Sphaerodes mycoparasitica biotrophic mycoparasite of 3-acetyldeoxynivalenol- and 15-acetyldeoxynivalenol-producing toxigenic Fusarium graminearum chemotypes

Fusarium spp. are economically important crop pathogens and causal agents of Fusarium head blight (FHB) of cereals worldwide. Of the FHB pathogens, Fusarium graminearum 3-acetyldeoxynivalenol (3-ADON) and 15-acetyldeoxynivalenol (15-ADON) are the most aggressive mycotoxigenic chemotypes, threatening food and feed quality as well as animal and human health. The objective of the study was to evaluate host specificity and fungal-fungal interactions of Sphaerodes mycoparasitica- a recently described mycoparasite - with F. graminearum 3- and 15-ADON strains by employing in vitro, microscopic and PCR techniques. Results obtained in this study show that the germination of mycoparasite ascospore in the presence of F. graminearum 3- and 15-ADON filtrates was greatly improved compared with Fusarium proliferatum and Fusarium sporotrichioides filtrates, suggesting a compatible interaction. Using quantitative real-time PCR with Fusarium-specific (Fg16N) and trichothecene Tri5 (Tox5-1/2)-specific primer sets, S. mycoparasitica was found to reduce the amount of F. graminearum 3-ADON and 15-ADON DNAs under separate coinoculation assays. Sphaerodes mycoparasitica was not only able to germinate in the presence of F. graminearum filtrates, but also established biotrophic mycoparasitic relations with two F. graminearum chemotypes and suppressed Fusarium growth.     

PCR detection assays for the trichothecene-producing species Fusarium graminearum, Fusarium culmorum, Fusarium poae, Fusarium equiseti and Fusarium sporotrichioides

Contamination of small-grain cereals with the fungal species Fusarium graminearum, F. culmorum, F. poae, F. sporotrichioides and F. equiseti is an important source of trichothecenes, Zearalenone and other mycotoxins which cause serious diseases in human and animals. Additionally, these species contribute to Fusarium Head Blight, a disease which produces important losses in cereal yield. Early detection and control of these Fusarium species is crucial to prevent toxins entering the food chain and a useful tool in disease management practices. We describe the development of specific PCR assays to F. graminearum, F. culmorum, F. poae, F. sporotrichioides and F. equiseti using DNA from pure fungal cultures as well as from naturally infected wheat seeds, using in this case a rapid and easy protocol for DNA isolation. The specific primers were designed on the basis of IGS sequences (Intergenic Spacer of rDNA), a multicopy region in the genome that permits to enhance the sensitivity of the assay in comparison with PCR assays based on single-copy sequences.     

The role of chitinese of Serratia marcescens in controlling the production of zearalenone by Fusarium graminearum

An experiment was designed to determine the role of the chitinase of S. marcescens in controlling the production of zearalenone by F. graminearum isolated from diseased wheat plants. Fusarium graminearum, F. avenaceum, F. equiseti, F. culmorum, and F. solani were isolated from diseased wheat plant. F. graminearum culture materials were highly pathogenic for wheat seedlings, toxic to ducklings and produced high levels of zearalenone. S. marcescens was grown on the cell wall of F. graminearum and its components, chitin and laminarin as a sole carbon source. The release of N-acetyl-D-glucosamine from chitin, F. graminearum cell wall and living or drying mycelium indicated substrate degradation. S. marcescens applied to F. graminearum infested wheat kernels decreased greatly the occurrence of zearalenone after 4 weeks of incubation. F. graminearum culture materials treated with S. marcescens proved to be non-toxic to ducklings and wheat seed germination.     

小麦赤霉病镰孢菌毒素的生物合成及其分子调控

禾谷镰刀菌是小麦赤霉病的主要致病菌,其真菌次生代谢产生的单端孢霉烯类B型毒素,如雪腐镰刀菌烯醇(nivalenol,NIV)、脱氧雪腐镰刀菌烯醇(deoxynivalenol,DON)和其它乙酰化衍生物等污染小麦籽粒后对人畜健康构成威胁。综述了近年来国内外对小麦赤霉病镰孢菌单端孢霉烯类B型毒素生物合成的主要途径及分子调控研究进展,对毒素合成过程中的重要调控基因如TRI5、TRI7和TRI13在农业中的应用进行了阐述。     

A mitogen-activated protein kinase gene (MGV1) in Fusarium graminearum is required for female fertility,heterokaryon formation,and plant infection

Fusarium graminearum is an important pathogen of small grains and maize in many areas of the world. Infected grains are often contaminated with mycotoxins harmful to humans and animals. During the past decade, F. graminearum has caused several severe epidemics of head scab in wheat and barley. In order to understand molecular mechanisms regulating fungal development and pathogenicity in this pathogen, we isolated and characterized a MAP kinase gene, MGV1, which is highly homologous to the MPS1 gene in Magnaporthe grisea. The MGV1 gene was dispensable for conidiation in F. graminearum but essential for female fertility during sexual reproduction. Vegetative growth of mgv1 deletion mutants was normal in liquid media but reduced on solid media. Mycelia of the mgv1 mutants had weak cell walls and were hypersensitive to cell wall degrading enzymes. Interestingly, the mgv1 mutants were self-incompatible when tested for heterokaryon formation, and their virulence was substantially reduced. The ability of the mutants to accumulate trichothecene mycotoxins on inoculated wheat was also greatly reduced. Our data suggest that MGV1 in F. graminearum is involved in multiple developmental processes related to sexual reproduction, plant infection, and cell wall integrity.     

A microsatellite based method for quantification of fungi in decomposing plant material elucidates the role of Fusarium graminearum DON production in the saprophytic competition with Trichoderma atroviride in maize tissue microcosms

Common PCR assays for quantification of fungi in living plants cannot be used to study saprophytic colonization of fungi because plant decomposition releases PCR-inhibiting substances and saprophytes degrade the plant DNA which could serve as internal standard. The microsatellite PCR assays presented here overcome these problems by spiking samples prior to DNA extraction with mycelium of a reference strain. PCR with fluorescent primers co-amplifies microsatellite fragments of different length from target and reference strains. These fragments were separated in a capillary sequencer with fluorescence detection. The target/reference ratio of fluorescence signal was used to calculate target biomass in the sample. Such PCR assays were developed for the mycotoxin deoxynivalenol (DON)-producing wheat and maize pathogen Fusarium graminearum and the biocontrol agent Trichoderma atroviride, using new microsatellite markers. In contrast to real-time PCR assays, the novel PCR assays showed reliable fungal biomass quantification in samples with differentially decomposed plant tissue. The PCR assays were used to quantify the two fungi after competitive colonization of autoclaved maize leaf tissue in microcosms. Using a DON-producing F. graminearum wild-type strain and its nontoxigenic mutant we found no evidence for a role of DON production in F. graminearum defense against T. atroviride. The presence of T. atroviride resulted in a 36% lower wild-type DON production per biomass.     

Development of a PCR assay to detect the potential production of nivalenol in Fusarium poae

Fusarium species can produce mycotoxins, which can contaminate cereal-based food producing adverse effects for human and animal health. In recent years, the importance of Fusarium poae has increased within the Fusarium head blight complex. Fusarium poae is known to produce trichothecenes, especially nivalenol, a potent mycotoxin able to cause a variety of toxic effects. In this study, a specific primer pair was designed based on the tri7 gene to detect potential nivalenol-producing F.?poae isolates. A total of 125 F.?poae, four F.?cerealis, two F.?culmorum, one F.?langsethiae, one F.?sporotrichioides and seven F.?graminearum, plus F.?austroamericanum, F.?meridionale, F.?graminearum sensu stricto and F.?cortaderiae from the NRRL collection were analysed, and only F.?poae isolates gave a positive result for the presence of a 296-bp partial tri7 DNA fragment. Moreover, the primer set was tested from cereal seed samples where F.?poae and other Fusarium species with a negative result for the specific reaction ( F.?graminearum, F.?oxysporum, F.?chlamydosporum, F.?sporotrichioides, F.?equiseti and F.?acuminatum) were isolated, and the expected fragment was amplified. We developed a rapid and reliable PCR assay to detect potential nivalenol-producing F.?poae isolates.     

Mycological survey of Korean cereals and production of mycotoxins by Fusarium isolates.

The fungal species isolated from Korean cereals (barley, polished barley, wheat, rye, and malt) were Alternaria spp., Aspergillus spp., Chaetomium spp., Drechslera spp., Epicoccum sp., Fusarium spp., and Penicillium spp., etc. The number of Fusarium strains isolated was 36, and their ability to produce Fusarium mycotoxins on rice was tested. Nivalenol (NIV) was produced by Fusarium graminearum (7 of 9 isolates), Fusarium oxysporum (3 of 10 isolates), and Fusarium spp. (7 of 15 isolates). Of 15 isolates of Fusarium spp., 6 formed deoxynivalenol (DON). Fusarenon-X and 3-acetyl-DON were produced by most NIV- and DON-forming isolates, respectively. Zearalenone was produced by 3 isolates of F. graminearum, 1 isolate of Fusarium equiseti, and 11 isolates of Fusarium spp. T-2 toxin was not produced by any Fusarium isolates. The highest concentrations of mycotoxins produced by Fusarium isolates were 77.4 (NIV), 5.3 (DON), 138.3 (fusarenon-X), 40.6 (3-acetyl-DON), and 23.2 (zearalenone) micrograms/g.     

Mycological survey of Korean cereals and production of mycotoxins by Fusarium isolates

The fungal species isolated from Korean cereals (barley, polished barley, wheat, rye, and malt) were Alternaria spp., Aspergillus spp., Chaetomium spp., Drechslera spp., Epicoccum sp., Fusarium spp., and Penicillium spp., etc. The number of Fusarium strains isolated was 36, and their ability to produce Fusarium mycotoxins on rice was tested. Nivalenol (NIV) was produced by Fusarium graminearum (7 of 9 isolates), Fusarium oxysporum (3 of 10 isolates), and Fusarium spp. (7 of 15 isolates). Of 15 isolates of Fusarium spp., 6 formed deoxynivalenol (DON). Fusarenon-X and 3-acetyl-DON were produced by most NIV- and DON-forming isolates, respectively. Zearalenone was produced by 3 isolates of F. graminearum, 1 isolate of Fusarium equiseti, and 11 isolates of Fusarium spp. T-2 toxin was not produced by any Fusarium isolates. The highest concentrations of mycotoxins produced by Fusarium isolates were 77.4 (NIV), 5.3 (DON), 138.3 (fusarenon-X), 40.6 (3-acetyl-DON), and 23.2 (zearalenone) micrograms/g.     

Antagonistic potentiality of Trichoderma harzianum towards seed-borne fungal pathogens of winter wheat cv. Protiva in vitro and in vivo

The antagonistic effect of Trichoderma harzianum on a range of seed-borne fungal pathogens of wheat (viz. Fusarium graminearum, Bipolaris sorokiniana, Aspergillus spp., and Penicillium spp.) was assessed. The potential of T. harzianum as a biocontrol agent was tested in vitro and under field conditions. Coculture of the pathogens and Trichoderma under laboratory conditions clearly showed dominance of T. harzianum. Under natural conditions, biocontrol effects were also obtained against the test fungi. One month after sowing, field emergence (plant stand) was increased by 15.93% over that obtained with the control treatment, and seedling infection was reduced significantly. Leaf blight severity was decreased from 22 to 11 at the heading stage, 35 to 31 at the flowering stage, and 86 to 74 at the grain filling stage. At harvest, the number of tillers per plant was increased by 50%, the yield was increased by 31.58%, and the 1,000-seed weight was increased by 21%.     

RAS2 regulates growth and pathogenesis in Fusarium graminearum

Fusarium graminearum is a ubiquitous pathogen of cereal crops, including wheat, barley, and maize. Diseases caused by F. graminearum are of particular concern because harvested grains frequently are contaminated with harmful mycotoxins such as deoxynivalenol (DON). In this study, we explored the role of Ras GTPases in pathogenesis. The genome of F. graminearum contains two putative Ras GTPase-encoding genes. The two genes (RAS1 and RAS2) showed different patterns of expression under different conditions of nutrient availability and in various mutant backgrounds. RAS2 was dispensable for survival but, when disrupted, caused a variety of morphological defects, including slower growth on solid media, delayed spore germination, and significant reductions in virulence on wheat heads and maize silks. Intracellular cAMP levels were not affected by deletion of RAS2 and exogenous treatment of the ras2 mutant with cAMP did not affect phenotypic abnormalities, thus indicating that RAS2 plays a minor or no role in cAMP signaling. However, phosphorylation of the mitogen-activated protein (MAP) kinase Gpmk1 and expression of a secreted lipase (FGL1) required for infection were reduced significantly in the ras2 mutant. Based on these observations, we hypothesize that RAS2 regulates growth and virulence in F. graminearum by regulating the Gpmk1 MAP kinase pathway.     

Fusarium graminearum and deoxynivalenol contamination in the durum wheat area of Argentina

Fusarium graminearum head blight of wheat is a destructive disease of the world's wheat-growing areas. This work was performed to analyze the distribution and contamination of deoxynivalenol (DON) and its relationship with F. graminearum kernel invasion in Argentina durum wheat area during two consecutive harvests. A total of 147 samples (cultivars and lines) of durum wheat from 5 locations of the major cropping area (Southern Buenos Aires Province) were analyzed. Percentage of F. graminearum kernel infection was evaluated following the blotter test (ISTA method) and fusarotoxins were analyzed by thin layer chromatography. None of the varieties and lines were free of F. graminearum infection. In the first harvest fungal invasion was very low. From 40 samples, 55% showed DON contamination but only 4 samples (10%) were higher than 2 ppm. In the second harvest, a crop year conducive to scab development, the highest level of F. graminearum kernel invasion observed was 42% on a sample from the humid area (eastern Buenos Aires Province) DON was detected in 47 (78.2%) of 60 samples analyzed and 19 (31.6%) showed levels of DON higher than those established in the guidelines in Canada and USA for food and feedstuff. In both years all locations situated in the humid area showed levels ranging from 0 to > 8 ppm. Within the durum wheat area differences among locations were found. This analysis indicates the need for more information on the problem and distribution of Fusarium mycotoxins in durum wheat grown in Argentina.     

The Type 2C protein phosphatase FgPtc1p of the plant fungal pathogen Fusarium graminearum is involved in lithium toxicity and virulence

Type 2C protein phosphatases (PP2Cs) are monomeric protein serine/threonine phosphatases that play various roles in eukaryotic organisms. In this study, we characterized the PP2C encoded by FgPTC1 in Fusarium graminearum , the major causal agent of Fusarium head blight on wheat and barley. We found that deletion of FgPTC1 delays the mycelium growth of F. graminearum in response to lithium. Consistently, FgPTC1 complemented the function of ScPTC1 in lithium toxicity in Saccharomyces cerevisiae . Furthermore, we showed that deletion of FgPTC1 attenuated the virulence of F. graminearum on wheat. Therefore, FgPTC1 plays an important role in regulating the hyphal growth and virulence of F. graminearum .     

Involvement of trichothecenes in fusarioses of wheat, barley and maize evaluated by gene disruption of the trichodiene synthase (Tri5) gene in three field isolates of different chemotype and virulence

Fusarium graminearum is the main causative agent of Fusarium head blight on small grain cereals and of ear rot on maize. The disease leads to dramatic yield losses and to an accumulation of mycotoxins. The most dominant F. graminearum mycotoxins are the trichothecenes, with deoxynivalenol and nivalenol being the most prevalent derivatives. To investigate the involvement of trichothecenes in the virulence of the pathogen, the gene coding for the initial enzyme of the trichothecene pathway was disrupted in three field isolates, differing in chemotype and in virulence. From each isolate three individual disruption mutants were tested for their virulence on wheat, barley and maize. Despite the different initial virulence of the three wild-type progenitor strains on wheat, all disruption mutants caused disease symptoms on the inoculated spikelet, but the symptoms did not spread into other spikelets. On barley, the trichothecene deficient mutants showed no significant difference compared to the wild-type strains: all were equally aggressive. On maize, mutants derived from the NIV-producing strain caused less disease than their wild-type progenitor strain, while mutants derived from DON-producing strains caused the same level of disease as their progenitor strains. These data demonstrate that trichothecenes influence the virulence of F. graminearum in a highly complex manner, which is strongly host as well as moderately chemotype specific.     

Microbiological and SYBR green real-time PCR detection of major Fusarium head blight pathogens on wheat ears

Fusarium head blight (FHB) caused by several Fusarium species is one of the most serious diseases affecting wheat throughout the world. The efficiency of microbiological assays and real-time PCRto quantify major FHB pathogens in wheat ears after inoculation with F. graminearum, F. culmorum, F. avenaceum and F. poae undergreenhouse and field conditions were evaluated. The frequency of infected kernel, content of fungal biomass, disease severity and kernel weight were determined. To measure the fungal biomass an improved DNA extraction method and a SYBR Green real-time PCR were developed. The SYBR Green real-time PCR proved to be highly specific for individual detection of the species in a matrix including fungal and plant DNA. The effect of Fusarium infection on visible FHB severity, frequency of infected kernels and thousand-kernel mass (TKM) significantly depended on the Fusarium species/isolate. F. graminearum resulted in highest disease level, frequency of infected kernels, content of fungal biomass, and TKM reduction followed by F. culmorum, EF avenaceum and F. poae, respectively. The comparison of frequency and intensity of kernel colonization proved differences in aggressiveness and development of the fungi in the kernels. Only for F. graminearum, the most aggressive isolate, application of microbiological and real-time PCR assays gave similar results. For the other species, the intensity of kernel colonization was lower than expected from the frequency of infection.     

Real-time PCR assay to quantify Fusarium graminearum wild-type and recombinant mutant DNA in plant material

Fusarium graminearum (teleomorph, Gibberella zeae) is the predominant causal agent of Fusarium head blight (FHB) of wheat resulting in yearly losses through reduction in grain yield and quality and accumulation of fungal generated toxins in grain. Numerous fungal genes potentially involved in virulence have been identified and studies with deletion mutants to ascertain their role are in progress. Although wheat field trials with wild-type and mutant strains are critical to understand the role these genes may play in the disease process, the interpretation of field trial data is complicated by FHB generated by indigenous species of F. graminearum. This report describes the development of a SYBR green-based real time PCR assay that quantifies the total F. graminearum genomic DNA in a plant sample as well as the total F. graminearum genomic DNA contributed from a strain containing a common fungal selectable marker used to create deletion mutants. We found our method more sensitive, reproducible and accurate than other similar recently described assays and comparable to the more expensive probe-based assays. This assay will allow investigators to correlate the amount of disease observed in wheat field trials to the F. graminearum mutant strains being examined.     

Method evaluation of Fusarium DNA extraction from mycelia and wheat for down-stream real-time PCR quantification and correlation to mycotoxin levels

Identification of Fusarium species by traditional methods requires specific skill and experience and there is an increased interest for new molecular methods for identification and quantification of Fusarium from food and feed samples. Real-time PCR with probe technology (Taqman) can be used for the identification and quantification of several species of Fusarium from cereal grain samples. There are several critical steps that need to be considered when establishing a real-time PCR-based method for DNA quantification, including extraction of DNA from the samples. In this study, several DNA extraction methods were evaluated, including the DNeasy Plant Mini Spin Columns (Qiagen), the Bio robot EZ1 (Qiagen) with the DNeasy Blood and Tissue Kit (Qiagen), and the Fast-DNA Spin Kit for Soil (Qbiogene). Parameters such as DNA quality and stability, PCR inhibitors, and PCR efficiency were investigated. Our results showed that all methods gave good PCR efficiency (above 90%) and DNA stability whereas the DNeasy Plant Mini Spin Columns in combination with sonication gave the best results with respect to Fusarium DNA yield. The modified DNeasy Plant Mini Spin protocol was used to analyse 31 wheat samples for the presence of F. graminearum and F. culmorum. The DNA level of F. graminearum could be correlated to the level of DON (r(2) = 0.9) and ZEN (r(2) = 0.6) whereas no correlation was found between F. culmorum and DON/ZEA. This shows that F. graminearum and not F. culmorum, was the main producer of DON in Swedish wheat during 2006.     

Multiplex PCR assay for the identification of nivalenol, 3- and 15-acetyl-deoxynivalenol chemotypes in Fusarium

The ability to rapidly distinguish trichothecene chemotypes in a given species/population of the genus Fusarium is important due to significant differences in the toxicity of these secondary metabolites. A multiplex PCR assay, based on primer pairs derived from the Tri3, Tri5 and Tri7 genes of the trichothecene gene cluster was established for the identification of the different chemotypes among Fusarium graminearum, F. culmorum and F. cerealis. Using the selected primers, specific amplification products of 625, 354 and 708 bp were obtained from Fusarium isolates producing nivalenol, 3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol, respectively. Moreover, the multiplex PCR was successfully used to identify the chemotype of the Fusarium species contaminating wheat kernels. Four picograms of fungal DNA were found to be necessary to obtain a visible amplification product.     

Xyloglucanases in the interaction between saprobe fungi and the arbuscular mycorrhizal fungus Glomus mosseae

We studied the production of xyloglucanase enzymes of pea and lettuce roots in the presence of saprobe and arbuscular mycorrhizal (AM) fungi. The AM fungus Glomus mosseae and the saprobe fungi Fusarium graminearum, Fusarium oxysporum-126, Trichoderma harzianum, Penicillium chrysogenum, Pleurotus ostreatus and Aspergillus niger were used. G. mosseae increased the shoot and root dry weight of pea but not of lettuce. Most of the saprobe fungi increased the level of mycorrhization of pea and lettuce, but only P. chrysogenum and T. harzianum inoculated together with G. mosseae increased the dry weight of pea and lettuce respectively. The AM and saprobe fungi increased the production of xyloglucanases by plant roots. The level of xyloglucanase activities and the number of xyloglucanolytic isozymes in plants inoculated with G. mosseae and most of the saprobe fungi tested were higher than when both microorganisms were inoculated separately. The possible relationship between xylogucanase activities and the ability of AM and saprobe fungi to improve the dry weight and AM root colonization of plants was discussed.     

东北地区小麦赤霉病菌DNA随机扩增多态性分析*

利用RAPD对我国东北地区引致小麦赤霉病的2种镰刀菌进行种群分析,并与江苏和西北的菌株进行了比较。共筛选出16个引物用于扩增反应,其扩增图谱显示供试菌株在种间和种内均具多态性。多数引物扩增出了种的特征性谱带,可用作种的鉴定。综合所有谱带系统聚类所得树状图明显地将Fusarium graminearum的34个菌株和F.avenaceum的5个菌株各聚为一类,每一类又可区分为不同组,以前者分为3组,后者分为2组差异显著,表明种内存在不同的菌系类型;组的划分与菌株致病力及其寄主品种间没有必然的相关性,但与菌株分布的大区域生态气候类型似有联系,供试的东北地区F.graminearum大多数菌株与西北的菌系有较大的遗传相似性,与江苏的菌系相距甚远。