| The sweet potato whitefly, Bemisia tabaci (Gennadius) (Homoptera:Aleyrodidae), is a notorious pest in the world. It damages crops by sucking phloem sap which causes nutrition loss in crop, secreting honeydew which lowers the rate of leaf photosynthesis, and moreover it transmits many kinds of plant viruses and causes huge economic losses. Meanwhile, Bemisia tabaci has developed resistance to most chemical insecticides and gives rise to pesticide residue and environment issues. Therefore biological control holds the advantages for control Bemisia tabaci in the future. Application of entomopathogenic fungi are an important aspect of biological control, Isaria fumosorosea is a worldwide distributed fungus. This research focused on the biological characterstics of a highly pathogenic strain of Isaria fumosorosea IF-1106 strains, its pathogenicity against Bemisia tabaci, the infection process and physiological and biochemical changes of Bemisia tabaci infected by Isaria fumosorosea. In addition we evaluate the safety of Isaria fumosorosea. The research probably presents some scientific theory basis for biological control of whiteflies in the future. The main results were as follows:1. Biological characterstics of Isaria fumosoroseaIsaria fumosorosea IF-1106 strains were cultured with solid and liquid mediums respectively. Dish culture was used to determine the mycelial diameter and sporulation in solid culture conditions and shake-flask culture was used to measure mycelial biomass and sporulation in liquid culture. The results showed that the best medium for mycelium growth was PDA medium, the optimum temperature for mycelium growth was 30℃, the optimum temperature for sporulation was 25 ℃, and the optimal pH was 7. The whole day bright (24 hours of illumination) were conducive to its mycelium growth, while 24 hours of total darkness were conducive to its sporulation. No sealing on the containers was the best way to sporulation and benefit for growth. In liquid cultures, sucrose and peptone were the most suitable carbon and nitrogen source, when the concentrations were 4% and 0.5% were conducive to sporulation. The preference temperature for the growth and spore production was between 20℃ and 30℃, the optimum was 25 ℃, and 24 hours of illumination were conducive to its mycelium growth, while 24 hours of total darkness were conducive to its sporulation. When the shaker rotation was 150 r/min and inoculum was 15% the sporulation reached the maximum. The optimum temperature for conidial germination was 26℃, the range of suitable temperature was 23-32 ℃, and the conidia germinated at a high relative humidity, when RH lowers than 75% the conidia were ungerminative. The conidia germination rates were high under the dark when the rate was low under the light. Under the condition of alternation of the light and the dark, the rate has a trend of growing with the longer light time. And the optimal pH was 5-7.0.05%-0.1% Tween-80 was used to prepare the suspension. With the treatment of heat stress and short-time ultraviolet radiation, the strong inhibition in conidia germination of Isaria fumosorosea was exhibited.2. Pathogenicity of Isaria fumosorosea against Bemisia tabaciPathogenicity of Isaria fumosorosea IF-1106 isolate against Bemisia tabaci was determined in laboratory. The results indicated that all stages of the insect were vulnerable to infection of Isaria fumosorosea IF-1106 isolate, the second instar of whitefly was the most sensitive stage to Isaria fumosorosea and the accumulative corrected mortality was high to 83.05%. The Isaria fumosorosea conidial suspension with the concentration of 1.0×107, 5.0×106, 1.0×106,5.0×105 and 1.O×105 conidial/ml were used to infect the second instar nymph, as the concentration increased, the accumulative corrected mortality was increased from 60.60% to 83.05% and LT50 was decreased from 5.86d to 4.47d. And as the treatment time increased, the LC50 was decreased; the LC50 was 2.61 ×104 conidial/ml. The effects of temperature (20,23,26,29,32 ℃) and relative humidity (53%,65%,75%,85%,95%) on conidial germination and pathogenicity of Isaria fumosorosea IF-1106 against 2nd instar nymphs of Bemisia tabaci were investigated. The cumulative corrected mortality of 2nd instar nymphs reached to a peak at 26 ℃. The pathogenicity of Isaria fumosorosea increased with relative humidity. The cumulative corrected mortality of the 2nd instar nymphs increased from 54.55% to 88.89% in RH range from 53% to 95%. It is concluded that the optimal conditions for Isaria fumosorosea IF-1106 to infect Bemisia tabaci were temperature at 26 ℃ and RH over 85%. The virulence of the same entomopathogenic fungi was always significant difference on different host plants. The effect of host plant on the virulence of Isaria fumosorosea to Bemisia tabaci were determined at different concentrations (1.0×107,5.0x106, 1.0×106,5.0×105 and 1.0×105conidia/ml) and compared the accumulative mortalities, the LC50 and LT50 values of B.tabaci on cucumber, eggplant, tomato and beans. The results showed that on the same host plant, when the concentration and processing time increased, the accumulative mortalities was upward. And the mortalities was significant different on the four species host plants in the same concentration and the same treatment time.3. Microscopic observation of Bemisia tabaci infected by Isaria fumosorosea and the physiological investitationScanning electron micrographs observation:at the 6th hours after inoculation the conidia of Isaria fumosorosea adhered on the cuticle of Bemisia tabaci, the conidia began to germinate, and at 24th hour, almost conidia germinated and appressoria were produced by the germinated conidia and adhered on the cuticle to grow. Within 48h, the hyphae grew and penetrated the cuticle. By the 72th hour after inoculation, the hyphae penetrated out the cuticle of host, formed conidiogenesis and new conidium and hyphae. By the 120th hour, extensive growth of hyphae formed net, and the hyphae covered the whole body of the host. The slide observation of Bemisia tabaci infected by Isaria fumosorosea:the conidia of Isaria fumosorosea adhered on the cuticle and then germinated, and germ tube was found which penetrated the inside of cuticle and entered the hemolymph, and mass propagation was shown in the hemolymph. As the inoculation time extended, there were more and more hyphae in the hemolymph, most tissues were disintegration and the nutrients were consumpted, the hemolymph was filled with hyphae which penetrated the cuticle of host and found outside the body. The activities of superoxide dismutase (SOD), perioxidase (POD), catalase (CAT), glutathione S-transferase (GSTs) and carboxylesterase (CarE) of adults and 3rd nymphs of Bemisia tabaci infected by Isaria fumosorosea were studied. The results indicated that activities of SOD, POD, CAT, GSTs and CarE were significantly affected by Isaria fumosorosea. And activities of SOD, POD, CAT, GSTs and CarE were increased after infection. The values were higher than control at the beginning of infection and reached their peaks in 48-60 h, then decreased and significantly low than control in 84 h. So the physiological activities of Bemisia tabaci were disturbed by the infection of Isaria fumosorosea.4. The evaluation on safety of Isaria fumosoroseaToxicological test methods of pesticides for registration (GB15670-1995) were used to obtain acute toxicity of Isaria fumosorosea, including poisoning through mouth and skin, acute inhalation toxicity, acute eye irritation test and dermal sensitization. The results showed that the acute oral toxicity of Isaria fumosorosea was LD50> 5000mg·kg-1, acute dermal toxicity LD50 (4h)>2000mg·kg-1, LC50 (2h)> 5000mg·m-3. No irritation was observed in rabbit eyes, and no dermal sensitization reaction was found on the treated rabbit skin. These results indicated that the acute oral toxicity, acute dermal toxicity and acute inhalation toxicity of Isaria fumosorosea were lower. There was no irritation to rabbit eye and skin, and it could be graded as a weak sensitizing.In conclusion, Isaria fumosorosea is a safe and effective agent for control of Bemisia tabaci. The best way to culture and the optimum condition for application of Bemisia tabaci were obtained, and preliminary investigation on infection process was conducted. Yet the pathology of the fungus in the whitefly needs to study in the future. |
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