Screening Of Metarhizium Anisopliae Uv-induced Mutants For Increased Virulence

Metarhizium anisopliae is one of the most important entomopathogenic fungi used as biological control agent. Here, the Uv-induced mutants with increased virulence against diamondback moth were isolated, The study on the biological characteristics of mutants were performed, such as, study on the 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 M. anisopliae strains for Uv-radiation treatment. The conidial suspension of 8 fungi strains at the concentration of 1.0 × 107 conidia /ml was prepared respecitively. Insect bioassays using the diamondback month, Pultella xylostella, were performed by dipping 2ndinstar larvae into fungal suspension for 10 seconds. The SM03 and SM04 strains were selected for UV-irradiation treatment on baise of the mortalities of insect larvae against fungi.2. Molecular identification of M. anisopliae. The genomic DNA of SM03 and SM04 strains were extracted using CTAB method. About 600 bp ITS fragment sequence was amplified by PCR with primers ITS4 / ITS5 using the genomic DNA as the template.Homology searching was performed using the BLAST algorithms against various databases of the Gen Bank, resulting high identiy(99%) to M. anisopliae. On base of fungal colony morphology characteristics combination with the molecular identification, SM03 and SM04 strains were identified as M. anisopliae.3. Screening of UV-induced mutants with high virulence. Mutants from SM03 and SM04 strains were produced by exposing harvested conidia to UV irradiation for 20, 40 and60 mintutes, and subsequently screened for high virulence strain. Insect bioassays using diamondback month revealed that the adjusted mortalities of the 2ndinstar larvae against SM04-UV40 mutants increased 51% / 37% at 3, 6d post treatment as compared to theparent. Similar to SM03-UV60 mutant, the adjusted mortalities increased 53% / 49% at 3,6d post treatment as compared to the parent. The mean lethal concentration(LC50) values of ethylacetate extracts from two mutants against diamondback month were calculated and were significantly lower than that from their parents. The virlence of SM04-UV40 and SM03-UV60 mutants against diamondback moth were increased significantly compared to their parent.4. Evaluation of the biological characteristics of the mutants. A comparison of the wile-type and UV-induced mutant(SM04-UV40) after growth on PDA at 26? for 11 days revealed faster vegetative growth of mutant than the wild type. Overall conidial production by the SM04-UV40 mutant(4.0 × 107conidia/ml) decreased 46% than that of the parent(7.4× 107conidia/ml). However, exposure to UV-irradiation for 40 min reduced germination to52% for the wild-type strain, but had no significant effect on the mutant which remaind at83.07%(84.60%).A comparison of the wile type and UV-induced mutant(SM03-UV60) after growth on PDA at 26? for 11 days showed no difference on vegetative growth between mutant and the parent. Overall conidial production by the SM03-UV60 mutant(5.67 × 107conidia/ml)decreased 13% than that of the parent(6.53 × 107conidia/ml). However, exposure to UV irradiation for 60 min reduced germination to 53% for the wild-type strain, but had no significant effect on the mutant which remaind at 68.15%(70.67%).5. Study on the metabolite productions of UV-irradiation on M. anisopliae. Protein and ethyl acetate extractions from metabolite productions of the wild type and UV-induced mutant culture supernatants were assessed. As determined by HPLC analysis, significant differences were seen in the metabolite profiles between the wild-type strain and UV-induced mutants. A wide peak(retention time 35-50 min), clearly containing numerous components, was evident in the wild type(SM04 and SM03) that was essentially absent in the mutant strain(SM04-UV40 and SM03-UV60). Conversely, a series of sharply defined peaks(retention time between 10-30 min) were seen in the SM04-UV40 mutant that was largely absent in the wild type, as for SM03 and SM03-UV60, resulting some few difference on peak numbers(retention time 10-30 min). Secreted protein from the wild type and mutant was examined over a time course of growth in PDA. Clear differences, and forthe most part, a reduced secreted protein, were seen for the mutant as compared to the wild type. At the early time points(3 and 4d) a few differences were noted, however, by 5d, the expression of a host of at least 5 major proteins with molecular masses ranging from 25 k Da to 20-35 k Da were seen in the wild type that were absent in the mutant.In conclusion, Mutants from SM03 and SM04 strains increased virulence by exposing harvested conidia to UV irradiation, resulting a series changes in primary and secondary metabolites, especially secreted protein and ethyl acetate extractions.