| Metarhizium anisopliae, a kind of entomopathogenic fungus widely applied in domestic and abroad, has an important role in biological control of insects. Compared with chemical insecticides,mycoinsecticides are not infective or toxic to human and do not pollute enviroment, but slow speed of kill is perceived as a potential drawback to the use of fungi against locusts. Studies on mechanisms of fungal pathogenesis and host defense may suggest strategies for the development of more efficient antilocust mycoinsecticides. Fungi can produce several extracellular disaccharide-hydrolysing enzymes including trehalose-hydrolysing enzymes once they get into the haemolymph of insects. Trehalose-hydrolysing enzymes can effectively hydrolyse trehalose in the haemolymph of insects, which play an important role in the pathogenesis of insects.In this paper, the changes in haemolymph disaccharides of Locusta migrateria manilensis infected with M. anisopliae var acridum CQMa102, culture conditions for the production of disaccharide-hydrolysing enzymes from CQMa102 and analysis of disaccharide-hydrolysing enzyme isozymes were studied systematically. These results provided detail knowledge for the purification of main disaccharide-hydrolysing enzyme isozymes and the selection of virulent-related gene, and was helpful for reconstructing strains to control pests by developing genetically enhanced microbial pesticides.The main results were as follows:1. Progression of infection of CQMa102 in L. migrateria manilensis had been observed by digital-optical microscope. No hyphal bodies were found in the haemolymph of 1 or 2d inoculated locusts. Fungal hyphal bodies were observed in the haemolymph of locusts three days after inoculation with CQMa102. A marked increase in the level of fungus occurred over days 4. 2. The levels of disacchrides and disaccharide-hydrolysing enzymes of L. migrateria manilensis infected with CQMa102 had been determined. The results indicated that the time in decrease of trehalose level coincides with that in increase of trehalose-hydrolysing enzyme activity in the haemolymph of the fungus-infected insects. However, haemolymph glucose decreases significantly during mycosis of locusts by M. anisopliae. Maltose level in the haemolymph of fungus-infected locusts decreased in comparison with controls, which was consistent with an increase in maltose-hydrolysing enzyme activity. 3. Disaccharide-hydrolysing enzyme activity of CQMa102 was determined by the amount of product (glucose released from the hydrolysis of trehalose or maltose) and optimum culture medium and culture conditions had been screened. The optimum medium for trehalose-hydrolysing enzyme production of CQMa102 was: 1.0g/L KH2PO4, 0.5g/L MgSO4, 1.0g/L KCl, 10g/L soluble starch, 5g/L mycological peptone, 5g/L yeast extract and 10ml/L trace elements (pH was adjusted to 6.0). When 0.5 ml conidial suspension (1.0×107spores/ml) was directly added to 50 ml optimum culture medium and shaken at 150rpm at 26℃ for three days,the total activity and specific activity of trehalose-hydrolysing enzymes were 14.125U and 9.588U/mg, respectively.The optimum medium for maltose-hydrolysing enzyme production of CQMa102 was: 1.0g/L KH2PO4, 0.5g/L MgSO4, 1.0g/L KCl, 10g/L maltose, 5g/L mycological peptone, 5g/L yeast extract and 10ml/L trace elements (pH was adjusted to 6.0). When 0.5 ml conidial suspension (1.0×107spores/ml) was directly added to 50 ml optimum culture medium and shaken at 150rpm at 26℃ for three days,the total activity and specific activity of maltose-hydrolysing enzymes were 12.051U and 7.749U/mg, respectively. 4. The localization of disaccharide-hydrolysing enzymes had been conducted successfully by comparison of enzyme activity between the mycelial extracts and culture filtrate. The result showed that trehalose-hydrolysing enzymes and maltose-hydrolysing enzymes are secretory proteins, and the majority of enzymes were secreted into liquid culture, only smaller amount of them were bound on cell wall.5. Disaccharide-hydrolysing enzyme isozym...
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