Screening And Characterization Of High Infection For Spodoptera Litura Body Wall Nomuraea Rileyi Strains

Nomuraea rileyi is one of the most important insect pathogenic fungi,particularly to Noctuidae（Lepidoptera）class has very strong pathogenicity,and can cause the field epidemic disease of insect.This fungi has strong pertinence,and be harmless to human beings and other non-target organisms including insect parasites and predators,has unique mode of infection and it is unlikely to cause pest resistance and other characteristics.It has a very good application and development prospect in the biological control of plant diseases and insect pests damage.In this study,it proceed with the pathogenicity and virulence difference factor analysis of 2nd and 4th instar larvae of Spodoptera litura.By observing and contrasting the attachment the attachment,germination and appressorium formation condition of Nomuraea rileyi on different instar of Spodoptera litura.Result analysis shows that cuticle degrading enzyme has very important influence for the virulence and correlation bettwen chitinase enzymes extracellular protease and virulence of Nomuraea rileyi.Therefore established the equation of linear regression between enzymes about body wall decomposition chitinase and extracellular protease and the virulence of Nomuraea rileyi strains,the higher correlation one was obtained with the help of the screening of highly virulent strains.Breeding Nomuraea rileyi by UV mutagenesis and got high virulence strains.In order to provided a large number of experimental data for the studies of Nomuraea rileyi and the relationship between decomposition enzyme activity of chitinases,proteases and virulence of strains,and provided the theoretical basis for effective screening and utilization of Nomuraea rileyi against Spodoptera litura.1.This study mearsured the virulence of 4 strains of Nomuraea rileyi from different hosts to the 2nd and 4th instar larvae of Spodoptera Litura and analysed experimental data by the probability analysis.Results showed that the pathogenicity of all strains to the test insects were strong but difference.Comprehensive comparison of cadaver rate,corrected mortality and LT₅₀,strain of Nr16 had the best comprehensive pathogenic effects on the Spodoptera litura larvaes,followed was Nr41 and Nr15 was the weakest for the pathogenicity of tested strains on 2nd instar larvae of Spodoptera litura.For 4th instar larvae,Nr16 had the highest virulence Nr12 followed and Nr15 was the weakest.Through comparing the irulence indicators of the same strain to different ages of the test insects founded that the pathogenicity of strains on the 2nd instar larvae of Spodoptera litura were stronger than 4th’s.2.The infection process of Nr16 on 2nd and 4th Spodoptera litura were observed by scanning electron microscope and found that spores in 2nd instar Spodoptera litura body surface whether the spore adherent amount,spore germination rate or the rate of appressorium formation were higher than that on the 4th instar larvae.The time that the germ tube penetrates through the body surface to enter the body cavity more quickly after spore germination on 2nd instar larva body surface.2 instar larvae’s death time was shorter than 4th and mycelium can easily break the insects’s body wall and growing on it.3.DNS method is used to test the chitinase activity of 4 strains.Experimental results showed that enzyme of the strains of living within the scope of 7.3-8.2 U.According to the enzyme activity to get rankings:Nr16>Nr41>Nr12>Nr15,Nr16 strains has the highest chitinase live for 8.1294±0.1728 U meanwhile Nr15 had the lowest chitinase activity of7.3188±0.8156 U.Folin-phenol method is used to test the protease activity of strains.Enzyme activity was not significantly different.Protease activities were in the range of1.40 to 1.57 U.Nr15 strain has the highest protease activity for 1.5664 U,Nr12 was 1.5494U,strain of Nr41 was 1.4924 U,Nr16 strain had the lowest was 1.4375 U.To do the the linear regression analysis between measured strains of chitinase activity,extracellular protease activity and strain half lethal time,and results showed that chitinase activity,proteinase activity has close relationship with virulence of strains.But the correlation cofficient of regression equation of chitinase activity were higher than protease.So in this article selected chitinase activity as biometric supplement indicators of strain virulence.4.Breeding Nomuraea rileyi by UV mutagenesis and determined the chitinase activity of 20 preliminary screening strains.Compared with the starting strain eight strains were chitinase producing mutant,chitinase activity of Nr1510-1 was 8.0841±0.1246 U and it was the highest in the positive mutant strains.Determination the solid spore production of screened chitinase producing mutant by solid fermentation method and strains of Nr1510-1and Nr1510-2 had significantly improved than the original strain.Taking chitinase activity and solid sporulation into consideration,Nr1510-1 and Nr1510-2 strains were target highly virulent strains.It was found that the LT₅₀of Nr1510-1 and Nr1510-2 strain that were selected from mutant Nr15 were 3.847 d and 3.981 d respectively,lethal time of 50%were raised 7.89%and 6.86%compared with the original strain’s respectively.