Selecting Of Beauveria Bassiana From Holcocerus Hippophaecolus And Chitinase Gene Cloning And Conidia Microencapsulating

Hippophae rhamnoides is one of the ecological and economic species in loess hilly area in China, which has a wide distribution area in Inner Mongolia and Liaoning. Holcocerus hippophaecolus is one of the borers attacking H. rhamnoides, which mainly harmed the roots and stems, causing death of H. rhamnoides in large areas. B. bassiana infected H. hippophaecolus larva and pupae and caused them dead. In order to use B. bassiana against H. hippophaecolus better, isolating, enzyme activity, chitinase gene clone, conidia germination accelerants, conodia microencapsulation and bioassay were studied as following:1. Selecting: Five isolates were obtained from H. hippophaecolus larva and pupae infected by B. bassiana. Superior isolate was selected by comparing growth rate, conidia production, conidia germination speed, conidia germination rate at different temperature and relative humidity conditions, high temperature treating and UV irradiation. The conidia germination rate of SJ-05 could reach to 60% at 76% humidity, which was 3 times higher than that of SJ-03. The germination rate of SJ-05 could reach to 48.77% after 18 mins heating at 50?water, which was 2.5 times higher than that of SJ-03. The rate was 33.92% after exposed to ultraviolet for 8 hours, which was twice higher than that of SJ-02. SJ-05 was the optimal isolate with certain drought resistance and thermotolerance and UV irradiation resistance.2. Enzyme activity and chitinase gene cloning: Extracellular protease and chitinase activity of five isolates were tested by Flat Determination method, Lowry and DNS determination method. Extracellular protease production of optimal isolate SJ-05 reached to 55.3 U/mL, and chitinase reached to 137.9 U/mL, which were 3.5 times and 4.3 times higher than those of SJ-03, respectively. Chitinase gene of B. bassiana was 1 047 bp and encoded 451 amino acids. The molecular weight and the isoelectric point were 36.9 kD and 5.94, respectively. It could be a new chitinase gene of B. bassiana.3. The influences of nutrient and pH and germinating accelerant and subculture on SJ-05: 1% peptone, 1%sucrose and 1% silkworm pupae could promote conidia germination. The optimum pH for conidia germination was 6.0?7.0. After the 5th subculture, the rate and the speed of conidia germination declined obviously. After cultured for 16h, the germination rates of the 3^rd, the 4^th and the 5^th culture were 97%, 87% and 40%, respectively. Germinating accelerant AA and BB had different effects on conidia germination rate. At the time of 14 h and 18 h, the germination rates in both 1.5ppm accelerant AA and 1.5ppm accelerant BB were 46% and 96%,respectively, which was 2.6 times and 2.5 times higher than that of control(without accelerant). Germinating accelerant CC had no positive effect on conidia germination rate, and with the increase of concentration accelerant CC had inhibitory action on conidia germination rate.4. Microencapsulation: Microencapsulation of SJ-05 conidia was obtained by interfacial polymerization. The optimal conditions were that C was 100%, E was 0.5%, F was 0.5M and 500 r/min selected through 4 factors 4 levels orthogonal experiment. The microencapsulation of SJ-05 could germinate at 61% and the germination rate was 33.85%, but the conidia could not germinate at the same humidity, the difference was significant. Both the conidia and microencapsulation germination rate were declined by ultraviolet (30W, 40 cm), but the microencapsulation had certain ultraviolet resistance. 26.91% capsule could germinate after exposed to ultraviolet for 10 h, conidia germination rate was only 3.92%.5. Bioassay: Field conditions was mimiced at laboratory, infections of both formulations to the larvae of H. hippophaecolus were studied. The larval mortality caused by the capsule and the conidia were 84.54% and 45.54%, respectively. The larval mortality of H. hippophaecolus caused by capsule was obviously higher than that of conidia. The virulence of SJ-05 and its microencapsulation against larvae of L. decempunctata Gebler, D. superans and H. hippophaecolus were tested at the laboratory. The LC₅₀ of SJ-05 conidia to three kinds of insect larvae were 3.45×10⁷ conidium/mL. 2.92×10⁷ conidium/mL and 9.79×10⁶ conidium/mL, respectively. The LC50 of SJ-05 capsule to D. superans and H. hippophaecolus were 1.03×10⁷ conidium/mL and 2.95×10⁶ conidium/mL, respectively. The results implied that the virulence of a entomopathogenic beauveria was host depending. Isolate from dead H. hippophaecolus was more virulent to H. hippophaecolus comparing to the other two insects. In addition, at the same concentration, the microencapsulation was more virulent than conidia solution.