Studies On Regulation And The Molecular Mechanism Of Production Of Anti-Fungal Substances By The Mycoparasite Coniothyrium Minitans

Coniothyrium minitans (CM) is a promising biocontrol agent of Sclerotinia spp. including Sclerotinia sclerotiorum (SS). My research work mainly focused on carbon, nitrogen and ambient pH regulate the production of antifungal substances (AFS) by C. minitans and the molecular mechanism.We found that the strain Chy-1 of C. minitans used in our lab could consistently produce AFS in modified Czapek-Dox (MCD) broth; however, production of AFS by this strain of C. minitans was hardly detected in potato dextrose broth (PDB). In order to elucidate the difference of antifungal activity (AA) of the mycoparasite C. minitans against S. sclerotiorum (AAcm-ss) of MCDcm and PDBcm, we studied the effects of ambient pH and nutritional factors on antifungal activity of C. minitans. The results showed that production of AACM-ss by C. minitans was closely related to ambient pH. The carbon or nitrogen suit for AFS production was closely related to low ambient pH of the medium. AACM-ss was much higher in C. minitans cultures buffered at pH 3 than that buffered at pH 6.For the molecular mechanism of ambient pH regulate AFS production in C. minitans. We cloned a putative pH-responsive transcription factor gene (cmpacC) and a putative carbon repression gene (cmcreA) from strain Chy-1 of C. minitans by degenerate PCR and Rapid amplification of cDNA ends. The full-length DNA sequence of cmpacC (GenBank Ace. No. FJ176399) has one open reading frame (ORF) and two introns. cmpacC putatively encodes a polypeptide with 596 amino acid residues (GenBank Ace. No. ACN43303). The sequence of this polypeptide has three conserved zinc finger domains. Meanwhile, a 2,740 bp-long 5’ flanking sequence of cmpacC and a 2,730bp-long 3’flanking sequence of cmpacC was obtained by genome walking. It contains five copies of PacC binding sites (5’-GCCAAG-3’). Real-time RT-PCR analysis indicated that with the increase of ambient pH from 3 to 8, expression of cmpacC, as well as the chitinase gene (cmchl) and theβ-1,3-glucanase gene (cmgl), increased consistently in C. minitans. When C.minitans grows in the media with the carbon source of Monosaccharide, the expression of cmcreA is relative lower than grow in the media with the carbon source of polysaccharide. We knocked out the cmpalF gene in C. minitans by Agrobacterium tumefacies-mediated homologous recombination. We found that when ambient pH of the media was higher than 6, cmpalF grew more slowly than the wild type strain Chy-1. AACM-ss of AcmpalF mutant was higher than Chy-1. The expression of cmpacC in cmpalF was significantly lower than that in strain Chy-1. We analysed the differentially expressed genes of C. minitans grew in the media of different ambient pH (pH 3 and pH 6). There were a total of 1286 genes of C. minitans differentially expressed,596 genes up-regulated and 690 genes down-regulated.These results broadened our understanding of the interaction between C. minitans and S. sclerotiorum and laid a solid foundation for future studies to elucidate molecular mechanisms involved in AFS biosynthesis in C. minitans.