Correlation Between Pr1 Protease And Strain Virulence Of Isaria Fumosorosea And The Influence Of Cellular Apoptosis For Plutella Xylostella Larvae

In the biological control of pests, Isaria fumosorosea and Beauveria bassiana play an important role as two major entomopathogenic fungi. They degrade the body wall of the insect via protease. The fungus secretes a variety of ectoenzyme hydrolases to invade the hosts. In the experiment, we use Pf9606, Pf76?Pf904?Pf94?Pfl4 of Isaria fumosorosea and Beauveria bassiana as materials, compare the virulence among different strains. Then we use the method of specificity substrates determining Prl protease activity, detect gene expression of Prl protease by real-time quantitative PCR technique. Finally we use fluorescence microscope technology to establish apoptosis detection of insect cellular immuntity system. The result are as follows:1.According to the virulence test of I. fumosorosea and B. bassiana infect the green peach aphid and plutella xylostella larvae, we can get the following conclusion:different tested strains'virulence have obvious differences. The green peach aphid fatality rate changes in the range of 78.43?96.08%. LT50 changes in the range of 2.39?3.10 d. Among these strains, Pf 904 has the highest fatality rate of 96.08% to green peach aphid. The smallest value of LT5o is 2.39d; B. bassiana has the lowest fatality rate of 78.43% to green peach aphid. The largest value of LT50 is 3.10d. The rate of I. fumosorosea Pf904 infecting 3rd instar larvae fatality is 96.67%, LT50 is 1.37d; pathogenicity of 4th instar larvae of plutella xylostella is 40%, visible resistant ability of the 3rd instar larvae was significantly lower than that of the 4th instar larvae.2.Specific peptide substrate Sue-Ala-Ala-Pro-Phe-pNA was used for Prl protease activity assay of different strains. The results showed that different strains Prl enzyme activity is different. Prl protease enzyme activity from high to low is in turn:Isaria fumosorosea Pf904, Pf7606, Pf14, Pf9606, Pf941, Beauveria bassiana. Isaria fumosorosea Pf904 Prl enzyme activity level up to 0.0857 U/mg. Beauveria bassiana Prl enzyme activity had the lowest levels is only 0.0052 U/mg.3.Real-time quantitative PCR technical inspection of different strains Prl protease gene expression.At the same time analysis with SPSS software, fatality rate, Prl protease activity and expression of the connection above three. Isaria fumosorosea was obvious different from Beauveria bassiana.The lowest expression quantity is Beauveria bassiana,1.1 times of contrast. The highest expression quantity is Isaria fmuosoroseus,5.9 times of contrast. The expression quantity of Isaria fumosorosea Pf7606?Pf14?Pf 9606?Pf 941 followed by 5.8, 5.2,4.8,5.2 times.4.Using SPSS software to analyze the connection between the pathogenicity of I. fumosorosea and B. bassiana, Prl protease activity and gene expression:The Prl protease activity and virulence value about different strains were proven to be positively correlated, Prl protease expression and the direct proportional relationship between the Prl enzyme activity.Therefore Prl enzyme activity and Prl protease gene expression are a significant influence on mortality, determine the virulence of strains.5.To evaluate the usage of fluorescent microscope in analyzing cellular apoptosis. Blood cells of Normal Plutella xylostella 3rd and 4th larval shape, color are basically identical. In under the action of I. fumosorosea Pf904,they have different degrees of apoptosis.After the treatment of 12h, the 3rd larval showed early apoptotic cells and late apoptotic cells,after 72h all of 3rd larval blood cells was apoptotic;however the 4th larval began to emerge early apoptotic cells after 24h, half of 4th larval was apoptotic after 72h. Apoptosis occur obvious 3rd earlier than 4th instar larvae about 24 hours. The same processing time,the number of 3rd apoptotic cells apparently higher than 4th, and as the time accumulation, the number of apoptosis cells are increased, normal cells gradually reduced. Under the observation of fluorescent microscope, cells showed the features of apoptosis is pyknosis,chromatin aggregation and karyorrhexis, has the obvious difference with normal blood cells.