The Screening Of Metarhizium Acridum Mutation Strains Of Thermotolerance

Development of insect pathogenic fungi efficient insecticide formulations,large-scale production and field application stability is the key of friendly biocontrol.the virulence of the spores is limited factor of efficient fungal pesticides application inthe field. The virulence is determined by the spore itself characteristics and ability ofadaption to field environment. Therefore, the characteristics of stress toleranceassociated with adapting to the environment is important to the application andconstruction of the efficient insecticide engineered strain.Insect pathogenic fungi was regarded as a great potential and friendly biocontrolresources. However, its large-scale field applications is limited, attributing to the slowerrate of insecticide, vulnerable to environmental impact and short shelf life. In recentyears, with the development and use of modern molecular biology and other dvancedtechnologies, insect pathogenic fungi, especially Metarhizium spp., have made a seriesof basic theory achievements of the function concerning toxic gene. However, thebreeding progress of high-efficiency engineered fungi strains used to locusts biocontrolis not smooth. Therefore, it is necessary to use more effective means and techniques tobreeding high efficient locusticidal fungal engineered strain. Traditional mutationbreeding has its unique advantages because it directly induce the mutagenesis ofinsecticidal active ingredient in spores and directly improve their locusticide effect.Insect pathogenic fungi always encounter a variety of environmental stresses in theprocess of field application, such as heat, drought and UV. The high temperature is avery important factor that influences the germination and growth of spore, the shelf lifeof spore formulation, and the formation of epidemic after the formation of spores infield applications. Especially, the high temperature can induce genetic damage andmutation of spores. In addition, the target locust will inhibit the insect pathogenicfungus infection by changing its surface temperature. Therefore, the temperature has amajor impact in the whole process of biological control. Thus screeningthermotolerance insect pathogenic fungal strains is of great value.Heat shock mutagenesis breeding has its unique advantages. It can not be inductionheat shock mutagenesis, but also can be directional screening with heat conditions. Ithas the potential that efficiently screens the engineered strains of fungal insecticide.The main results were as follows: ①Confirming the limit temperature point of heat shockThe maximum temperature of the heat shock is selected on the basis of thetemperature point dealing with spores2h and then growing only a handful of singlecolonies at28℃, while there are not colony growth at higher temperature point of heatshock. Based on this standard, a series of experiments was performed. The maximumtemperature of the thermal shock assay ranged from a temperature gradient of45.0℃to50.0℃,53.0℃,55.0℃, so the final temperature point of the heat shock wasdetermined to57.2℃.②Selecting and screening the thermotolrance mutant strainsAfter57.2℃heat shock mutagenesis, the first screening acquired three mutationssingle colonies, named M1, M2and M3. Expanding culture, then we performed thesecond heat shock mutagenesis of M2’s mature spores and screening mutants, namedM2-1, M2-2, M2-3, M2-4, M2-5, M2-6, M2-7, M2-8, M2-9, M2-10and M2-11. we obtained M2-1,M2-2and M2-7resistant strains via the verification experiment of45.0℃heatresistance.③Measuring Resistant characteristics of the screening striansM2-1, M2-2and M2-7three resistant strains were confirmed with45℃heat shockexperiments, screening the best heat resistance strians M2-7. Then we assayed M2-7germination rate, UV-B resistance and other characteristics.