Pathogenecity Analysis On One T-DNA Insertion Mutant Isolate Of Botrytis Cinerea

The plant pathogenic fungus Botrytis cinerea causes grey mold of various crops and ornamental plants, which can cause serious economic lossing. B. cinerea belongs to Deuteromycotina and can live through the winter in sick deformed or soil by the mycelium, sclerotia or spores form. Because of lacking resistant germplasm resources caused by fast reproduction, genetic variation and high parasitic fitness of B. cinerea, gray mold is difficult to be prevented. Chemical method which is the main method combines with biological and ecological measures, agricultural measures and other measures to prevent gray mold.In this study, a pathogenic mutant strain of BCG-183 was screened from more than 400 ATMT transformants of B. cinerea from the previous work and was analyzed from the morphological phenotype: the hypha of mutant was more than that of the wild type. After culturing in PDA plates 7 days, the colony of mutant was white and didn't produce spores, while the wild-type colony was gray and produced a large amount of spores; the mutant and wild-type strains were respectively inoculated onto sterile tomato leaves and tomato fruits, the mutant formed a larger lesion area than the wild-type after cultured 3d in the moisture environment, which indicated that the virulence of the mutant was enhanced compared with that wild-type; the mutant and wild-type colony respectively cultured in producing cell wall degrading enzyme medium 3 days by shaking, then the activity of the cell wall degrading enzyme was detected by spectrophotometer, and the result was that cell wall-degrading enzyme activity of the mutant was better than that of the wild-type. Cucumber seeds and tomato seeds, which were soaked 1 day in the toxigenic filtrate of the mutant and wild-type strain respectively, were fostered on the wet filter paper 5 days. The result was that the germination ratio of the cucumber seeds and tomato seeds were both inhibited by the toxigenic filtrate of the mutant. After the toxin extracted from the mutant and wild-type strains was dripped onto sterile tomato leaves, the lesion area formed by the mutant was larger than that of the wild-type; Mutant's acid production was significantly enhanced compared with wild-type.The genomic DNA of wild type and BCG-183 were extracted by CTAB method. Subsequently, TAIL-PCR was used to amplify the flanking sequence of T-DNA insertion site from its genome to determine the disrupted gene. We confirm that T-DNA had inserted into the 3'-end of the hypothetical protein gene (BC1G₀7455) locating in the genome Super contig 42 (Supercontig 42) of B. cinerea by bioinformatics analysis. The gene BC1G₀7455 is 1 221 bp, encoding 406 amino acids, and the putative protein showed more than 97% homology with the Penicillium salicylate hydroxylase [GenBank accession No. 7023390],the FAD dependent oxidation reductase of the Aspergillus [GenBank accession No.4983596] and kynurenine 3-monooxygenase of Verticillium wilt fungus [GenBank accession No. 9532153].The transcription level of endopolygalacturonase encoding gene Bcpg1 and BcBOT2 of the mutant was higher than that of the wild-type strains. The expression of Bcpg1 and BcBOT2 was upregulated by the disruption of BC1G₀7455. It was indicated that BC1G₀7455 might play a negative regulatory role in the endopolygalacturonase and toxin production.