Analysis of infection mechanism of muscardine disease in the silkworm, Bombyx mori, using starvation stress genes

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Synthesis of the cuticle-degrading-enzymes including protease (Pr1) by the entomopathogenic fungi occurs rapidly during carbon and nitrogen derepression in minimal media. Pr1 levels from M. anisopliae were enhanced when minimal media were supplemented wit insect cuticle. Addition of more readily utilized metabolites (e. g. glucose) repressed Pr1 production confirming that production is repressible.Nested PCR using 4 primers on DNA from strains of M. anisopliae consistently gave a strong product of about 1.2Kbp and very few non-specific product. The product is significantly larger than the predicted size of the Pr1 PCR product using cDNA as template. This is because the Pr1 gene contain at least three introns. Apart from Sau 3A1, all of the endonucleases tested cut the Pr1 product showed multiple polymorphisms. Restriction patterns generated by these endnucleases produced 5 different patterns. The results were suggested that more than 5 different Pr1s were existed in M. anisoliae. Base on differences of restriction patterns, the strains of M. anisopliae were clusterd into 4 groups at 87% similarities.To determine the relationships of the phylogeny based on Pr1 and ribosomal DNA (rDNA) from M. anisopliae, sequence data of the rDNA were aligned and analysis of the ITS and 5.8S sequences using Pasimony analysis supports Metarhizium as a monophyletic group. Ribosomal DNA has proved an extremely useful identification region for Metarhizium. It may also eventually help to construct phylogenies between species. The phylogenies based on rDNA were similar to those based on Pr1 genes from M. anisopliae.