of isolates with the 3-ADON marker in the Upper Midwest between the older collection made in 1997 to 2000 and the newer collection made in 2008. This study provides information to barley-breeding programs for their selection of isolates of G. zeae for evaluating barley genotypes for resistance to FHB and DON accumulation.

Trichothecene Mycotoxins Associated with Potato Dry Rot Caused by Fusarium graminearum

Fusarium graminearum, a known producer of trichothecene mycotoxins in cereal hosts, has been recently documented as a cause of dry rot of potato tubers in the United States. Due to the uncertainty of trichothecene production in these tubers, a study was conducted to determine the accumulation and diffusion of trichothecenes in potato tubers affected with dry rot caused by F. graminearum. Potato tubers of cv. Russet Burbank were inoculated with 14 F. graminearum isolates from potato, sugar beet, and wheat and incubated at 10 to 12 degrees C for 5 weeks to determine accumulation of trichothecenes in potato tubers during storage. Twelve of the isolates were classified as deoxynivalenol (DON) genotype and two isolates were as nivalenol (NIV) genotype. Trichothecenes were detected only in rotted tissue. DON was detected in all F. graminearum DON genotype isolates up to 39.68 microg/ml in rotted potato tissue. Similarly, both NIV genotype isolates accumulated NIV in rotted potato tissue up to 18.28 microg/ml. Interestingly, isolates classified as genotype DON accumulated both DON and NIV in the dry rot lesion. Potato tubers were then inoculated with two isolates of F. graminearum chemotype DON and incubated up to 7 weeks at 10 to 12 degrees C and assayed for DON diffusion. F. graminearum was recovered from >53% of the isolations from inoculated tubers at 3 cm distal to the rotted tissue after 7 weeks of incubation but DON was not detected in the surrounding tissue. Based in this data, the accumulation of trichothecenes in the asymptomatic tissue surrounding dry rot lesions caused by F. graminearum is minimal in cv. Russet Burbank potato tubers stored for 7 weeks at customary processing storage temperatures.

Quantification of Tri5 gene, expression, and deoxynivalenol production during the malting of barley.

Fusarium can survive, grow, and produce mycotoxins during malting. We evaluated the percentage of barley kernels infected with Fusarium (FI) and deoxynivalenol (DON) concentration in three barley treatments (high-quality, naturally infected, and Fusarium graminearum inoculated barley) during various stages of malting. We also applied real-time polymerase chain reaction (real-time PCR) and real-time reverse transcriptase PCR (real-time RT-PCR) methods to quantify trichothecene-producing (Tri5) DNA concentration and expression, respectively. We observed that FI significantly (P<0.05) increased during the germination stage of malting in all barley treatments. Temperatures of 49°C and higher during kilning reduced the FI in high-quality barley treatments, but for inoculated treatments temperatures in excess of 60°C were needed to reduce FI. The Tri5 DNA concentration ranged from non-detectable to 3.9 ng/50mg, 0.1 to 109.8 ng/50mg and 3.4 to 397.5 ng/50 mg in malted high-quality, inoculated and naturally infected barley treatments respectively. Strong gene expression (Tri5) in naturally infected barley treatments was found during the third day of germination, when compared to high-quality and inoculated barley treatments during malting. Deoxynivalenol was present even at high kilning temperatures, as DON is heat stable. The average DON concentration ranged from non-detectable to 0.1 μg/g, non-detectable to 1.1 μg/g, and 1.5 to 45.9 μg/g during various stages of malting in high-quality, inoculated and infected barley and malt samples respectively. Overall, the last 2 days of germination and initial stages of kilning were peak stages for FI, Tri5 gene production, Tri5 gene expression and DON production.