Fusarium head blight (FHB) is an important disease of wheat worldwide caused mainly by Fusarium graminearum (syn. Gibberella zeae). This fungus can be highly aggressive and can produce several mycotoxins such as deoxynivalenol (DON), a well known harmful metabolite for humans, animals, and plants. The fungus can survive overwinter on wheat residues and on the soil, and can usually attack the wheat plant at their point of flowering, being able to infect the heads and to contaminate the kernels at the maturity. Contaminated kernels can be sometimes used as seeds for the cultivation of the following year. Poor knowledge on the ability of the strains of F. graminearum occurring on wheat seeds to be transmitted to the plant and to contribute to the final DON contamination of kernels is available. Therefore, this study had the goals of evaluating: (a) the capability of F. graminearum causing FHB of wheat to be transmitted from the seeds or soil to the kernels at maturity and the progress of the fungus within the plant at different growth stages; (b) the levels of DON contamination in both plant tissues and kernels. The study has been carried out for two years in a climatic chamber. The F. gramineraum strain selected for the inoculation was followed within the plant by using Vegetative Compatibility technique, and quantified by Real-Time PCR. Chemical analyses of DON were carried out by using immunoaffinity cleanup and HPLC/UV/DAD. The study showed that F. graminearum originated from seeds or soil can grow systemically in the plant tissues, with the exception of kernels and heads. There seems to be a barrier that inhibits the colonization of the heads by the fungus. High levels of DON and F. graminearum were found in crowns, stems, and straw, whereas low levels of DON and no detectable levels of F. graminearum were found in both heads and kernels. Finally, in all parts of the plant (heads, crowns, and stems at milk and vitreous ripening stages, and straw at vitreous ripening), also the accumulation of significant quantities of DON-3-glucoside (DON-3G), a product of DON glycosylation, was detected, with decreasing levels in straw, crown, stems and kernels. The presence of DON and DON-3G in heads and kernels without the occurrence of F. graminearum may be explained by their water solubility that could facilitate their translocation from stem to heads and kernels. The presence of DON-3G at levels 23 times higher than DON in the heads at milk stage without the occurrence of F. graminearum may indicate that an active glycosylation of DON also occurs in the head tissues. Finally, the high levels of DON accumulated in straws are worrisome since they represent additional sources of mycotoxin for livestock.
wheat; Fusarium graminearum; seed transmission; systemic growth; deoxynivalenol; deoxynivalenol-translocation; deoxynivalenol-3-glucoside
Fusarium species cause Fusarium head blight (FHB) and other important diseases of cereals. The causal agents produce trichothecene mycotoxins such as deoxynivalenol (DON). The dicotyledonous model species Arabidopsis thaliana has been used to study Fusarium-host interactions but it is not ideal for model-to-crop translation. Brachypodium distachyon (Bd) has been proposed as a new monocotyledonous model species for functional genomic studies in grass species. This study aims to assess the interaction between the most prevalent FHB-causing Fusarium species and Bd in order to develop and exploit Bd as a genetic model for FHB and other Fusarium diseases of wheat.
The ability of Fusarium graminearum and Fusarium culmorum to infect a range of Bd tissues was examined in various bioassays which showed that both species can infect all Bd tissues examined, including intact foliar tissues. DON accumulated in infected spike tissues at levels similar to those of infected wheat spikes. Histological studies revealed details of infection, colonisation and host response and indicate that hair cells are important sites of infection. Susceptibility to Fusarium and DON was assessed in two Bd ecotypes and revealed variation in resistance between ecotypes.
Bd exhibits characteristics of susceptibility highly similar to those of wheat, including susceptibility to spread of disease in the spikelets. Bd is the first reported plant species to allow successful infection on intact foliar tissues by FHB-causing Fusarium species. DON appears to function as a virulence factor in Bd as it does in wheat. Bd is proposed as a valuable model for undertaking studies of Fusarium head blight and other Fusarium diseases of wheat.
Fusarium; Brachypodium distachyon; wheat; deoxynivalenol; model-to-crop translation; disease resistance; host-pathogen interaction
Fusarium head blight (FHB; scab), primarily caused by Fusarium graminearum, is a devastating disease of wheat worldwide. FHB causes yield reductions and contamination of grains with trichothecene mycotoxins such as deoxynivalenol (DON). The genetic variation in existing wheat germplasm pools for FHB resistance is low and may not provide sufficient resistance to develop cultivars through traditional breeding approaches. Thus, genetic engineering provides an additional approach to enhance FHB resistance. The objectives of this study were to develop transgenic wheat expressing a barley class II chitinase and to test the transgenic lines against F. graminearum infection under greenhouse and field conditions. A barley class II chitinase gene was introduced into the spring wheat cultivar, Bobwhite, by biolistic bombardment. Seven transgenic lines were identified that expressed the chitinase transgene and exhibited enhanced Type II resistance in the greenhouse evaluations. These seven transgenic lines were tested under field conditions for percentage FHB severity, percentage visually scabby kernels (VSK), and DON accumulation. Two lines (C8 and C17) that exhibited high chitinase protein levels also showed reduced FHB severity and VSK compared to Bobwhite. One of the lines (C8) also exhibited reduced DON concentration compared with Bobwhite. These results showed that transgenic wheat expressing a barley class II chitinase exhibited enhanced resistance against F. graminearum in greenhouse and field conditions.
Chitinase; Fusarium graminearum; Fusarium head blight; transformation; wheat
Fusarium Head Blight (FHB) caused primarily by Fusarium graminearum (Fg) is one of the major diseases of small-grain cereals including bread wheat. This disease both reduces yields and causes quality losses due to the production of deoxynivalenol (DON), the major type B trichothecene mycotoxin. DON has been described as a virulence factor enabling efficient colonization of spikes by the fungus in wheat, but its precise role during the infection process is still elusive. Brachypodium distachyon (Bd) is a model cereal species which has been shown to be susceptible to FHB. Here, a functional genomics approach was performed in order to characterize the responses of Bd to Fg infection using a global transcriptional and metabolomic profiling of B. distachyon plants infected by two strains of F. graminearum: a wild-type strain producing DON (Fgdon+) and a mutant strain impaired in the production of the mycotoxin (Fgdon-).
Histological analysis of the interaction of the Bd21 ecotype with both Fg strains showed extensive fungal tissue colonization with the Fgdon+ strain while the florets infected with the Fgdon- strain exhibited a reduced hyphal extension and cell death on palea and lemma tissues. Fungal biomass was reduced in spikes inoculated with the Fgdon- strain as compared with the wild-type strain. The transcriptional analysis showed that jasmonate and ethylene-signalling pathways are induced upon infection, together with genes encoding putative detoxification and transport proteins, antioxidant functions as well as secondary metabolite pathways. In particular, our metabolite profiling analysis showed that tryptophan-derived metabolites, tryptamine, serotonin, coumaroyl-serotonin and feruloyl-serotonin, are more induced upon infection by the Fgdon+ strain than by the Fgdon- strain. Serotonin was shown to exhibit a slight direct antimicrobial effect against Fg.
Our results show that Bd exhibits defense hallmarks similar to those already identified in cereal crops. While the fungus uses DON as a virulence factor, the host plant preferentially induces detoxification and the phenylpropanoid and phenolamide pathways as resistance mechanisms. Together with its amenability in laboratory conditions, this makes Bd a very good model to study cereal resistance mechanisms towards the major disease FHB.
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Fusarium Head Blight; Brachypodium distachyon; Transcriptome; Metabolic profiling; Serotonin
Fusarium head blight (FHB; scab) caused mainly by Fusarium graminearum is a devastating disease of wheat and barley around the world. FHB causes yield reductions and contamination of grain with trichothecene mycotoxins such as deoxynivalenol (DON) which are a major health concern for humans and animals. The objective of this research was to develop an easy seed or seedling inoculation assay, and to compare these assays with whole plant resistance of twenty-nine Korean winter wheat cultivars to FHB. The clip-dipping assay consists of cutting off the coleoptiles apex, dipping the coleoptiles apex in conidial suspension, covering in plastic bag for 3 days, and measuring the lengths of lesions 7 days after inoculation. There were significant cultivar differences after inoculation with F. graminearum in seedling relative to the controls. Correlation coefficients between the lesion lengths of clip-dipping inoculation and FHB Type II resistance from adult plants were significant (r=0.45; P<0.05). Results from two other seedling inoculation methods, spraying and pin-point inoculation, were not correlated with adult FHB resistance. Single linear correlation was not significant between seed germination assays (soaking and soak-dry) and FHB resistance (Type I and Type II), respectively. These results showed that clip-dipping inoculation method using F. graminearum may offer a real possibility of simple, rapid, and reliable for the early screening of FHB resistance in wheat.
Fusarium head blight (FHB); seed germination assay; seedling inoculation assay; wheat
Fusarium head blight (FHB) of wheat, caused by Fusarium graminearum and other Fusarium species, is a major disease problem for wheat production worldwide. To combat this problem, large-scale breeding efforts have been established. Although progress has been made through standard breeding approaches, the level of resistance attained is insufficient to withstand epidemic conditions. Genetic engineering provides an alternative approach to enhance the level of resistance. Many defense response genes are induced in wheat during F. graminearum infection and may play a role in reducing FHB. The objectives of this study were (1) to develop transgenic wheat overexpressing the defense response genes α-1-purothionin, thaumatin-like protein 1 (tlp-1), and β-1,3-glucanase; and (2) to test the resultant transgenic wheat lines against F. graminearum infection under greenhouse and field conditions. Using the wheat cultivar Bobwhite, we developed one, two, and four lines carrying the α-1-purothionin, tlp-1, and β-1,3-glucanase transgenes, respectively, that had statistically significant reductions in FHB severity in greenhouse evaluations. We tested these seven transgenic lines under field conditions for percent FHB disease severity, deoxynivalenol (DON) mycotoxin accumulation, and percent visually scabby kernels (VSK). Six of the seven lines differed from the nontransgenic parental Bobwhite line for at least one of the disease traits. A β-1,3-glucanase transgenic line had enhanced resistance, showing lower FHB severity, DON concentration, and percent VSK compared to Bobwhite. Taken together, the results showed that overexpression of defense response genes in wheat could enhance the FHB resistance in both greenhouse and field conditions.
Fusarium head blight; Wheat; Fusarium graminearum; Transgenic wheat; Triticum aestivum
Fusarium graminearum clade pathogens cause Fusarium head blight (FHB) or scab of wheat and other small cereal grains, producing different kinds of trichothecene mycotoxins that are detrimental to human and domestic animals. Type B trichothecene mycotoxins such as deoxynivalenol, 3-acetyldeoxynivalenol (3-AcDON), 15-acetyldeoxynivalenol (15-AcDON) and nivalenol (NIV) are the principal Fusarium mycotoxins reported in China, as well as in other countries. A genomic polymerase chain reaction (PCR) to predict chemotypes was developed based on the structural gene sequences of Tri13 genes involved in trichothecene mycotoxin biosynthesis pathways. A single pair of primers derived from the Tri13 genes detected a 583 bp fragment from 15-AcDON-chemotypes, a 644 bp fragment from 3-AcDON-chemotypes and an 859 bp fragment from NIV-producing strains. Fusarium strains from China, Nepal, USA and Europe were identified by this method, revealing their mycotoxin chemotypes identical to that obtained by chemical analyses of HPLC or GC/MS and other PCR assays. The mycotoxin chemotype-specific fragments were amplified from a highly variable region located in Tri13 genes with three deletions for 15-AcDON-chemotypes, two deletions for 3-AcDON-chemotypes and no deletion for NIV-producers. This PCR assay generated a single amplicon and thus should be more reliable than other PCR-based assays that showed the absence or presence of a PCR fragment since these assays may generate false-negative results. The results with strains from several different countries as well as from different hosts further indicated that this method should be globally applicable. This is a rapid, reliable and cost-effective method for the identification of type B trichothecene mycotoxin chemotypes in Fusarium species and food safety controls.
Fusarium graminearum clade; trichothecene; mycotoxin chemotype; 3-AcDON; 15-AcDON; NIV
The mycotoxin producing fungal pathogen Fusarium graminearum is the causal agent of Fusarium head blight (FHB) of small grain cereals in fields worldwide. Although F. graminearum is highly investigated by means of molecular genetics, detailed studies about hyphal development during initial infection stages are rare. In addition, the role of mycotoxins during initial infection stages of FHB is still unknown. Therefore, we investigated the infection strategy of the fungus on different floral organs of wheat (Triticum aestivum L.) under real time conditions by constitutive expression of the dsRed reporter gene in a TRI5prom::GFP mutant. Additionally, trichothecene induction during infection was visualised with a green fluorescent protein (GFP) coupled TRI5 promoter. A tissue specific infection pattern and TRI5 induction were tested by using different floral organs of wheat. Through combination of bioimaging and electron microscopy infection structures were identified and characterised. In addition, the role of trichothecene production for initial infection was elucidated by a ΔTRI5-GFP reporter strain.
The present investigation demonstrates the formation of foot structures and compound appressoria by F. graminearum. All infection structures developed from epiphytic runner hyphae. Compound appressoria including lobate appressoria and infection cushions were observed on inoculated caryopses, paleas, lemmas, and glumes of susceptible and resistant wheat cultivars. A specific trichothecene induction in infection structures was demonstrated by different imaging techniques. Interestingly, a ΔTRI5-GFP mutant formed the same infection structures and exhibited a similar symptom development compared to the wild type and the TRI5prom::GFP mutant.
The different specialised infection structures of F. graminearum on wheat florets, as described in this study, indicate that the penetration strategy of this fungus is far more complex than postulated to date. We show that trichothecene biosynthesis is specifically induced in infection structures, but is neither necessary for their development nor for formation of primary symptoms on wheat.
Fusarium head blight (FHB), mainly caused by Fusarium graminearum, substantially reduces wheat grain yield and quality worldwide. Proteins play important roles in defense against the fungal infection. This study characterized differentially expressed proteins between near-isogenic lines (NILs) contrasting in alleles of Fhb1, a major FHB resistance gene in wheat, to identify proteins underlining FHB resistance of Fhb1.
The two-dimensional protein profiles were compared between the Fusarium-inoculated spikes of the two NILs collected 72 h after inoculation. The protein profiles of mock- and Fusarium-inoculated Fhb1+NIL were also compared to identify pathogen-responsive proteins.
Eight proteins were either induced or upregulated in inoculated Fhb1+NIL when compared with mock-inoculated Fhb1+NIL; nine proteins were either induced or upregulated in the Fusarium-inoculated Fhb1+NIL when compared with Fusarium-inoculated Fhb1−NIL. Proteins that were differentially expressed in the Fhb1+NIL, not in the Fhb1−NIL, after Fusarium inoculation included wheat proteins for defending fungal penetration, photosynthesis, energy metabolism, and detoxification.
Coordinated expression of the identified proteins resulted in FHB resistance in Fhb1+NIL. The results provide insight into the pathway of Fhb1-mediated FHB resistance.
Deoxynivalenol (DON) is the most prevalent trichothecene in Europe and its occurrence is associated with infections of Fusarium graminearum and F. culmorum, causal agents of Fusarium head blight (FHB) on wheat. Resistance to FHB is a complex character and high variability occurs in the relationship between DON content and FHB incidence. DON conjugation to glucose (DON-3-glucoside, D3G) is the primary plant mechanism for resistance towards DON accumulation. Although this mechanism has been already described in bread wheat and barley, no data are reported so far about durum wheat, a key cereal in the pasta production chain. To address this issue, the ability of durum wheat to detoxify and convert deoxynivalenol into D3G was studied under greenhouse controlled conditions. Four durum wheat varieties (Svevo, Claudio, Kofa and Neodur) were assessed for DON-D3G conversion; Sumai 3, a bread wheat variety carrying a major QTL for FHB resistance (QFhs.ndsu-3B), was used as a positive control. Data reported hereby clearly demonstrate the ability of durum wheat to convert deoxynivalenol into its conjugated form, D3G.
masked mycotoxins; fusarium head blight; pasta; deoxynivalenol; virulence factor
Gibberella zeae is one of the most devastating pathogens of barley and wheat in the United States. The fungus also infects noncereal crops, such as potatoes and sugar beets, and the genetic relationships among barley, wheat, potato, and sugar beet isolates indicate high levels of similarity. However, little is known about the toxigenic potential of G. zeae isolates from potatoes and sugar beets. A total of 336 isolates of G. zeae from barley, wheat, potatoes, and sugar beets were collected and analyzed by TRI (trichothecene biosynthesis gene)-based PCR assays. To verify the TRI-based PCR detection of genetic markers by chemical analysis, 45 representative isolates were grown in rice cultures for 28 days and 15 trichothecenes and 2 zearalenone (ZEA) analogs were quantified using gas chromatography-mass spectrometry. TRI-based PCR assays revealed that all isolates had the deoxynivalenol (DON) marker. The frequencies of isolates with the 15-acetyl-deoxynivalenol (15-ADON) marker were higher than those of isolates with the 3-acetyl-deoxynivalenol (3-ADON) marker among isolates from all four crops. Fusarium head blight (FHB)-resistant wheat cultivars had little or no influence on the diversity of isolates associated with the 3-ADON and 15-ADON markers. However, the frequency of isolates with the 3-ADON marker among isolates from the Langdon, ND, sampling site was higher than those among isolates from the Carrington and Minot, ND, sites. In chemical analyses, DON, 3-ADON, 15-ADON, b-ZEA, and ZEA were detected. All isolates produced DON (1 to 782 μg/g) and ZEA (1 to 623 μg/g). These findings may be useful for monitoring mycotoxin contamination and for formulating FHB management strategies for these crops.
WOR1 is a gene for a conserved fungal regulatory protein controlling the dimorphic switch and pathogenicity determents in Candida albicans and its ortholog in the plant pathogen Fusarium oxysporum, called SGE1, is required for pathogenicity and expression of key plant effector proteins. F. graminearum, an important pathogen of cereals, is not known to employ switching and no effector proteins from F. graminearum have been found to date that are required for infection. In this study, the potential role of the WOR1-like gene in pathogenesis was tested in this toxigenic fungus. Deletion of the WOR1 ortholog (called FGP1) in F. graminearum results in greatly reduced pathogenicity and loss of trichothecene toxin accumulation in infected wheat plants and in vitro. The loss of toxin accumulation alone may be sufficient to explain the loss of pathogenicity to wheat. Under toxin-inducing conditions, expression of genes for trichothecene biosynthesis and many other genes are not detected or detected at lower levels in Δfgp1 strains. FGP1 is also involved in the developmental processes of conidium formation and sexual reproduction and modulates a morphological change that accompanies mycotoxin production in vitro. The Wor1-like proteins in Fusarium species have highly conserved N-terminal regions and remarkably divergent C-termini. Interchanging the N- and C- terminal portions of proteins from F. oxysporum and F. graminearum resulted in partial to complete loss of function. Wor1-like proteins are conserved but have evolved to regulate pathogenicity in a range of fungi, likely by adaptations to the C-terminal portion of the protein.
Plant pathogenic fungi can have devastating effects on crop yield and quality. In addition, these fungi may generate mycotoxins that pose health risks when the contaminated crops are consumed. The pathogen Fusarium graminearum infects wheat heads and grows through the rachis by synthesizing trichothecene toxins. The mechanisms and environmental cues triggering the production of trichothecene toxins have been studied for many years. Here, we describe a fungal gene, Fgp1, that is absolutely required for pathogenicity and mycotoxin synthesis during infection and in culture. Fgp1 is not required for vegetative growth of F. graminearum but is important for reproductive development and potentially for a putative switch from a vegetative to a pathogenic phase. Deletion of Fgp1 results in reduced expression of the trichothecene biosynthetic gene cluster and genes specifically expressed during toxin synthesis in planta and in vitro. Fgp1 contains a conserved N-terminal domain and a divergent in the C-terminal region. The corresponding C-terminus from a sister species, F. oxysporum, is unable to function fully in F. graminearum when fused to the conserved F. graminearum N-terminal domain, suggesting the gene function is highly species specific.
Fusarium graminearum, one of the causal agents of Fusarium Head Blight (FHB, scab), leads to severe losses in grain yield and quality due to the production of mycotoxins which are harmful to human and livestock. Different traits for FHB resistance in wheat were identified for common wheat (Triticum aestivum L.) while the sources of FHB resistance in durum wheat (Triticum turgidum ssp. Durum), one of the cereals most susceptible to F. graminearum infection, have not been found. New lines of evidence indicate that content and composition of cell wall polymers affect the susceptibility of the wall to degrading enzymes produced by pathogens during infection and can play a role in the outcome of host-pathogen interactions. The objective of our research is to identify potential cell wall biochemical traits linked to Fusariosis resistance to be transferred from a resistant common wheat to a susceptible durum wheat line.
A detailed analysis of cell wall composition in spikes isolated from a highly resistant common wheat accession “02-5B-318”, a breeding line derived from the FHB-resistant Chinese cv. Sumai-3 and a high susceptible durum wheat cv. Saragolla was performed. Significant differences in lignin monolignols composition, arabinoxylan (AX) substitutions and pectin methylesterification were found between resistant and susceptible plants. We isolated and characterized a pectin methylesterase gene WheatPME1, which we found being down regulated in the FHB-resistant line and induced by fungal infection in the susceptible wheat.
Our results indicate cell wall traits differing between the FHB sensitive and resistant wheat genotypes, possibly related to FHB-resistance, and identify the line 02-5B-318R as a potential resource of such traits. Evidence suggests that WheatPME1 is involved in wheat response to F. graminearum.
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Fusarium Head Blight resistance; Wheat; Pectin methylesterase; Cell wall; Fusarium graminearum
Deoxynivalenol (DON) is a mycotoxin produced by the plant pathogenic fungi Fusarium graminearum and F. culmorum. These and other closely related fungi cause a disease known as Fusarium head blight (FHB) in small grain cereals. Other mycotoxins produced by FHB-causing fungi include nivalenol, T-2 toxin, and zearalenone. Ingestion of mycotoxin-contaminated food and feed can lead to toxicosis in humans and animals, respectively. DON is the predominant and most economically important of these mycotoxins in the majority of small grain-producing regions of the world. This review examines the factors that influence DON accumulation in small grain cereals from an agricultural perspective. The occurrence and economic importance of FHB and DON in small grain cereals, epidemiological factors and cereal production practices that favor FHB development and DON accumulation in grain under field conditions, and regulatory/advisory standards for DON in food and feed are discussed. This information can be used to develop strategies that reduce DON accumulation in grain before harvest and to mitigate the human and animal health risks associated with DON contamination of food and feed.
Fusarium head blight; deoxynivalenol; small grain cereals; environmental factors; growth stage; inoculum dosage; pathogen aggressiveness; chemotype; lodging; tillage system; cultivar resistance; fungicide application; regulatory/advisory standards
Wheat is one of the main crops in Mediterranean countries, and its cultivation has an important role in the Syrian economy. In Syria, Fusarium head blight (FHB) has not been reported so far. Mycological analysis of 48 samples of wheat kernels collected from cultivation areas with different climatic conditions were performed in 2009 and 2010. Fungal isolates were identified at the genus level morphologically; Fusarium species were characterized morphologically and by species-specific PCR. The most frequent fungal genera found were Alternaria spp. and Cladosporium spp., with frequencies of 24.7% and 8.1%, respectively, while the frequency of Fusarium spp. was 1.5% of kernels. Most frequent Fusarium species were F. tricinctum (30% of all Fusarium isolates), F. culmorum (18%), F. equiseti (14%) and F. graminearum (13%). The mycotoxin production potential of selected Fusarium isolates was assessed by HPLC-MS analysis of rice cultures; chemotyping by PCR was carried out for comparison. All six F. graminearum strains tested produced small amounts (<3 mg/kg) of nivalenol (NIV). All ten F. culmorum strains tested produced large amounts of trichothecenes (>100 mg/kg); four strains produced NIV and six strains produced deoxynivalenol (DON) and 3-acetyl-deoxynivalenol (3Ac-DON). PCR chemotyping lead to an oversimplified picture, because all 3Ac-DON chemotype strains produced more DON than 3Ac-DON; furthermore, the strongest NIV producers produced significant amounts of DON. All tested strains of F. culmorum, F. graminearum, F. pseudograminearum (two strains) and most F. equiseti strains (five of six strains) produced zearalenone. Grains of durum wheat were more frequently colonized by Fusarium spp. than grains of soft wheat. Incidence of Fusarium spp. in irrigated fields was higher than in rainfed fields. The incidence of Fusarium strains producing mycotoxins raises concerns about the risk of Fusarium head blight to Syria and its consequences for public health.
Syria; wheat kernels; Fusarium chemotypes; trichothecenes
We developed a PCR-based assay to quantify trichothecene-producing Fusarium 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 Fusarium species. Amounts of the trichothecene-producing Fusarium and 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-producing Fusarium 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 head blight is a very important disease of small grain cereals with F. graminearum as one of the most important causal agents. It not only causes reduction in yield and quality but from a human and animal healthcare point of view, it produces mycotoxins such as deoxynivalenol (DON) which can accumulate to toxic levels. Little is known about external triggers influencing DON production.
In the present work, a combined in vivo/in vitro approach was used to test the effect of sub lethal fungicide treatments on DON production. Using a dilution series of prothioconazole, azoxystrobin and prothioconazole + fluoxastrobin, we demonstrated that sub lethal doses of prothioconazole coincide with an increase in DON production 48 h after fungicide treatment. In an artificial infection trial using wheat plants, the in vitro results of increased DON levels upon sub lethal prothioconazole application were confirmed illustrating the significance of these results from a practical point of view. In addition, further in vitro experiments revealed a timely hyperinduction of H2O2 production as fast as 4 h after amending cultures with prothioconazole. When applying H2O2 directly to germinating conidia, a similar induction of DON-production by F. graminearum was observed. The effect of sub lethal prothioconazole concentrations on DON production completely disappeared when applying catalase together with the fungicide.
These cumulative results suggest that H2O2 induced by sub lethal doses of the triazole fungicide prothioconazole acts as a trigger of DON biosynthesis. In a broader framework, this work clearly shows that DON production by the plant pathogen F. graminearum is the result of the interaction of fungal genomics and external environmental triggers.
Hessian fly (Mayetiola destructor), a member of the gall midge family, is one of the most destructive pests of wheat (Triticum aestivum) worldwide. Probing of wheat plants by the larvae results in either an incompatible (avirulent larvae, resistant plant) or a compatible (virulent larvae, susceptible plant) interaction. Virulent larvae induce the formation of a nutritive tissue, resembling the inside surface of a gall, in susceptible wheat. These nutritive cells are a rich source of proteins and sugars that sustain the developing virulent Hessian fly larvae. In addition, on susceptible wheat, larvae trigger a significant increase in levels of amino acids including proline and glutamic acid, which are precursors for the biosynthesis of ornithine and arginine that in turn enter the pathway for polyamine biosynthesis.
Following Hessian fly larval attack, transcript abundance in susceptible wheat increased for several genes involved in polyamine biosynthesis, leading to higher levels of the free polyamines, putrescine, spermidine and spermine. A concurrent increase in polyamine levels occurred in the virulent larvae despite a decrease in abundance of Mdes-odc (ornithine decarboxylase) transcript encoding a key enzyme in insect putrescine biosynthesis. In contrast, resistant wheat and avirulent Hessian fly larvae did not exhibit significant changes in transcript abundance of genes involved in polyamine biosynthesis or in free polyamine levels.
The major findings from this study are: (i) although polyamines contribute to defense in some plant-pathogen interactions, their production is induced in susceptible wheat during interactions with Hessian fly larvae without contributing to defense, and (ii) due to low abundance of transcripts encoding the rate-limiting ornithine decarboxylase enzyme in the larval polyamine pathway the source of polyamines found in virulent larvae is plausibly wheat-derived. The activation of the host polyamine biosynthesis pathway during compatible wheat-Hessian fly interactions is consistent with a model wherein the virulent larvae usurp the polyamine biosynthesis machinery of the susceptible plant to acquire nutrients required for their own growth and development.
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Polyamines; Wheat; Hessian fly; Compatible; Incompatible; RT-qPCR; Odc; Samdc; Spds
Deoxynivalenol (DON) is the major mycotoxin produced by Fusarium fungi in grains. Food and feed contaminated with DON pose a health risk to humans and livestock. The risk can be reduced by enzymatic detoxification. Complete mineralization of DON by microbial cultures has rarely been observed and the activities turned out to be unstable. The detoxification of DON by reactions targeting its epoxide group or hydroxyl on carbon 3 is more feasible. Microbial strains that de-epoxidize DON under anaerobic conditions have been isolated from animal digestive system. Feed additives claimed to de-epoxidize trichothecenes enzymatically are on the market but their efficacy has been disputed. A new detoxification pathway leading to 3-oxo-DON and 3-epi-DON was discovered in taxonomically unrelated soil bacteria from three continents; the enzymes involved remain to be identified. Arabidopsis, tobacco, wheat, barley, and rice were engineered to acetylate DON on carbon 3. In wheat expressing DON acetylation activity, the increase in resistance against Fusarium head blight was only moderate. The Tri101 gene from Fusarium sporotrichioides was used; Fusarium graminearum enzyme which possesses higher activity towards DON would presumably be a better choice. Glycosylation of trichothecenes occurs in plants, contributing to the resistance of wheat to F. graminearum infection. Marker-assisted selection based on the trichothecene-3-O-glucosyltransferase gene can be used in breeding for resistance. Fungal acetyltransferases and plant glucosyltransferases targeting carbon 3 of trichothecenes remain promising candidates for engineering resistance against Fusarium head blight. Bacterial enzymes catalyzing oxidation, epimerization, and less likely de-epoxidation of DON may extend this list in future.
Deoxynivalenol; Detoxification; De-epoxidation; Epimerization; Feed additive; Resistance engineering
Fusarium head blight (FHB), caused by Fusarium graminearum, is a devastating disease of small grain cereal crops. FHB causes yield reductions and contamination of grain with trichothecene mycotoxins such as deoxynivalenol (DON). DON inhibits protein synthesis in eukaryotic cells and acts as a virulence factor during fungal pathogenesis, therefore resistance to DON is considered an important component of resistance against FHB. One mechanism of resistance to DON is conversion of DON to DON-3-O-glucoside (D3G). Previous studies showed that expression of the UDP-glucosyltransferase genes HvUGT13248 from barley and AtUGt73C5 (DOGT1) from Arabidopsis thaliana conferred DON resistance to yeast. Over-expression of AtUGt73C5 in Arabidopsis led to increased DON resistance of seedlings but also to dwarfing of transgenic plants due to the formation of brassinosteroid-glucosides. The objectives of this study were to develop transgenic Arabidopsis expressing HvUGT13248, to test for phenotypic changes in growth habit, and the response to DON. Transgenic lines that constitutively expressed the epitope-tagged HvUGT13248 protein exhibited increased resistance to DON in a seed germination assay and converted DON to D3G to a higher extent than the untransformed wild-type. By contrast to the over-expression of DOGT1 in Arabidopsis, which conjugated the brassinosteriod castasterone with a glucoside group resulting in a dwarf phenotype, expression of the barley HvUGT13248 gene did not lead to drastic morphological changes. Consistent with this observation, no castasterone-glucoside formation was detectable in yeast expressing the barley HvUGT13248 gene. This barley UGT is therefore a promising candidate for transgenic approaches aiming to increase DON and Fusarium resistance of crop plants without undesired collateral effects.
Deoxynivalenol; Fusarium head blight; trichothecenes; UDP-glucosyltransferase
Fungicide application is a key factor in the control of mycotoxin contamination in the harvested wheat grain. However, the practical results are often disappointing. In 2000-2004, 2006-2008 and 2007 and 2008, three experiments were made to test the efficacy of fungicide control on Fusarium Head Blight (FHB) in wheat and to find ways to improve control of the disease and toxin contamination. In a testing system we have used for 20 years, tebuconazole and tebuconazole + prothioconazole fungicides regularly reduced symptoms by about 80% with a correlating reduction in toxin contamination. Averages across the years normally show a correlation of r = 0.90 or higher. The stability differences (measured by the stability index) between the poorest and the best fungicides are about 10 or more times, differing slightly in mycotoxin accumulation, FHB index (severity) and Fusarium damaged kernels (FDK). The weak fungicides, like carbendazim, were effective only when no epidemic occurred or epidemic severity was at a very low level. Similar fungicide effects were seen on wheat cultivars which varied in FHB resistance. In this study, we found three fold differences in susceptibility to FHB between highly susceptible and moderately resistant cultivars when treated with fungicides. In the moderately resistant cultivars, about 50% of the fungicide treatments lowered the DON level below the regulatory limit. In the most susceptible cultivars, all fungicides failed to reduce mycotoxin levels low enough for grain acceptance, in spite of the fact that disease was significantly reduced. The results correlated well with the results of the large-scale field tests of fungicide application at the time of natural infection. The Turbo FloodJet nozzle reduced FHB incidence and DON contamination when compared to the TeeJet XR nozzle. Overall, the data suggest that significant decreases in FHB incidence and deoxynivalenol contamination in field situations are possible with proper fungicide applications. Additionally, small plot tests can be used to evaluate the quality of the field disease and toxin production.
Gibberellazeae; F.culmorum; scab; AUDPC; FDK; DON; prothioconazole; tebuconazole; carbendazim
Fusarium head blight (FHB) caused by Fusarium species like F. graminearum is a devastating disease of wheat (Triticum aestivum) worldwide. Mycotoxins such as deoxynivalenol produced by the fungus affect plant and animal health, and cause significant reductions of grain yield and quality. Resistant varieties are the only effective way to control this disease, but the molecular events leading to FHB resistance are still poorly understood. Transcriptional profiling was conducted for the winter wheat cultivars Dream (moderately resistant) and Lynx (susceptible). The gene expressions at 32 and 72 h after inoculation with Fusarium were used to trace possible defence mechanisms and associated genes. A comparative qPCR was carried out for selected genes to analyse the respective expression patterns in the resistant cultivars Dream and Sumai 3 (Chinese spring wheat).
Among 2,169 differentially expressed genes, two putative main defence mechanisms were found in the FHB-resistant Dream cultivar. Both are defined base on their specific mode of resistance. A non-specific mechanism was based on several defence genes probably induced by jasmonate and ethylene signalling, including lipid-transfer protein, thionin, defensin and GDSL-like lipase genes. Additionally, defence-related genes encoding jasmonate-regulated proteins were up-regulated in response to FHB. Another mechanism based on the targeted suppression of essential Fusarium virulence factors comprising proteases and mycotoxins was found to be an essential, induced defence of general relevance in wheat. Moreover, similar inductions upon fungal infection were frequently observed among FHB-responsive genes of both mechanisms in the cultivars Dream and Sumai 3.
Especially ABC transporter, UDP-glucosyltransferase, protease and protease inhibitor genes associated with the defence mechanism against fungal virulence factors are apparently active in different resistant genetic backgrounds, according to reports on other wheat cultivars and barley. This was further supported in our qPCR experiments on seven genes originating from this mechanism which revealed similar activities in the resistant cultivars Dream and Sumai 3. Finally, the combination of early-stage and steady-state induction was associated with resistance, while transcript induction generally occurred later and temporarily in the susceptible cultivars. The respective mechanisms are attractive for advanced studies aiming at new resistance and toxin management strategies.
Mycotoxin contamination associated with head blight of wheat and other grains caused by Fusarium culmorum and F. graminearum is a chronic threat to crop, human, and animal health throughout the world. One of the most important toxins in terms of human exposure is deoxynivalenol (DON) (formerly called vomitoxin), an inhibitor of protein synthesis with a broad spectrum of toxigenicity against animals. Certain Fusarium toxins have additional antimicrobial activity, and the phytotoxin fusaric acid has recently been shown to modulate fungus-bacterium interactions that affect plant health (Duffy and Défago, Phytopathology 87:1250-1257, 1997). The potential impact of DON on Fusarium competition with other microorganisms has not been described previously. Any competitive advantage conferred by DON would complicate efforts to control Fusarium during its saprophytic growth on crop residues that are left after harvest and constitute the primary inoculum reservoir for outbreaks in subsequent plantings. We examined the effect of the DON mycotoxin on ecological interactions between pathogenic Fusarium and Trichoderma atroviride strain P1, a competitor fungus with biocontrol activity against a wide range of plant diseases. Expression of the Trichoderma chitinase genes, ech42 and nag1, which contribute to biocontrol activity, was monitored in vitro and on crop residues of two maize cultivars by using goxA reporter gene fusions. We found that DON-producing F. culmorum and F. graminearum strains repressed expression of nag1-gox. DON-negative wild-type Fusarium strains and a DON-negative mutant with an insertional disruption in the tricothecene biosynthetic gene, tri5, had no effect on antagonist gene expression. The role of DON as the principal repressor above other pathogen factors was confirmed. Exposure of Trichoderma to synthetic DON or to a non-DON-producing Fusarium mutant resulted in the same level of nag1-gox repression as the level observed with DON-producing Fusarium. DON repression was specific for nag1-gox and had no effect, either positive or negative, on expression of another key chitinase gene, ech42. This is the first demonstration that a target pathogen down-regulates genes in a fungal biocontrol agent, and our results provide evidence that mycotoxins have a novel ecological function as factors in Fusarium competitiveness.
Fusarium graminearum sensu stricto (s.s.) is an ubiquitous pathogen of cereals. The economic impact of Fusarium head blight (FHB) is characterized by crop losses and mycotoxin contamination. Our objective was to associate SNP diversity within candidate genes with phenotypic traits. A total of 77 F. graminearum s.s. isolates was tested for severity of fungal infection (= aggressiveness) and deoxynivalenol (DON) production in an inoculated field experiment at two locations in each of two years. For seven genes known to control fungal growth (MetAP1, Erf2) or DON production (TRI1, TRI5, TRI6 TRI10 and TRI14) single nucleotides polymorphic sites (SNPs) were determined and evaluated for the extent of linkage disequilibrium (LD). Associations of SNPs with both phenotypic traits were tested using linear mixed models.
Decay of LD was in most instances fast. Two neighboring SNPs in MetAP1 and one SNP in Erf2 were significantly (P < 0.05) associated with aggressiveness explaining proportions of genotypic variance (pG) of 25.6%, 0.5%, and 13.1%, respectively. One SNP in TRI1 was significantly associated with DON production (pG = 4.4).
We argue that using the published sequence information of Fusarium graminearum as a template to amplify comparative sequence parts of candidate genes is an effective method to detect quantitative trait loci. Our findings underline the potential of candidate gene association mapping approaches to identify functional SNPs underlying aggressiveness and DON production for F. graminearum s.s populations.
Aggressiveness; Association mapping; DON; Fusarium graminearum; FHB; Linkage disequilibrium; QTL; Triticum aestivum; SNP
Fusarium head blight (FHB), caused mainly by Fusarium graminearum (Fg) Schwabe (teleomorph: Gibberellazeae Schwble), brings serious damage to wheat production. Chinese wheat landrace Wangshuibai is one of the most important resistance sources in the world. The knowledge of mechanism underlying its resistance to FHB is still limited.
To get an overview of transcriptome characteristics of Wangshuibai during infection by Fg, a high-throughput RNA sequencing based on next generation sequencing (NGS) technology (Illumina) were performed. Totally, 165,499 unigenes were generated and assigned to known protein databases including NCBI non-redundant protein database (nr) (82,721, 50.0%), Gene Ontology (GO) (38,184, 23.1%), Swiss-Prot (50,702, 30.6%), Clusters of orthologous groups (COG) (51,566, 31.2%) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) (30,657, 18.5%), as determined by Blastx search. With another NGS based platform, a digital gene expression (DGE) system, gene expression in Wangshuibai and its FHB susceptible mutant NAUH117 was profiled and compared at two infection stages by inoculation of Fg at 24 and 48 hour, with the aim of identifying genes involved in FHB resistance.
Pathogen-related proteins such as PR5, PR14 and ABC transporter and JA signaling pathway were crucial for FHB resistance, especially that mediated by Fhb1. ET pathway and ROS/NO pathway were not activated in Wangshuibai and may be not pivotal in defense to FHB. Consistent with the fact that in NAUH117 there presented a chromosome fragment deletion, which led to its increased FHB susceptibility, in Wangshuibai, twenty out of eighty-nine genes showed changed expression patterns upon the infection of Fg. The up-regulation of eight of them was confirmed by qRT-PCR, revealing they may be candidate genes for Fhb1 and need further functional analysis to confirm their roles in FHB resistance.
Fusarium head blight; Wangshuibai; NAUH117; Resistance; Transcriptome; Digital gene expression