Yanni Yin

Biological control of aflatoxin contamination of crops

Aflatoxins produced primarily by two closely related fungi, Aspergillus flavus and Aspergillus parasiticus, are mutagenic and carcinogenic in animals and humans. Of many approaches investigated to manage aflatoxin contamination, biological control method has shown great promise. Numerous organisms, including bacteria, yeasts and nontoxigenic fungal strains of A. flavus and A. parasiticus, have been tested for their ability in controlling aflatoxin contamination. Great successes in reducing aflatoxin contamination have been achieved by application of nontoxigenic strains of A. flavus and A. parasiticus in fields of cotton, peanut, maize and pistachio. The nontoxigenic strains applied to soil occupy the same niches as the natural occurring toxigenic strains. They, therefore, are capable of competing and displacing toxigenic strains. In this paper, we review recent development in biological control of aflatoxin contamination.

Involvement of a Velvet Protein FgVeA in the Regulation of Asexual Development, Lipid and Secondary Metabolisms and Virulence in Fusarium graminearum

The velvet protein, VeA, is involved in the regulation of diverse cellular processes. In this study, we explored functions of FgVeA in the wheat head blight pathogen, Fusarium graminearum,using a gene replacement strategy. The FgVEA deletion mutant exhibited a reduction in aerial hyphae formation, hydrophobicity, and deoxynivalenol (DON) biosynthesis. Deletion of FgVEA gene led to an increase in conidial production, but a delay in conidial germination. Pathogencity assays showed that the mutant was impaired in virulence on flowering wheat head. Sensitivity tests to various stresses exhibited that the FgVEA deletion mutant showed increased resistance to osmotic stress and cell wall-damaging agents, but increased sensitivity to iprodione and fludioxonil fungicides. Ultrastructural and histochemical analyses revealed that conidia of FgVeA deletion mutant contained an unusually high number of large lipid droplets, which is in agreement with the observation that the mutant accumulated a higher basal level of glycerol than the wild-type progenitor. Serial analysis of gene expression (SAGE) in the FgVEA mutant confirmed that FgVeA was involved in various cellular processes. Additionally, six proteins interacting with FgVeA were identified by yeast two hybrid assays in current study. These results indicate that FgVeA plays a critical role in a variety of cellular processes in F. graminearum.

Characterization of Sterol Demethylation Inhibitor-Resistant Isolates of Fusarium asiaticum and F. graminearum Collected from Wheat in China

Fusarium asiaticum and F. graminearum are the primary causal agents of Fusarium head blight (FHB) of wheat in China. In this study, sensitivities of 159 F. asiaticum and F. graminearum isolates to a benzimidazole fungicide carbendazim (MBC) and to sterol demethylation inhibitors (DMIs) tebuconazole and prochloraz were determined. Among the 159 isolates, 9 were resistant to MBC and designated as MBC-R isolates. Three showed resistance to tebuconazole and prochloraz and designated as DMI-R isolates. There was no cross-resistance between MBC and DMI. Genetic analysis by microsatellite-primed polymerase chain reaction (PCR) showed that MBC-R or DMI-R isolates had different genotypes, which indicated that they originated from different wild-type parents. Analysis of two 14alpha-demethylase (cyp51) homologous genes (cyp51A and cyp51B) showed that the F. asiaticum isolates could be distinguished from F. graminearum isolates based on the sequence of cyp51A. Analysis of deduced amino acid sequence of cyp51A and cyp51B suggested that no mutations were associated with DMI resistance. Real-time PCR analysis showed that the DMI resistance was not related to the expression of cyp51A and cyp51B in F. asiaticum and F. graminearum, but expressions of both genes were induced greatly by the tebuconazole. Results of this study indicated that cyp51A would be an informative marker for analysis of population structure of F. asiaticum and F. graminearum, and the existence of homologous cyp51 genes in F. asiaticum and F. graminearum could provide new insights into DMI resistance in phytopathogenic fungi.

The escalating threat of Rhizoctonia cerealis, the causal agent of sharp eyespot in wheat

Rhizoctonia cerealis, the causal agent of sharp eyespot on wheat, was not considered to be an important pathogen for many years. Recently, the disease has become endemic in many countries except for South America. The disease has created a new threat to world wheat production because the damage of wheat sharp eyespot has become increasingly severe. In this paper, previous studies on this pathogen, including the disease geographical distribution, pathogen identification, life cycle, symptoms, favourable environmental conditions, effects on wheat yield and control strategy, are reviewed. Such information will be helpful in management of sharp eyespot.

Gene transcription profiling of Fusarium graminearum treated with an azole fungicide tebuconazole

Using a deep serial analysis of gene expression (DeepSAGE) sequencing approach, we profiled the transcriptional response of Fusarium graminearum to tebuconazole, a most widely used azole fungicide. By comparing the expression of genes in F. graminearum treated and untreated with tebuconazole, we identified 324 and 155 genes showing more than a 5-fold increase and decrease, respectively, in expression upon tebuconazole treatment. These genes are involved in a variety of cell functions including egrosterol biosynthesis, transcription, and cellular metabolism. The validity of DeepSAGE results were confirmed by real-time PCR analysis of expression of 20 genes with different expression levels in the DeepSAGE analysis. The results from this study provide useful information in understanding the mechanisms for the responses of F. graminearum to azole fungicides.

Identification and characterization of carbendazim-resistant isolates of Gibberella zeae.

Sensitivity of Gibberella zeae to carbendazim was determined by measuring mycelial growth in fungicide-amended media. Among 1,529 isolates tested, 31 isolates showed a high level of resistance (HR) to carbendazim (fungicide concentration that results in 50% inhibition of mycelial growth [EC50] of 10.35 to 30.26 mg a.i. liter-1) and 10 isolates were moderately resistant (MR) (EC50 of 4.50 to 7.28 mg a.i. liter-1). The remaining 1,488 isolates were sensitive to carbendazim and were unable to grow on potato dextrose agar amended with carbendazim at 2 mg a.i. liter-1. Analysis of DNA sequences of the β2-tubulin (Tub2) gene showed that all 10 MR isolates had a point mutation at codon 198 causing a replacement of glutamic acid by glutamine. At the codon position 167, the amino acid phenylalanine was replaced by tyrosine in 28 of 31 HR isolates. The remaining three HR isolates had a point mutation at codon 200 which converted phenylalanine to tyrosine. Based on these point mutations in the Tub2 gene, allele-specific polymerase chain reaction primers were developed for rapid detection of the point mutations. The rapid molecular method will be a valuable tool for the monitoring of carbendazim resistance in G. zeae. Additionally, deletion of the β1-tubulin gene (Tub1) in the HR isolate GJ33 resulted in increased resistance to carbendazim. These results indicate that Tub1 plays a role in the sensitivity of G. zeae to carbendazim.

Histone H3K4 methylation regulates hyphal growth, secondary metabolism and multiple stress responses in Fusarium graminearum

Histone H3 lysine 4 methylation (H3K4me) is generally associated with actively transcribed genes in a variety of eukaryotes. The function of H3K4me in phytopathogenic fungi remains unclear. Here, we report that FgSet1 is predominantly responsible for mono-, di- and trimethylation of H3K4 in Fusarium graminearum. The FgSET1 deletion mutant (ΔFgSet1) was crippled in hyphal growth and virulence. H3K4me is required for the active transcription of genes involved in deoxynivalenol and aurofusarin biosyntheses. Unexpectedly, FgSet1 plays an important role in the response of F. graminearum to cell wall-damaging agents via negatively regulating phosphorylation of FgMgv1, a core kinase in the cell wall integrity pathway. In addition, ΔFgSet1 exhibited increased resistance to the transcription elongation inhibitor mycophenolic acid. Yeast two-hybrid assays showed that FgSet1 physically interacts with multiple proteins including FgBre2, FgSpp1 and FgSwd2. FgBre2 further interacts with FgSdc1. Western blotting analyses showed that FgBre2 and FgSdc1 are associated with H3K4me. Both proteins are also involved in regulating deoxynivalenol biosynthesis and in responses to mycophenolic acid and cell wall-damaging agents. Taken together, these data indicate that H3K4me plays critical roles not only in regulation of fungal growth and secondary metabolism but also in multiple stress responses in F. graminearum.

Involvement of FgERG4 in ergosterol biosynthesis, vegetative differentiation and virulence in Fusarium graminearum.

The ergosterol biosynthesis pathway is well understood in Saccharomyces cerevisiae, but currently little is known about the pathway in plant-pathogenic fungi. In this study, we characterized the Fusarium graminearum FgERG4 gene encoding sterol C-24 reductase, which catalyses the conversion of ergosta-5,7,22,24-tetraenol to ergosterol in the final step of ergosterol biosynthesis. The FgERG4 deletion mutant ΔFgErg4-2 failed to synthesize ergosterol. The mutant exhibited a significant decrease in mycelial growth and conidiation, and produced abnormal conidia. In addition, the mutant showed increased sensitivity to metal cations and to various cell stresses. Surprisingly, mycelia of ΔFgErg4-2 revealed increased resistance to cell wall-degrading enzymes. Fungicide sensitivity tests revealed that ΔFgErg4-2 showed increased resistance to various sterol biosynthesis inhibitors (SBIs), which is consistent with the over-expression of SBI target genes in the mutant. ΔFgErg4-2 was impaired dramatically in virulence, although it was able to successfully colonize flowering wheat head and tomato, which is in agreement with the observation that the mutant produces a significantly lower level of trichothecene mycotoxins than does the wild-type progenitor. All of these phenotypic defects of ΔFgErg4-2 were complemented by the reintroduction of a full-length FgERG4 gene. In addition, FgERG4 partially rescued the defect of ergosterol biosynthesis in the Saccharomyces cerevisiae ERG4 deletion mutant. Taken together, the results of this study indicate that FgERG4 plays a crucial role in ergosterol biosynthesis, vegetative differentiation and virulence in the filamentous fungus F. graminearum.

Simultaneous detection of Fusarium asiaticum and Fusarium graminearum in wheat seeds using a real-time PCR method

Aims:  To develop a PCR‐based method for quantitative detection of Fusarium asiaticum (Fa) and Fusarium graminearum (Fg) in wheat seeds.

A small molecule species specifically inhibits Fusarium myosin I

Fusarium head blight (FHB) caused by Fusarium graminearum is a devastating disease of cereal crops worldwide. Recently, a novel fungicide JS399-19 has been launched into the marketplace to manage FHB. It is compelling that JS399-19 shows highly inhibitory activity towards some Fusarium species, but not to other fungi, indicating that it is an environmentally compatible fungicide. To explore the mode of action of this species-specific compound, we conducted a whole-genome transcript profiling together with genetic and biochemical assays, and discovered that JS399-19 targets the myosin I of F. graminearum (FgMyo1). FgMyo1 is essential for F. graminearum growth. A point mutation S217L or E420K in FgMyo1 is responsible for F. graminearum resistance to JS399-19. In addition, transformation of F. graminearum with the myosin I gene of Magnaporthe grisea, the causal agent of rice blast, also led to JS399-19 resistance. JS399-19 strongly inhibits the ATPase activity of the wild-type FgMyo1, but not the mutated FgMyo1(S217L/E420K) . These results provide us a new insight into the design of species-specific antifungal compounds. Furthermore, our strategy can be applied to identify novel drug targets in various pathogenic organisms.