| Using Metarhizium anisopliae offers an important approach in the integrated management of Anoplophora glabripennis adults and larvae. In order to reveal the pathogenic mechanism of M. anisopliae on A. glabripennis and its application potentials, and also to clarify the interaction between M. anisopliae and A. glabripennis larvae, a M. anisopliae isolate MS01 highly virulent to A. glabripennis larvae was screened from the isolates baited from the soil using"Tenebrio molitor Bait Method". The biological characteristics, the pathogenicity against A. glabripennis and impact factors to the pathogenicity (temperature, humidity, passage manners and passage times), as well as infection and pathogenicity process of M. anisopliae on A. glabripennis larvae, defense response of the host against M. anisopliae and the pathological changes of A. glabripennis infected by M. anisopliae were studied. The results of this study indicated that:Eight isolates of M. anisopliae were baited and isolated from different soil samples using Tenebrio molitor as bait. Bioassays of the isolates were conducted on A. glabripennis larvae. The results showed that the pathogenicity of the isolates to 50 d A. glabripennis larvae differed from each other and the isolate MS01 was the most virulent one. The corrected mortality, the infection rate and LT50 were 100%, 83.33% and 2.69 d respectively. Furthermore, bioassays of MS01 and commercially available fungus powder were conducted on 95 d Anoplophora glabripennis larvae after insect rejuvenation. The results indicated that the pathogenicity of the rejuvenated strain of MS01 was higher than that of the commercially available strain. Up to the 8th day the corrected mortality, the infection rate and LT50 were 100%, 90.4% and 5.25 d respectively. The LC50 of the MS01 isolate to 95 d Anoplophora glabripennis larvae was defined to be 1.68×106conidia·mL-1. Therefore, M. anisopliae isolate MS01 was confirmed to be more pathogenical to A. glabripennis larvae.The most proper culture media for the mycelia growth and conidia production of isolate MS01 was determined to be PPDA. The strain presented the best nutritional growth at 26℃. Furthermore, the strain showed highest conidia production, fastest conidia germination and highest germination rate at 28℃. The optimal relative humidity for the nutritional growth and conidia production of the isolate was ranged from 95% to 100%. The conidia germinating speed and germination rate were positively correlated to the relative humidity, and reached maximum when relative humidity was 100%.The oviposition amount, the incubation rate and the survival rate of incubated larvae were significantly reduced after A. glabripennis adults had been inoculated by M. anisopliae isolate MS01 in low, medium and high concentrations. The oviposition amount and the survival rate of incubated larvae were negatively correlated to inoculation concentration. The optimal temperature and humidity for the MS01 isolate to be pathogenical to A. glabripennis larvae were 26℃and 100% respectively. There was no significant impact to the pothogenicity of the strain after 2 times passage on PPDA culture media, however, 4 times passage resulted in lower pothogenicity. On the other hand, the pathogenicity of the strains was enhanced at different degrees when the passage had been performed via A. glabripennis larvae.Examination of the invasion process by using scanning and transmission electron microscope showed that M. anisopliae attacked A. glabripennis larvae mainly through abdominal intersegmental membrane, at where more attached conidia, faster germination, higher germination and penetration rates were observed. The next actively intruded area was the area around the valves. The two places were vulnerable to M. anisopliae invasion. Conidia germinated and grew gemmiform protuberance on the cuticle of A. glabripennis larvae within 12 h post-inoculation, then germ tube and various attaching structure were produced. The host cuticle was invaded by a few hyphal bodies 16 h post-inoculation. Up to 24 h hyphae have widely penetrated into host cuticle. The penetration was successful as a result of joint efforts by mechanical pressure and enzymatic activity produced by the germ tube. Conidia penetrated the integument into the haemocoele of A. glabripennis larvae from 36 h to 48 h post-inoculation. The hyphae and hyphal bodies invaded all organs and tissues via hemolymph circulation. Along with the quick and vast reproduction of the hyphae and hyphal bodies in the midgut epithelium, the midgut tissues were gradually destroyed, the midgut microvilli exfoliated, vacuole formed in the midgut epithelium, the peritrophic membrane was decomposed and disappeared, eventually the midgut tissues collapsed and disintegrated from 48 h to 72 h.When the larvae had been inoculated by M. anisopliae strains descended with different passage manners, though the phenoloxidase activity in the haemolymph fluctuated, all tested samples showed a maximum value. It indicated that, when the host was stimulated by M. anisopliae invasion, the immune interaction pushed the phenoloxidase activity to the maximum. However, the phenoloxidase activity decreased in a fluctuated manner along with the gradual enhancement of the adaptability of the invading strains. The analysis of significance of difference was performed on the phenoloxidase activity in the haemolymph of larvae inoculated with different strains. The result showed that, compared to those strains descended through hosts, the strains descended through culture media would trigger higher phenoloxidase activity after inoculated on A. glabripennis larvae.The hemocytes of A. glabripennis larvae were able to resist the invasion of M. anisopliae to some extent. Granulocytes and plasmatocytes were the main hemocytes involved in the defense. During peak defense period (48 h～72 h), the total amount of hemocytes increased sharply, while the ratio of granulocytes and plasmatocytes changed. The defense activities of the hemocytes mainly included adhesion, aggregation, phagocytosis, encapsulation and nodule formation. Due to fast reproduction of the hyphal bodies in the blood, the defense mechanism of the hemocytes inhibited and suppressed the invasion only temporarily, and was eventually overrun by M. anisopliae.The food consumption of A. glabripennis adults remarkably decreased after inoculation with M. anisopliae strain MS01 of low, medium and high concentrations. The food consumption was negatively correlated with inoculation concentration. The food consumption and weight of A. glabripennis larvae were all lower than those of the control after infected by M. anisopliae. The content of the proteins in the hemolymph showed a generally decreasing trend although fluctuated significantly. Total 17 free amino acids were found in the hemolymph of A. glabripennis larvae. During different post-infection periods, the total contents of free amino acids fell first and rose later. The contents of different amino acids fluctuated differently.The activities of the main protective enzymes and detoxifying enzymes changed at different degrees due to invasion of M. anisopliae. During the initial infection stage, the activities of SOD, POD and CAT increased quickly, while during the later stage, the activities of SOD and POD decreased at different degrees but that of CAT rose to a high level. The CarE activity rose first and then gradually decreased. The trend of changes of GSTs activity could be summarized as activated→suppressed→activated→suppressed. The impact on AchE activity could be summarized as suppressed→activated→suppressed→activated→suppressed.The histopathological changes of A. glabripennis larvae after infection and the development process of M. anisopliae in the host body were clarified. The invading M. anisopliae first separated the host cuticle and the epithelium. Along with the vast proliferation of hyphal bodies in the host haemocoel, apparent pathological changes occured at all infected organs and tissues, e.g. fat bodies loosened, cracks appeared in musles, trachea tissue disintegrated, malpighian tubes deformed and digestive tract disintegrated.
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