Analysis Of RAPD Of Beauveria Bassiana Strains’ Degrdation

Beauveria bassiana is the current research and the application of the most a broad range of entomogenous fungi in the world. The pathogenic force is powerful to injurious insect and insecticidal spectrum is broad. It is harmless to warm-blooded animals and easy to raise. Applied types and area of Beauveria bassiana which control of agriculture and forestry pests is the world’s largest in our county. However, production traits often become vestigial after gontinuous passage culture, for example, spore production decrease and insecticidal poison become lower, etc, that impact on application effect of pest biological control of Beauveria bassiana seriously in research and the application process. The problems of spawn degeneration have been bothering microbiology workers, also is many scientific research workers has devoted to solve the problem for many years. Research workers is usually just from the strain preservation methods and training conditions to improve, control the number of gontinuous passage, forced to form heterokaryons artificially, screening stable high toxic force single spore strains, etc to control the degradation speed of strains have not solve the problem fundamentally. Strains variation is mainly due to genetic essence of strains, the most effective measures are taken biological engineering technology. For the effective control degradation of strains, to know the degradation mechanism detailedly.This study for the materials with Beauveria bassiana that is highly pathogenic to the larvae A.germari.By using RAPD that is preliminary study for genetic variation between normal strains and strains of degradation . The main research results are as follows:1.Rejuvenate and passage of Bb00 strains of laboratory. After three times rejuvenation in ivio of host the spore yields much more and the growth rate become slower. 20 generations strain from passage, the results also show that the growth rate become faster and the spore yields of strains becomes fewer along with the passage. There are some strains have no spore yield which suggested that the strains have degenerated at the macro level.2. For 20 generations strain from passage to measure the growth rate and spore yield.The results show that the growth rate of strains become faster along with passage. The growth rate of thirteen was set to reach its maximum at fourteen days. For the strains, above diameter of 8 centimeters is generation of 11-15、17、19、20. The spore yield becomes fewer and fewer along with passage, spores layer is thinner.Spore yield of the strain Bb00 is 2.64×108/cm2 in the first generation that is most,and is 0.61×108/cm2 in the 20th generation that is least.3. Pathogenicity of different successive reisolates of Bb00 to the larvae A.germari. The results show that all the passage strains can kill experimental larvae A.germari. But the infection rate is different. This is show that the pathogenicity to the larvae A.germari becomes fewer and fewer along with passage.After seven days infection rate of the first generation is 91.46%,and infection rate of the 20th generation is 26.83%.4. The research of RAPD-PCR reaction system. By using L16 (45) orthogonal experiment and the single factor of the annealing temperature and recycle number for the optimized combination of factors of reaction system optimization. Results show that the optimum combination of all factors in 20uL polymerase chain reaction (PCR) system and response procedures include 2μL of 10×Buffer, of 2.4uL MgCl2(25mmol/L), 0.8uL of four dNTPs(each 2.5mmol/L), 1.4uL of random primer (10μmol/L), 0.4uL of Taq polymerase(5U/uL), 1uL of template DNA(10ng/uL). Reaction conditions is Pre denaturation temperature as 94℃for 2min, denaturation temperature as 94℃for 30 s, annealing temperature as 38℃for 40s, extensions temperature as 72℃for 1min, recycle number for 40 times, extensions temperature as 72℃for 5min.5. RAPD analysis of DNA diversity of degradation strains. Through the similar rate calculate result. The genetic distances between first generation and 6th generation are largest that is 94.86%,and between first generation and 12th generation are minimum that is 42.04%,and between first generation and 14th,16th,18th,20th generation are over 70%.It is show that before 6th generation,genome don’t variate almost, the genic mutation of 12th generation is largest.after 14th generation, the genic mutation become fewer. It suggested that genetic variation has happened, but the whole genome mutations degree is not great.Genomic DNA of strains has changed at 12th successive times.