Leiyan Yan

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.

The mitogen-activated protein kinase kinase kinase BcOs4 is required for vegetative differentiation and pathogenicity in Botrytis cinerea.

The high-osmolarity glycerol signal pathway plays an important role in the response of fungi to various environmental stresses. In this study, we characterized a mitogen-activated protein kinase kinase kinase gene BcOS4 in Botrytis cinerea, which is homologous to Saccharomyces cerevisiae SSK2/SSK22. The BcOS4 deletion mutant was significantly impaired in vegetative growth and conidial formation. The mutant exhibited increased sensitivity to the osmotic, oxidative stresses and to the fungicides iprodione and fludioxonil. Western blot analysis showed that BcSak1, a putative downstream component of BcOs4, was not phosphorylated in the mutant. In addition, the BcOS4 mutant was unable to infect leaves of rapeseed and cucumber, and grape fruits, although it can cause disease on apple fruits. All the defects were restored by genetic complementation of the BcOS4 deletion mutant with the wild-type BcOS4 gene. The data of this study indicate that BcOS4 is involved in vegetative differentiation, virulence, adaption to hyperosmotic and oxidative stresses, and to fungicides in B. cinerea.

Molecular characterization of toxigenic and atoxigenic Aspergillus flavus isolates, collected from peanut fields in China

Aims:  The objectives of this study were to assess the genetic relationships between toxigenic and atoxigenic isolates of Aspergillus flavus collected from peanut fields in China, and to analyse deletions within the aflatoxin biosynthetic gene cluster for the atoxigenic isolates.

The mitogen-activated protein kinase kinase BOS5 is involved in regulating vegetative differentiation and virulence in Botrytis cinerea

We present a characterization of bos5 from Botrytis cinerea, a gene that encodes a mitogen-activated protein kinase kinase (MAPKK), which is homologous to OS-5 of Neurospora crassa. The bos5 gene deletion mutant exhibited reduced vegetative growth and strongly impaired conidiation. The mutant also exhibited increased sensitivity to the dicarboximide fungicide iprodione and to osmotic stress mediated by NaCl or KCl. Western-blot analysis showed that the BcSAK1 protein, the putative downstream component of BOS5, was not phosphorylated in the mutant. Plant inoculation tests showed that the mutants were unable to infect cucumber leaves. All of these defects were restored by genetic complementation of the Deltabcos5-21 mutant with the wild-type bos5 gene. These results indicated that BOS5 is involved in the regulation of vegetative differentiation, virulence, adaptation to iprodione and ionic stress in B. cinerea.

Development of a real‐time PCR assay for the detection of Cladosporium fulvum in tomato leaves

Aims:  The aim of this study was to develop a sensitive real‐time polymerase chain reaction (PCR) assay for the rapid detection of Cladosporium fulvum in tomato leaves.

Molecular characterization of an atoxigenic Aspergillus flavus strain AF051

An atoxigenic Aspergillus flavus strain AF051 collected from a peanut field in Jiangsu province, P. R. China was characterized by analysis of aflatoxin gene cluster in this study. By using a thermal asymmetric interlaced PCR (TAIL-PCR) and conventional PCR techniques, an 89.59-kb deletion was found in the cluster, and this deletion was replaced by a 3.83-kb insert, which was located at 300-bp upstream ver1 gene and 2594-bp downstream a putative gluconolactone oxidase gene. Based on the DNA sequence at the breakpoint, a nested-PCR method was developed for the rapid and sensitive detection of AF051 strain in soil and peanut samples once the strain is used as a biological agent.

Involvement of a putative response regulator Brrg-1 in the regulation of sporulation, sensitivity to fungicides, and osmotic stress in Botrytis cinerea.

The response regulator protein is a core element of two-component signaling pathway. In this study, we investigated functions of BRRG-1 of Botrytis cinerea, a gene that encodes a putative response regulator protein, which is homologous to Rrg-1 in Neurospora crassa. The BRRG-1 gene deletion mutant ΔBrrg1-62 was unable to produce conidia. The mutant showed increased sensitivity to osmotic stress mediated by NaCl and KCl, and to oxidative stress generated by H2O2. Additionally, the mutant was more sensitive to the fungicides iprodione, fludioxonil, and triadimefon than the parental strain. Western-blot analysis showed that the Bos-2 protein, the putative downstream component of Brrg-1, was not phosphorylated in the ΔBrrg1-62. Real-time polymerase chain reaction assays showed that expression of BOS-2 also decreased significantly in the mutant. All of the defects were restored by genetic complementation of the ΔBrrg1-62 with the wild-type BRRG-1 gene. Plant inoculation tests showed that the mutant did not show changes in pathogenicity on rapeseed leaves. These results indicated that Brrg-1 is involved in the regulation of asexual development, sensitivity to iprodione, fludioxonil, and triadimefon fungicides, and adaptation to osmotic and oxidative stresses in B. cinerea.

Heterologous Expression of the CYP51 Gene of the Obligate Fungus Blumeria graminis in the Necrotrophic Fungus Botrytis cinerea

ABSTRACT. As it is extremely difficult to make DNA transformation for the obligate fungus, Blumeria graminis f. sp. tritici (Bgt), we developed a heterologous expression system for characterization of a Bgt gene, CYP51, which encodes 14α‐demethylase. The CYP51 gene from Bgt was transformed into the necrotrophic fungus, Botrytis cinerea. Reverse transcription polymerase chain reaction showed that the Bgt CYP51 was transcribed in B. cinerea. Green fluorescence was observed in the transformants of B. cinerea carrying the Bgt CYP51‐GFP fusion cassette, suggesting that its translation was successful. Fungicide sensitivity tests revealed that B. cinerea transformed with Bgt CYP51 showed reduced sensitivity to a sterol demethylation inhibitor triadimefon, but not to a benzimidazole fungicide carbendazim. These results indicated that this heterologous expression system can be used for functional analysis of other Bgt genes.

The FgSsb-FgZuo-FgSsz complex regulates multiple stress responses and mycotoxin production via folding the soluble SNARE Vam7 and β2-tubulin in Fusarium graminearum

Hsp70 proteins play important roles in protein folding in the budding yeast, but their functions in pathogenic fungi are largely unknown. Here, we found that Fusarium graminearum Hsp70 proteins FgSsb, FgSsz and their cochaperone FgZuo formed a complex. This complex was required for microtubule morphology, vacuole fusion and endocytosis. More importantly, the β2-tubulin FgTub2 and SNARE protein FgVam7 were identified as targeting proteins of this complex. We further found that the complex FgSsb-FgZuo-FgSsz controlled sensitivity of F. graminearum to the antimicrotubule drug carbendazim and cold stress via regulating the folding of FgTub2. Moreover, this complex assisted the folding of FgVam7, subsequently modulated vacuole fusion and responses to heavy metal, osmotic and oxidative stresses. In addition, the deletion of this complex led to dramatically decreased deoxynivalenol biosynthesis. This study uncovers a novel regulating mechanism of Hsp70 in multiple stress responses in a filamentous fungus.