Biological Characteristics Of Metarhizium Anisopliae Isolates LD65 And LF68 And Their Pathogenesis To Plutella Xylostella

This research focused on the biological characteristics of Metarhizium anisopliae isolates LD65 and LF68, their virulence against Plutella xylostella and factors affecting the pathogenicity, infection process and histopathological changes of P. xylostella infected by M. anisopliae, as well as the defense response of the host to the pathogen. The purposes of this study were to establish the pathogenesis of M. anisopliae to the diamondback moth and interaction between M. anisopliae and P. xylostella, to evaluate the potential of the fungus as a biocontrol agent for P. xylostella, which provide the basis for the application of the pathogen in the integrated control of diamondback moth and provide new exemplum for the research of interaction between host and pathogens.Biological research showed the two isolates had good adaptability to the environmental conditions such as temperatures, illumination and pH values. The proper cultural conditions for the two isolates were 25℃~30℃ , pH5~9, with some illumination. The germination of the conidia was affected by temperatures and humidities, the optimum condition for germination were 25℃ ~30℃, RH>90%, and the isolate LF68 germinated faster than LD65. The two isolates were both tolerant to high temperatures and sensitive to UV light. The extracellular protease and chitinase of the two isolates showed different levels in different times and temperatures. The protease of LD65 appeared earlier than LF68 and LF68 had higher output in the late stage. LF68 produced more protease than LD65 at 20℃~30℃ whereas LD65 produced more protease than LF68 at 15℃ and 35℃. The chitinase appeared later than the protease and isolate LD65 produced more chitinase than LF68.The isolates LD65 and LF68 of M. anisopliae were both virulent to the larvae of diamondback moth and could infect the pupae and adults under high concentration. In this study, the virulences of the two isolates against 2nd to 4th instar larvae of P. xylostella were tested with 3 doses at 4 temperatures. The results showed both isolates were highly virulent and had quick lethal action to P. xylostella larvae. The median lethal times of both isolates to 2nd to 4th instar larvae were shorter than 2.5 days when inoculated with 108 conidia/ml at 25℃~30℃. Temperatures and humidities had marked effects on the pathogenicity, among them, the humidities played key roles in it and interactions between temperatures and humidities existed. The virulences of both isolates were not affected when cultured at 15℃~30℃ or on PPDA medium with pH4~11 or through 9 times passages. The extracelluar protease levels were consistent to the virulence at proper temperatures for M. anisopliae growth and propagation while the chitinase didn't show the same trend. Larvae of Plutella xylostella infected with 107 conidia/ml and 108 conidia/ml ate 77%~94% less leaf tissue than control larvae.Larval cuticle of P. xylostella was divided into three types according to their surface topography. We found the surface topography of host cuticle affected infection behavior by M. anisopliae, especially appressoria production. There were more appressoria formed and longer germ tubes grew before penetrating in gently and spinous surface topography than in acanthiod surface topography, and the germ tubes of LF68 isolate were shorter than that of LD65 isolate before the appressoria produced or penetration occurred in all kinds of surface topography. Conidia germinated within 7h and activepenetrations were observed in 10h~13h and 13h~19h after inoculation for LF68 and LD65 isolates, respectively. Transmission electron microscopy observation revealed that the penetration of M. anisopliae into the cuticle of P. xylostella were the combination of mechanical pressure and enzymatic degradation. Histopathological changes of infected larvae of diamondback moth were observed. Paraffin sections showed the separation of epidermis from the endocuticle soon after the penetration. After entering into the hemocoel, the penetrating hypha invaded the fat body and muscles nearby, but mo...