| Entomogenous fungi are widely used in biological control of pests, since they have better safety, environment friendliness and non resistance. Myconsecticide is a potential alternative of chemical insecticides. Soil with the huge fungal library, is very important for research and development of mycopesticide to isolate and screen entomogenous fungal strains. Genetic modification is an effectively technology to improve the insecticidal effect of entomogenous fungi. Constructing combinant to express the ds RNA fragments targeting at host insect's RNAi sensitive gene is becoming a new way of genetic modification of entomogenous fungal species.In current research, 26 soil samples were collected from Inner Mongolia district, and26 fungal strains were isolated and identificated from the soil samples. Among them, five strains were selected for bioassey against B-biotype of Bemisia tabaci(Gennadius). A combinant strain of Beauveria bassiana was constructed for express ds RNA targeting to the Sl Dorsal gene of Spodoptera litura. The main research results are listed as follows:There were 26 entomogenous fungal strains isolated from 26 soil samples collected from different areas of the Inner Mongolia with the 100% isolation rate(i.e., one strain was obtained from each soil sample). The all strains were respectively preserved in the PDA medium slants and sand-tubes.The all strains were identified mainly on the basis of morphology and molecular biology including the characteristics of colony, conidia and sporulation structure, and the sequence homology of ITS1-5.8S-ITS4 r DNA gene and ?-tubulin gene. Among them,MS-10, MS-12 and MS-15 strains were identified as Paecilomyces lilacinus; MS-25 strain was B. bassiana; MS-01, MS-06 and MS-19 strains were Lecanicillium lecanicillium;MS-02, MS-03, MS-04, MS-08, MS-13, MS-16, MS-18, MS-22, MS-23 and MS-24 strains were Lecanicillium muscarium; MS-14 strain was Peniophora incarnata; MS-20 strain was Schizophyllum commune; MS-04, MS-07, MS-21 strains were Cladosporium and MS-26 strains was Acremonium sp; MS-09 and MS-17 strains were morphologically like Paecilomyces, but not obtain the corresponding gene sequence.The five strains, MS-25, MS-12, MS-15, MS-18, MS-10, were subjected to a bioassay against the second-instar nymph of B-biotype whitefly(Bemisia tabaci). At a dosage of108 spores/m L, the strains MS-12, MS-15, MS-18, MS-25, MS-10 killed respectively the pests of 92.4%, 91.6%, 89.9%, and the 86.7%, after treatment 10 d. Meanwhile, the LT50 s were recorded 4.5 d for MS-18 and MS-15, 5.5 d for MS-10, and 6 d for MS-10 and MS-12 strains.The protoplast transformation technology was selected to construct a recombinant strain based on the wild Beauveria bassiana strain Bb01. Under the optimal conditions of 1%snail enzyme+2% cellulase enzyme, at a temperature of 30 ?, p H7.0 and 0.7 mol/L Na Cl final infiltration liquid of enzyme for 5 hours, the most protoplasts were obtained. Then, the protoplasts were transformed by recombinant vector Sl Dorsal mediated by 30% PEG4000.Subsequently, a genetic stable recombinant strain Bb01-Sl Dorsalwas selected. It was preserved in China Center for Type Culture Collection with the accession no. CCTCC M2015721.By means of dipping method, recombinant strain Bb01-Sl Dorsal was subjected to a bioassay against to the second instar larvae of Spodoptera litura. The results indicated that after treatment 10 d, the Bb01-Sl Dorsal had a LC50 of 0.34×106 spores/m L and a LT50 of 5d at a concentration of 5×107 spores/m L, Bb01-Sl Dorsal, while the original strain Bb01 had a LC50 of 0.72×106 spores/m L and a LT50 of 6 d at a concentration of 5×107 spores/m L. It is suggested that the recombinant strain Bb01-Sl Dorsal than the original strain Bb01 has higher virulence to Spodoptera litura. |
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