| Metarhizium anisopliae is one of the most important entomopathogenic fungi used as biological control agent. The UV-induced mutants with increased virulence against diamondback moth were isolated, the study on the biological characteristics of mutants was performed, such as, colony growth, conidial production and spore germination rate, anti ultraviolet ability, thermal tolerances, and variation of the secondary metabolites and protein derived from mutants by HPLC-MS and SDS-PAGE. All the experiments were performed as follows: 1. Screening of UV-induced mutant of M. anisopliae strains with high virulence. Mutants from Ma01 and Ma02 strains were produced by exposing harvested conidia to UV irradiation for 20, 40 and 60 minutes, and subsequently screened for high virulence strain. The number of UV-induced colonies decreased with increasing UV-irradiation time. The colonies with stable morphology and faster growth were selected for insect bioassay. Insect bioassays using diamondback month revealed that the mortalities of the 2nd instar larvae against 5 mutant strains were higher than that of larvae treated by their parent with the values of 81.85 % and 91.48 % in 6 day. 2. Effect of UV-irradiation on the biological characteristics of the mutants.A comparison of the wild-type strains and UV-induced mutants after growth on PDA, CZA, SDA and SDAY at 26? for 11 days revealed that there were no difference on the vegetative growth among the mutants and their parent when growing on one media, however, the colony morphology of Ma01 and UV01 b on four medium were differ different. Overall conidial productions by the mutant stains were significant lower than that of the parent. 3. Effect of UV-irradiation on the secondary metabolite production of M. anisopliaeThe mean lethal concentration(LC50) values of ethyl acetate extracts from two mutants against diamondback month were calculated and were significantly lower than that from their parent. The virulence of Ma01 and UV01 b, Ma02 and UV02 b fungal strains against diamondback moth were 13.3 mg/L and 3.9 mg/L,56.5 mg/L and 7.1 mg/L,indicating the virulence of mutants higher than their parent. The ethyl acetate extractions from the secondary metabolite productions of wild-type strains and UV-induced mutants culture supernatants were assessed. As determined by HPLC analysis, significant differences were seen in the metabolite profiles between the wild-type strains and UV-induced mutants. It indicated the secondary metabolite production changed by UV-irradiation. 4. Effect of UV-irradiation on the primary metabolite productions of M. anisopliaeThe protein from the primary metabolite productions of wild-type strains and UV-induced mutants culture supernatants were assessed. Secreted protein from the wild type and UV mutant was examined over a time course of growth in PDA. Clear differences, and for the most part, a increased secreted protein, were seen for the mutant as compared to the wild type. The expression of a host of at least 2 major proteins were seen in the mutant that were absent in the wild-type strain, especially for UV02 b with molecular masses 31 k Da. It indicated secreted protein in fungal fermentation broth changed by UV-irradiation. 5. Bioassay of mutant strain against UV-irradiationGermination of mutants and their parents at 18, 21, 24 and 27 h were tested after conidia exposure to UV-irradiation. Exposure to UV-irradiation for 40 min reduced germination 40 %-60 % for the wild-type strain, but there were no difference of the spore germination between the UV01 b / UV02 b mutants and their parent.In conclusion, Mutants from Ma01 and Ma02 strains increased anti-UV and virulence by exposing harvested conidia to UV irradiation, resulting a series changes in primary and secondary metabolites, especially secreted protein and ethyl acetate extractions. |
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