P. Nicholson

Development of PCR assays to Tri7 and Tri13 trichothecene biosynthetic genes, and characterisation of chemotypes of Fusarium graminearum, Fusarium culmorum and Fusarium cerealis

Abstract Fusarium graminearum, Fusarium culmorum and Fusarium cerealis are major causal agents of Fusarium Head Blight (scab) which is a disease of global significance in all cereal growing areas. These fungi produce trichothecene mycotoxins, principally nivalenol (NIV) and deoxynivalenol (DON). Genes Tri13 and Tri7 from the trichothecene biosynthetic gene cluster convert DON to NIV (Tri13) and NIV to 4-acetyl-NIV (Tri7). We have developed positive–negative PCR assays based on these two genes, which accurately indicate a DON or NIV chemotype in F. graminearum, F. culmorum and F. cerealis. These assays are useful in assessing the risk of trichothecene contamination, and can be informative in epidemiological studies. All NIV chemotype isolates studied have functional copies of both Tri13 and Tri7, and all DON-producing isolates have both genes disrupted or deleted. We have identified several mutations in these genes, which are conserved across F. graminearum lineage, RAPD and SCAR groupings and between the three species. There appears to be evidence of inter-species hybridisation within the trichothecene biosynthetic gene cluster.

Differential control of head blight pathogens of wheat by fungicides and consequences for mycotoxin contamination of grain

Fusarium head blight of wheat is caused by a disease complex comprised of toxigenic pathogens, predominantly Fusarium spp., and a non-toxigenic pathogen Microdochium nivale, which causes symptoms visually indistinguishable from Fusarium and is often included as a causal agent of Fusarium head blight. Four field trials are reported here, including both naturally and artificially inoculated trials in which the effect of fungicide treatments were noted on colonisation by Fusarium and Microdochium, and on the production of deoxynivalenol (DON) mycotoxin. The pathogen populations were analysed with quantitative PCR and samples were tested for the presence of the mycotoxin DON. Application of fungicides to reduce Fusarium head blight gave a differential control of these fungi. Tebuconazole selectively controlled F. culmorum and F. avenaceum and reduced levels of DON, but showed little control of M. nivale. Application of azoxystrobin, however, selectively controlled M. nivale and allowed greater colonisation by toxigenic Fusarium species. This treatment also lead to increased levels of DON detected. nobreak Azoxystrobin application two days post-inoculation increased the production of DON mycotoxin per unit of pathogen in an artificially inoculated field trial. This result indicates the potential risk of increased DON contamination of grain following treatment with azoxystrobin to control head blight in susceptible wheat cultivars. This is the first study to show differential fungicidal control of mixed natural pathogen populations and artificial inoculations in field trials.

Action and reaction of host and pathogen during Fusarium head blight disease

The Fusarium species Fusarium graminearum and Fusarium culmorum, which are responsible for Fusarium head blight (FHB) disease, reduce world-wide cereal crop yield and, as a consequence of their mycotoxin production in cereal grain, impact on both human and animal health. Their study is greatly promoted by the availability of the genomic sequence of F. graminearum and transcriptomic resources for both F. graminearum and its cereal hosts. Functional genomic, proteomic and metabolomic studies, in combination with targeted mutagenesis or transgenic studies, are unravelling the complex mechanisms involved in Fusarium infection, penetration and colonization of host tissues, and host avoidance thereof. This review illuminates and integrates emerging knowledge regarding the molecular crosstalk between Fusarium and its small-grain cereal hosts. An understanding of the complexity of the host-pathogen interactions will be instrumental in designing new efficient strategies for the control of FHB disease.

Variation in pathogenicity associated with the genetic diversity of Fusarium graminearum

We screened 188 isolates of Fusarium graminearum, which originated from northwest Europe, the USA and Nepal, for genetic diversity using a sequence-characterised amplified region polymorphism (SCAR). On the basis of this analysis, 42 of the 118 isolates were selected for random amplified polymorphic DNA (RAPD) analysis. Three groups were identified, two of which, A and B, contained the isolates from Nepal, and a third, group C, contained the isolates from Europe and the USA. In pathogenicity tests on wheat and maize seedlings, group C isolates were more pathogenic than the group A and B isolates. The isolates were assigned chemotypes based on their ability to produce the trichothecene mycotoxins nivalenol (NIV) and deoxynivalenol (DON). Isolates from group A were equally likely to produce NIV or DON while group B isolates produced predominantly NIV, and group C isolates produced predominantly DON. Within group A, isolates of the two chemotypes were equally pathogenic to wheat but isolates with the NIV chemotype were significantly more pathogenic to maize. The results confirm that distinct genetic groups exist within F. graminearum and demonstrate that these groups have different biological properties, especially with respect to their pathogenicity to two of the most economically important hosts of this pathogen.

Community Ecology of Fungal Pathogens Causing Wheat Head Blight

Research on the pathogen components involved in Fusarium head blight (FHB) along with the effects of their interactions on disease development and mycotoxin accumulation is reviewed. The fungal components within the FHB complex differ significantly in different environments. Individual species may respond differently to, and be differentially influenced by, particular disease control measures. Almost all published co-inoculation studies on wheat spikes or grains show that competitive interactions among FHB pathogens are the rule when fungal/disease development is considered. However, the fungi with the competitive advantage do not usually gain any advantage from the presence of other weaker competing fungi. Total mycotoxin production in mixed inoculations may decrease, increase, or remain unchanged compared with single-isolate inoculations, depending on the fungal species concerned and environmental conditions. A few recent studies, where each individual fungal component was quantified using molecular methods, suggest that mycotoxin productivity in mixed inoculations generally increases.

Molecular Characterization of Rht-1 Dwarfing Genes in Hexaploid Wheat

The introduction of the Reduced height (Rht)-B1b and Rht-D1b semidwarfing genes led to impressive increases in wheat (Triticum aestivum) yields during the Green Revolution. The reduction in stem elongation in varieties containing these alleles is caused by a limited response to the phytohormone gibberellin (GA), resulting in improved resistance to stem lodging and yield benefits through an increase in grain number. Rht-B1 and Rht-D1 encode DELLA proteins, which act to repress GA-responsive growth, and their mutant alleles Rht-B1b and Rht-D1b are thought to confer dwarfism by producing more active forms of these growth repressors. While no semidwarfing alleles of Rht-A1 have been identified, we show that this gene is expressed at comparable levels to the other homeologs and represents a potential target for producing novel dwarfing alleles. In this study, we have characterized additional dwarfing mutations in Rht-B1 and Rht-D1. We show that the severe dwarfism conferred by Rht-B1c is caused by an intragenic insertion, which results in an in-frame 90-bp insertion in the transcript and a predicted 30-amino acid insertion within the highly conserved amino-terminal DELLA domain. In contrast, the extreme dwarfism of Rht-D1c is due to overexpression of the semidwarfing Rht-D1b allele, caused by an increase in gene copy number. We show also that the semidwarfing alleles Rht-B1d and Rht-B1e introduce premature stop codons within the amino-terminal coding region. Yeast two-hybrid assays indicate that these newly characterized mutations in Rht-B1 and Rht-D1 confer “GA-insensitive” dwarfism by producing DELLA proteins that do not bind the GA receptor GA INSENSITIVE DWARF1, potentially compromising their targeted degradation.

Identification of QTLs for resistance to Fusarium head blight, DON accumulation and associated traits in the winter wheat variety Arina

Fusarium head blight (FHB) of wheat has become a serious threat to wheat crops in numerous countries. In addition to loss of yield and quality, this disease is of primary importance because of the contamination of grain with mycotoxins such as deoxynivalenol (DON). The Swiss winter cultivar Arina possesses significant resistance to FHB. The objective of this study was to map quantitative trait loci (QTL) for resistance to FHB, DON accumulation and associated traits in grain in a double haploid (DH) population from a cross between Arina and the FHB susceptible UK variety Riband. FHB resistance was assessed in five trials across different years and locations. Ten QTL for resistance to FHB or associated traits were detected across the trials, with QTL derived from both parents. Very few of the QTL detected in this study were coincident with those reported by authors of two other studies of FHB resistance in Arina. It is concluded that the FHB resistance of Arina, like that of the other European winter wheat varieties studied to date, is conferred by several genes of moderate effect making it difficult to exploit in marker-assisted selection breeding programmes. The most significant and stable QTL for FHB resistance was on chromosome 4D and co-localised with the Rht–D1 locus for height. This association appears to be due to linkage of deleterious genes to the Rht-D1b (Rht2) semi-dwarfing allele rather than differences in height per se. This association may compromise efforts to enhance FHB resistance in breeding programmes using germplasm containing this allele.

Novel Fusarium head blight pathogens from Nepal and Louisiana revealed by multilocus genealogical concordance

This study was conducted to assess evolutionary relationships, species diversity and trichothecene toxin potential of five Fusarium graminearum complex (FGSC) isolates identified as genetically novel during prior Fusarium head blight (FHB) surveys in Nepal and Louisiana. Results of a multilocus genotyping (MLGT) assay for B-trichothecene species determination indicated these isolates might represent novel species within the FGSC. GCPSR-based phylogenetic analyses of a 12-gene dataset, comprising portions of seven loci totaling 13.1 kb of aligned DNA sequence data, provided strong support for the genealogical exclusivity of the Nepalese and Louisianan isolates. Accordingly, both species are formally recognized herein as novel FGSC species. Fusarium nepalense was resolved as the sister lineage of Fusarium ussurianum+Fusarium asiaticum within an Asian subclade of the FGSC. Fusarium louisianense was strongly supported as a reciprocally monophyletic sister of Fusarium gerlachii+F. graminearum, suggesting that this subclade might be endemic to North America. Multilocus Bayesian species tree analyses augment these results and provide evidence for a distinct lineage within F. graminearum predominately from the Gulf Coast of Louisiana. As predicted by the MLGT assay, mycotoxin analyses demonstrated that F. nepalense and F. louisianense could produce 15ADON and nivalenol, respectively, in planta. In addition, both species were only able to induce mild FHB symptoms on wheat in pathogenicity experiments.

Molecular tools to study epidemiology and toxicology of fusarium head blight of cereals

Fusarium head blight (FHB) of cereals is a disease complex. Fusarium graminearum is the major pathogen worldwide, while F. culmorum, F. avenaceum and F. poae are also associated with this disease. In addition to the true Fusarium species, Microdochium nivale may also cause head blight and is particularly prevalent where cooler, wetter conditions prevail. Other species such as F. sporotrichioides, F. equiseti and even F. verticillioides may also be of significance in particular situations. FHB is of particular concern because of the ability of the Fusarium species to produce mycotoxins in the grain that are harmful to human and animal consumers. The predominant mycotoxins within cereals are the trichothecenes, chiefly deoxynivalenol, nivalenol and their acetylated derivatives, along with T-2, HT-2, diacetoxyscirpenol and neosolaniol. This paper reviews the use of molecular techniques to identify the individual causal agents and to quantify their relative amounts within plant tissue. Diagnostic and quantitative polymerase chain reaction assays have been developed to detect and quantify individual fungal species within the disease complex and, where relevant, to differentiate between chemotypes within a single species. Assays to determine the type of toxin produced, or monitor the regulation of toxin production also provide valuable tools for understanding this disease. These techniques are being used to dissect the disease complex into its component parts in order to study interactions between the pathogens and their host and between the pathogens themselves as well as to determine the influence of environmental factors on the disease and the toxins produced by these fungi.

Detection and Differentiation of Trichothecene and Enniatin-Producing Fusarium Species on Small-Grain Cereals

A large number of Fusarium species are associated with Fusarium head blight of wheat and other small-grain cereals as well as seedling blight and brown foot rot. Different Fusarium species tend to predominate under different environmental conditions and in different regions. In addition to causing disease, these fungi are of particular significance because they produce a number of mycotoxins including the trichothecenes and enniatins that contaminate infected grain. The nature and amount of the mycotoxins that accumulate will alter according to the species or even the particular isolates involved in the infection. It is highly desirable to be able to analyse such complex infections to determine which species and, preferably, which chemotypes are present, in order to understand the factors that affect the pathogenicity of each species and to evaluate the potential risk for contamination of grain with mycotoxins. This paper reports the development of molecular methods, based upon the polymerase chain reaction (PCR), for the detection of mycotoxigenic fungi. Several of the Fusarium species involved are closely related, making the development of specific assays problematic. We describe the development of primers specific to individual species and discuss how this work provides insight into fungal populations and relates to taxonomic studies. In some instances, it is desirable to detect the presence of potential mycotoxin producers rather than individual fungal species. Generic assays have been produced for several genes involved in trichothecene biosynthesis and for enniatin synthetase in order to permit the detection of species able to produce the associated mycotoxins. Additional work is under way to refine assays to enable detection related to the class of trichothecene and chemotype of isolate because of the potential risk posed to human and animal consumers by different trichothecenes.