The Technical Research Of Metarhizium Anisopliae Isolates For Biological Control Of Red Palm Weevil, Rhynchophorus Ferrugineus

Red palm weevil (RPW), Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae), as Red palm weevil).Red Palm Weevil will cause harm to19kinds of palms from15genuses. The Pest Risk Analysis shows that the RPW are high-risk pests in many regions of our country in which are vulnerable to colonize and cause huge threat to the palm plants. The application of Metarhizium anisopliae to combat the Red Palm Weevil opens up a new way for the integrated control of the Red Palm Weevil. In order to reveal the pathogenic process and the preventing and controlling potential of Metarhizium anisopliae Prevention, and uncover the pathological changes of Metarhizium anisopliae infecting red palm Weevil larvae clearly, we can find the research results as follows:1.Isolation and identification of the Metarhizium anisopliae14strains separated from Red Palm Weevil cadaver which were collected from the wild would be isolated, purified and identified, then we obtained the following information:the integrity region sequence of ITS1-58S-the ITS2of14strains were about600bp length. After comparison on the NCBI we found13of them were Metarhizium anisopliae spore variants, but the cultural characteristics and pathogenicity differed. And YX5strain was a new species of Metarhizium genus, only91%of it can be found same in the whole database. The length of the amplified region is685bp. The sequence was submitted to GenBank, and the sequence accession number is JN377427. Based on the14strains, we used DNAMAN phylogenetic tree analysis to establish and analysis the phylogenetic relationships between the14strains and other types in Metarhizium anisopliae.2. Bioassay for red palm weevil fourth instar larvae with Metarhizium anisopliaeThe virulence of14strains Metarhizium anisopliae on the red palm weevil larvae was evaluated by spraying indoors, after preliminary screening we found:the virulence of different strains on RPW4th instar larvae differed, abd Red2, Red3, and Gre1have the highest virulence. The mortality of red palm weevil larvae after processing was about90%, half lethal time LT50was5d. Using Red2, Red3and Gre1for the assessment of the concentration gradient on4th instar larvae, we obtained:LC50of Red2strain was7.32×106conidia/mL, the difference was significantly higher than Red3and Gre1strains, which were10.60×106and11.24×106conidia/mL. As the representative of the highest virulence, we compared Red2aqueous suspension and oil suspensions and found:half lethal time in the oil suspension was significantly shortened, but the difference of death rate was not significant. This offered us a certain reference in the field application and discharge of pharmaceutical. And I designed a new type of conveying bacteria device of pathogenic and pheromones in the field based on Metarhizium anisopliae infect cycle.3. Histopathological study on Metarhizium anisopliae infecting the Red Palm WeevilBy observing the frozen section of the red palm weevil larvae, and scanning by electron microscope of the red palm weevil adults, we revealed clearly the basic process of Metarhizium anisopliae infection on Red Palm Weevil:firstly the Metarhizium anisopliae contacted with the red palm weevil, and adhered at the weak point, it germinated in suitable conditions and the hyphae grew to penetrate the epidermis of the host body and then infected the cavity. The Metarhizium anisopilae began to multiply until the host immune system dead. After the Red palm weevil adults were inoculated for24hours, the Metarhizium anisopliae, began to germinate, and the conidiophore and germ tube structure issued at the adhesion; it could be seen the proliferation of the mycelium, which covered on the epidermis, invade in the weak point, and break through the host epidermis defense;72hours later the Metarhizium anisopliae mycelium covered the body trachea, Malpighian tubules, fat, muscle tissue and head organs, a large number of sporophyte reproduction diffusion can be seen on the external epidermis. The leg tarsus, pores, hair nest and interosseous membrane were all destroyed and been filled with mycelium. Eventually the body of Red Palm Weevil was nothing left but the hard chitin shell, the nutrients were broken down and exhausted, the body cavity were all replaced by hyphae.