| East Asian locust, Locusta migratoria manilensis (Meyen), is listed as major pest of crops and pastures in China. Locust has been mainly controlled with large amount of broad-spectrum chemical pesticides that usually cause pollution of environment and damage to human beings, as well as result in an exacerbation of locust problems due to the loss of natural enemies. In order to reduce both locust damage and insecticide application, it is important to develop biological alternative agents as part of a program of integrated pest management (IPM) of locusts and grasshopper.Metarhizium anisopliae, an entomopathogenic fungus, play an important role in biological control of pests for its target-specific and pest resistance difficultly produced. Metarhizium anisopliae became one of the most promising biological agents in controlling locusts and grasshoppers and widely applied as safety commodity commended by Food and Agriculture Organization of the United Nations (FAO).However, Metarhizium anisopliae did not apply to control Locusts in large scale in China for lack of registered Metarhizium anisopliae agents and commercially marketed. There are some obstacles inhibited the application in large scale and development of the mycoinsecticide in mycoinsecticide, such as lag in research of process and lack of quality control system. One of the main reasons was the lack of methods to quick and accurate assessment of virulence. The gradually proliferation of entomopathogenic fungus in host is preliminary of pathopoiesis and the proliferation quantity and velocity affect directly to the pathogenic process and the pesticide efficiency. It will meet the needs for research and quality assessment of agents and virulence assessment during supervising to set up the methods of quantified determination to the early infecting stage of entomopathogenic fungi in insect. The methods will supply technologic support to the production, research and determination of mycoinsecticide.In this research, the entomopathogenic fungus strain CQMa102 distinguished as M. anisopliae var. acridum was isolated from cadivour of Ceracris Kiangsu (Orthoptera: Acrididae) in China. The research items were included a) Study on biological characters of strain CQMa102, b) Optimization the fermentation of strain CQMa102 industrialization on liquid-Solid phase, c) Establishment of formulation and assessment safety on environment, d) Application technology of strain CQMa102 in field, and d) Setup real-time fluorescent PCR system which detect strain CQMa102 in locusts. The main results achieved in the following aspects. 1. In the biological characteristic reseach of CQMa102,the insecticidal activity controlling L. migratoria manilensis (Meyen), heat-resistance of conidia and ultraviolet (UV)-resistance were compared with that of strain IMI330189 in laboratory. The results show there are no significant difference in heat-resistance and ultraviolet (UV)- resistance of conidia between two strains, but insecticidal activity controlling L. migratoria manilensis (Meyen) using strain CQMa102 is higher than that of strain IMI330189. Time of efficacy over 90% in the field cages test shortened 1-2 days. Otherwise, strain CQMa102 can infect several kinds of locusts and grasshoppers.2. The optimum culture medium, which was used in solid-state fermentation of strain CQMa102, was based on rice supplemented with 0.01-0.02% sporlulation agent CuSO4 1%NaNO3 and 1% liquid dissolving various trace elements. The optimum culture conditions of strain CQMa102 were among 25-28℃, initial pH5.5-6.5 of medium, higher ambient humidity in fermentation prophase and low humidity in culture period of 12-15 days. In optimum culture medium and condition, the yields reach 1.9×109 conidia/g of conidia of CQMa102 in ventilated ferment bag and increased about 20% compared with that in plastic bag. In addition, the illumination had no effect on sporulation of M. anisopliae strain CQMa102.3. The fine suspension plant oil-based formulation of Metarhizium anisopliae were formulated 1×1010 conidia/ml using soybean oil containing 3 % vaseline. The germination rate of conidia in formulation was 88% after 12 months storage at 26℃. The percentage of formulation suspending was 92.3% after 3 months storage at 26℃. The percent of mortality of locust reached 80% after treatment 5 days using formulated conidia of CQMa102 and was higher than that of unformulated conidia powder and liquid suspending conidia. The results of toxicity test of show that oil-based formulation of M. anisopliae belong to almost non-toxicity insecticide. The storage stability of conidia mixed with 5-10% soybean is significantly better than that of dry-conidia. The ultraviolet radiation resistance and heat-stability of oil-conidia mixed with diluentent of mycoinsecticide were higher that of dry-conidia and water-conidia.4,The 1% beta-cypermethrin exhibit strong synergism with 1×1010 conidia/ml M. anisopliae oil formulation. The syntoxicity coefficient of the combination is high up to 263. The efficacy of the combination reached >90% at 5 day after treatment against L. migratoria manilensis(Meyen) and shorten 7-8 days compared with M. anisopliae in oil-based formulation using spray. The dosage of beta-cypermethrin in the combination, which was mixed with diluentent of mycoinsecticide at 1:4 ratio before application, is lower than that of chemical insecticide of beta-cypermethrin. Accordingly beta-cypermethrin synergism with M. anisoplia reduced the effect on non-target biology in the field due to greatly reducing chemical application dosage.5. 4 Value of hydrophilic-lipophilic balance (HLB) of plant oil-water system was determined. The diluentent of mycoinsecticide, which hasten germination of conidia and enhance virulence of M. anisoplia against locusts, is consist in water (40%), soybean oil (58.5%) and composite emulsification (1.5%) chosen according to the principle of HLB. Results show that the concentration of spores of in locust is 2.41×107 spores /ml at 4 day after inoculating M. anisoplia mixing with the diluentent on locust, and approximate 50 times of that of M. anisoplia.6. In a wide variety of vegetation and weather condition, 2-4 instars nymph of Locusta migratoria manilensis (Meyen) were effectively controlled by strain CQMa102 of Metarhizium anisopliae formulated 1×1010 conidia/ml oil based formulation, which was mixed with diluentent of myco-insecticide with the ratio of 1:2, using a ULV. In ground spray trial against L. m. manilensis (Meyen), it took 9-15 days to caused 90% mortality. In the aerial spray against L. migratoria manilensis (Meyen), the final percent survivals of locusts lowered than 10% at 14-15 days. The strain CQMa102 of Metarhizium anisopliae formulated oil furmulation and powder effectively declined quantity of Ceracris Kiangsu (Orthoptera: Acrididae) in nature. The efficacy reached over 80% in 11 days after treatment.7. According to the internal transcription spacer(ITS) sequence of 23S rDNA and the gene sequence of 5.8S rDNA of Metarhizium anisopliae var. acridum CQMa102, a specific primer pair of fluorescence quantitative PCR was designed, and a modified extraction method of DNA from the infected haemolymph of male adult locust was optimized. By combination of the specific primer pairs and the DNA extraction method, a pre-mycoses quantitative detection approach for rDNA of insect pathogen in the haemolymph of infected locust was established using real-time fluorescent quantity PCR (FQ-PCR) which is of speediness, sensitiveness and specificity. With this method, rDNA of Metarhizium anisopliae in the haemolymph of locust could be detected 36 hrs post-inoculation.
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