Simon G. Edwards

Quantification of Trichothecene-Producing Fusarium Species in Harvested Grain by Competitive PCR To Determine Efficacies of Fungicides against Fusarium Head Blight of Winter Wheat

ABSTRACT We developed a PCR-based assay to quantify trichothecene-producingFusarium based on primers derived from the trichodiene synthase gene (Tri5). The primers were tested against a range of fusarium head blight (FHB) (also known as scab) pathogens and found to amplify specifically a 260-bp product from 25 isolates belonging to six trichothecene-producing Fusariumspecies. Amounts of the trichothecene-producing Fusariumand the trichothecene mycotoxin deoxynivalenol (DON) in harvested grain from a field trial designed to test the efficacies of the fungicides metconazole, azoxystrobin, and tebuconazole to control FHB were quantified. No correlation was found between FHB severity and DON in harvested grain, but a good correlation existed between the amount of trichothecene-producing Fusarium and DON present within grain. Azoxystrobin did not affect levels of trichothecene-producingFusarium compared with those of untreated controls. Metconazole and tebuconazole significantly reduced the amount of trichothecene-producing Fusarium in harvested grain. We hypothesize that the fungicides affected the relationship between FHB severity and the amount of DON in harvested grain by altering the proportion of trichothecene-producing Fusarium within the FHB disease complex and not by altering the rate of DON production. The Tri5 quantitative PCR assay will aid research directed towards reducing amounts of trichothecene mycotoxins in food and animal feed.

Fusarium mycotoxin content of UK organic and conventional oats

Every year between 2002 and 2005 approximately 100 samples of oats from fields of known agronomy were analysed by GC/MS for 10 trichothecenes: deoxynivalenol (DON), nivalenol, 3-acetylDON, 15-acetylDON, fusarenone X, T-2 toxin (T2), HT-2 toxin (HT2), diacetoxyscirpenol, neosolaniol and T-2 triol. Samples were also analysed for moniliformin and zearalenone by HPLC. Of the 10 trichothecenes analysed from 458 harvest samples of oat only three, 15-acetylDON, fusarenone X and diacetoxyscirpenol, were not detected. Moniliformin and zearalenone were absent or rarely detected, respectively. HT2 and T2 were the most frequently detected fusarium mycotoxins, present above the limit of quantification (10 µg kg−1) in 92 and 84% of samples, respectively, and were usually present at the highest concentrations. The combined mean and median for HT2 and T2 (HT2 + T2) was 570 and 213 µg kg−1, respectively. There were good correlations between concentrations of HT2 and all other type A trichothecenes detected (T2, T2 triol and neosolaniol). Year and region had a significant effect on HT2 + T2 concentration. There was also a highly significant difference between HT2 + T2 content in organic and conventional samples, with the predicted mean for organic samples five times lower than that of conventional samples. This is the largest difference reported for any mycotoxin level in organic and conventional cereals. No samples exceeded the legal limits for DON or zearalenone in oats intended for human consumption. Legislative limits for HT2 and T2 are currently under consideration by the European Commission. Depending on the limits set for unprocessed oats intended for human consumption, the levels detected here could have serious consequences for the UK oat-processing industry.

Strategies for the Control of Fusarium Head Blight in Cereals

Fusarium head blight (FHB) is a widespread and destructive disease of small grained cereals caused by a number of Fusarium species and Microdochium nivale. In addition to causing significant reductions in grain yield, FHB can result in the reduction of grain quality, either by affecting grain processing qualities or by producing a range of toxic metabolites that have adverse effects on humans and livestock. Control of FHB can be achieved by a number of cultural, biological and chemical strategies along with the exploitation of host plant resistance. In recent years, much of the research undertaken for the control of FHB has been concentrated on understanding and exploiting the genetic resistance of cereal plants to FHB-causing pathogens. Although, a brief overview of genetic resistance is presented, this review seeks to summarise the significance of FHB and review the effectiveness of cultural, biological and chemical control strategies that have been investigated for the control the disease.

PCR-based detection and quantification of mycotoxigenic fungi*

Mycotoxins are secondary metabolites produced by many phytopathogenic and food spoilage fungi including Penicillium, Fusarium and Aspergillus. The toxicity and carcinogenicity of many of these mycotoxins, and their potential to contaminate foods and animal feedstuffs is a cause of serious concern globally, both from a food safety and food trade standpoint. Thus the rapid identification of mycotoxigenic fungi would be desirable, such that early intervention steps could be applied to help limit the amounts of contaminated materials, particularly cereals and cereal-based products, gaining access to the human food chain. With this in mind a number of PCR-based methodologies have been developed for the identification of mycotoxin biosynthetic genes in different fungal genera, together with assays developed using other genes or random amplification of polymorphic DNA (RAPD) methodologies for the identification of specific toxigenic fungi. In addition, reverse transcription (RT)-PCR, competitive PCR and Real Time quantitative PCR methodologies have also been developed for this purpose. The development of each of these techniques, their usefulness, limitations and adaptability will be discussed together with descriptions of specific examples where these techniques have been utilised in different experimental settings.

Relationship Between the Fungal Complex Causing Fusarium Head Blight of Wheat and Environmental Conditions

ABSTRACT Over 4 years, the environmental conditions and the causal agents of Fusarium head blight (FHB) disease of wheat were determined in field sites in four European countries: Hungary, Ireland, Italy, and the United Kingdom. Polymerase chain reaction-based methods were used to detect each species causing FHB and quantify its DNA (as a measurement of fungal abundance) in the samples. Canonical correspondence analysis (CCA) was used to determine the relationship of the incidence and abundance of each species with weather variables. CCA indicated that little variability in the species prevalence data was explained by the weather variables. In contrast, a greater proportion of variability in abundance data was accounted for by the weather variables. Most samples contained two or more species and statistical analysis suggested that these species tended to coexist at field sites. CCA also indicated that there were differences in the relationships of the prevalence and abundance of the six FHB species with environmental variables. Fusarium poae was associated with relatively drier and warmer conditions, whereas F. graminearum was associated with warmer/humid conditions. F. avenaceum and F. culmorum were both associated with niches of cooler/wet/humid conditions. Two Microdochium species were associated with regions of relatively cool/moderate temperatures and frequent rainfalls of short duration. The results also suggested that environmental conditions differentially affect the infection and colonization processes, and the comparative abundance of the six species.

Effect of dose rate of azoxystrobin and metconazole on the development of Fusarium head blight and the accumulation of deoxynivalenol (DON) in wheat grain

Glasshouse studies were undertaken to determine if fungicides used for the control of Fusarium head blight (FHB) result in elevated concentrations of the trichothecene mycotoxin, deoxynivalenol (DON) in harvested wheat grain. Metconazole and azoxystrobin, at double, full, half or quarter the manufacturers recommended dose rate, were applied to ears of wheat (cv. Cadenza), artificially inoculated with conidia of either Fusarium culmorum or F. graminearum. Metconazole demonstrated high activity against both pathogens, reducing significantly the severity of FHB and the DON concentrations at each of the four dose rates tested when compared to untreated controls. Applications of azoxystrobin significantly reduced FHB and DON compared to unsprayed controls. However, their effectiveness was significantly less than that of metconazole and no dose rate response was observed. Quantification of the amount of trichothecene-producing Fusarium present in harvested grain was determined using a competitive PCR assay based on primers derived from the trichodiene synthase gene (Tri5). Simple linear regression analyses revealed strong relationships between the amount of trichothecene-producing Fusarium present in grain and the DON concentrations (r2=0.72–0.97). It is concluded that fungicides, applied for the control of FHB, affect DON concentrations indirectly by influencing the amount of trichothecene-producing Fusarium species present in wheat grain. There was no evidence that fungicide applications directly increase the concentration of DON in grain.

Occurrence and fate of Fusarium mycotoxins during commercial processing of oats in the UK

The commercial processing of oats is different from that of other cereals, such as wheat and maize. In northwest Europe, oats also appear to be more susceptible to contamination with HT-2 and T-2 toxins than other cereals. Mycotoxins, such as deoxynivanol and zearalenone, in cereals are already controlled by EU legislation. With regard to additional, impending legislation, this study examined HT-2 and T-2 toxins together with zearalenone, deoxynivalenol and other related toxins in a commercial oat mill and how the concentrations varied from raw oats to the final prepared oat flakes. Concentrations of each Fusarium mycotoxin fell by 90–95% during the process, with the major loss being a physical distribution occurring at the de-hulling stage. Initial studies of losses occurring at other stages, such as kilning or de-branning of prepared oat groats, suggest these to be small. The use of colour sorting after kilning showed higher concentrations of each mycotoxin in the discoloured groats. The feasibility of developing a predictive tool for the oat industry is examined.

Quantification of an arbuscular mycorrhizal fungus, Glomus mosseae, within plant roots by competitive polymerase chain reaction

An assay based on the competitive polymerase chain reaction (PCR) was developed to quantify Glomus mosseae , an arbuscular mycorrhizal (AM) fungus, within plant roots. Using previously designed G. mosseae specific primers, a heterologous internal standard was constructed by amplifying Pseudomonas DNA under low stringency annealing conditions. Co-amplification of G. mosseae and internal standard DNA within leek root extracts provided accurate quantification of target DNA. Colonization of leek roots by G. mosseae was monitored in a comparative study by competitive PCR and microscopy, a conventional method of quantification. These two methods gave closely parallel data for G. mosseae colonization from three different inoculum levels over a 6 week period. Results indicate that competitive PCR is a sensitive and accurate method of quantification. The major advantage of competitive PCR over microscopy is that it can quantify specific AM fungi.

Mode of antagonism of Brevibacillus brevis against Botrytis cinerea in vitro

Aims: To assess the activity of Brevibacillus brevis (formerly Bacillus brevis) Nagano and the antibiotic it produces, gramicidin S, against the plant pathogen Botrytis cinerea.

Within-field variability of Fusarium head blight pathogens and their associated mycotoxins

Within-field variability in the Fusarium head blight (FHB) and its associated mycotoxins was studied in four European countries. At each of 14 sites, each FHB pathogen and associated mycotoxins were quantified in 16 quadrat samples at harvest. Overall, the incidence of quadrat samples with detectable and quantifiable pathogen DNA was significantly lower in the grain than in the corresponding chaff. Deoxynivalenol (DON) was the most frequently detected toxin in the samples and its accumulation was most strongly associated with the presence of Fusarium graminearum. Nivalenol (NIV) accumulation was significantly associated only with the presence of F. culmorum. Zearalenone (ZON) accumulation was strongly associated with the presence of all three pathogens (F. graminearum, F. culmorum and F. poae). The levels of both DON and ZON concentrations were positively related to the amount of F. graminearum DNA in the grain or in the chaff. The presence/absence of FHB pathogens within a single quadrat appeared to be independent of each other. The presence of a particular FHB pathogen and the amount of its DNA, as well as the associated mycotoxin(s), varied greatly among samples at each site. This study demonstrated the large extent of within-field variability of FHB and its associated mycotoxins, and the importance of representative sampling in FHB studies.