Basic Study On Application Of Metarhizium Against Dendrolimus Punctatus Walker

Nine strains of the entomopathogenic fungi belonging to Metarhizium used to efficient biocontrol of Dendrolimus punctatus were studied in this thesis.The researches focused on screening of high virulent strains and effects of temperature and humidity on their pathogenicity, host pest's physiological response,cultivation and stability,compatibility with chemical insecticides,Metarhizium anisopliae mixed with other insecticides and synergism,as well as effects of M.anisopliae on the diversity of arthropod community in Masson pine forest,and aimed to evaluate the potential of Metarhizium as a biocontrol agent for D.punctatus and to provide the theoretical basis for the applications.The main results are as follows:1.The pathogenicity of nine swains of Metarhizium spp.(including M₁₀₃,M₁₀₄,M₁₃₁,M₃₃₇, M₁₈₇,M₃₃₆,M₁₁₅,M₃₃₅and M₁₈₉)to larvae of D.punctatus were studied in the laboratory.Two strains(M₁₀₄,M₃₃₇)showed pathogenicity to the larvae.The bioassays for toxicity of strains M₁₀₄ and M₃₃₇were tested to D.punctatus respectively and showed the virulence of M₃₃₇was stronger than that of M₁₀₄,and the Strain M₃₃₇was significantly more effective to control D.punctatus. Nutrition and other conditions for the two swains' growth,sporulation and conidia germination were determined,and the results showed that the two strains had good adaptability to the conditions such as temperature,humidity and pH value.The suitable sporulation conditions were 25—28℃,pH 6.5—7.0.The germination of the conidia was remarkably affected by temperature, humidity and pH value.The optimum condition for germination were 25—28℃,pH 6.5—7.5, RH＞92.5%,however,M₃₃₇germinated faster than strain M₁₀₄and showed more tolerance to high temperature and low humidity,and higher conidia germination and production rate.These implied that the strain M₃₃₇had a great potential of exploitation and application.2.The virulence of M₁₀₄and M₃₃₇were tested and analyzed by contrasting with the biocontrol agent Beauveria bassiana(including Bb₀₁and Bb₀₂),which showed that there were no difference of virulence to D.punctatus among M₃₃₇,Bb₀₁and Bb₀₂,however,the virulence of M₁₀₄was lower than those of Bb₀₁and Bb₀₂.The strain M₃₃₇of M.anisopliae was more tolerant to high temperature and drought than strain Bb₀₁of B.bassiana.In the conditions of high temperature and low humidity,the control effects of M.anisopliae on D.punctatus were higher also.Field test indicated that M.anisopliae was great superior to control the first generation of D.punctatus.3.The research on the host insect physiological response to the pathogen showed that the total hemocyte counts and the concentration of hemolymph soluble protein were significantly higher in infected larvae than in non-infected ones,from 1d to 4d after infection.By means of polyacrylamide gel electrophoresis technique,the physiological changes of proteins and peroxidases in hemolymph and body wall of the D.punctatus which 3d,4d and 6d post infection were analyzed.The results revealed that M anisopliae had some effect on the metabolism of the host's proteins,and the peroxidase activities showed a tendency to decline.4.The solid-submerge fermentation technology of strain M₃₃₇of M.anisopliae was studied, and the optimal control for fermentation process were determined.The optimized medium of solid state fermentation was obtained,which would be composed of corn flour(2 portion),wheat bran(2 portion)and rice hull(2 portion).The fermentation period for liquid seed was 72h and the appropriate initial inoculation amount was 25%.The culture temperature and humidity were at 25—28℃and 83—94%,respectively.Meanwhile,in order to control the contamination of mould, the suitable covering material was selected necessarily.The period of solid state fermentation was adjusted to 11d,and drying temperature was manipulated at 35℃for 24h,and then the production cycle would be shortened.In addition,the influence of two chemical germicides(including 30%Cuprum oxychloride and Naiketong)on growth and sporulation of strain M₃₃₇of M.anisopliae,and inhibitory effect on competed moulds were investigated.The results showed that the two chemical germicides,viz. 30%Cuprum oxychloride and Naiketong had no obvious effect on colony recovery of M. anisopliae at the concentration of 0.05—0.15%.Naiketong at the concentration of 0.05—0.15% had increased conidial output,while it also shortened the sporulation time of M.anisopliae.The two germicides inhibited the colony growth of competed moulds(Rhizopus sp.,Penicillium sp. Aspergillus sp.)strongly,and the inhibition became stronger when concentration increased.5.Six strains belonging to Metarhizium(including M₃₃₇,M₁₀₃,M₁₀₄,M₁₁₅,M₃₃₅and M₃₃₆) were cultivated in submerged culture.The results indicated that the submerged conidia could be produced by means of microcyclic sporogenesis for strains M₃₃₇,M₁₀₃,M₁₀₄and M₁₁₅,perhaps, the occurrence of microcyclic sporogenesis depended on the nature of strains.Under solid-state and liquid-state cultivations,the entomopathogenic fungus M.anisopliae produced different types of spores.The morphologic characteristics of submerged cortidia of strain M₃₃₇are ovoid to subspherical,monocellular,with the size of 3.17—4.51μm×2.51—3.34μm,quite different from the aerial conidia.Nutrition and other conditions for the growth and sporulation of M₃₃₇were emphatically studied in submerged culture.The results showed that the submerged sporulation depended on a delicate equilibrium in the composition of the medium.Various carbon and nitrogen sources had significant effect on the production of submerged conidia.Sucrose was ideal carbon source for submerged sporulation.Peanut cake powder and bean cake powder were effective for sporulation of submerged conidia.The production of submerged conidia was correlated with the properties of nitrogen sources,which complex nitrogen source for sporulation of submerged conidia was more favorable to simple nitrogen source.Liquid medium containing sucrose and peanut cake powder produced large numbers of submerged conidia(14.13×10⁹ spores·L^-1).The experiment also showed that the output of submerged conidia was relationship with carbon-to-nitrogen(C:N) ratio of medium.Highest yield of submerged conidia was obtained in medium containing C:N ratio of 3:1.Trace elements and Vitamins had remarkable impacts on submerged sporulation. Molybdenum was the most important element.Zinc was stimulatory factor.When all 6 elements (including B,Cu,Fe,Mn,Mo and Zn)were added together,highest yield of submerged conidia could be obtained.VB₆+VH and compound vitamin B were more effective for producing submerged conidia.Various amino acids had important influence on growth and sporulation of submerged conidia.Liquid medium containing L-asparagine or L-alanine produced large numbers of submerged conidia.Illumination had no effect on mycelia growth and submerged spomlation directly.The cultural temperature and pH value had significant effect on the mycelia growth and output of submerged conidia.The proper temperatures and pH values for submerged culture were 25—28℃and pH 6.5—6.8,respectively.The maximum yield of submerged conidia was obtained in the condition of temperature at 28℃and pH 6.8.Biomass and submerged conidia yield were measured for three strains of Metarhizium spp. (including M₃₃₇,M₁₀₃and M₁₁₅)grown in liquid media containing different concentrations of Tween 80.The data was showed that concentrations of Tween 80 had effect on sporulation of Metarhizium spp.significantly.The submerged conidia yield was highest in liquid media containing 0.6—1.0%Tween 80.Three strains of M.anisopliae(M₃₃₇,M₁₀₃,M₁₀₄)and one strain of Metarhizium guizhouense (M₁₁₅)were cultivated in submerged culture by magnetized water media,and the biomagnetic effect of magnetized water on submerged sporulation of Metarhizium spp.were preliminary analyzed.The results indicated that the submerged conidia yields were affected by magnetized water,but the biomass were unaffected.The outputs of submerged conidia of strains M₃₃₇and M₁₀₃were increased remarkably in proper intensity of magnetizing water.But the experiment also showed that there existed obvious difference in different strains for biological effects of magnetizing water.This results provided an experimental basis for further research on the biological effects and applications of the magnetized water.In addition,The virulence of submerged conidia of strain M₃₃₇was tested against D. punctatus in laboratory.Results showed that submerged conidia had stronger virulence to the larvae of D.punctatus.There were no apparent differences in the toxicity between submerged conidia and aerial cortidia,but the virulence of submerged conidia to larvae of D.punctatus was slightly lower.Therefore,it is of great value for further research on submerse fermentation of Metarhizium.6.The subcultures of M₃₃₇were studied in this experiment.Effects of 6 regular media,4 carbon and nitrogen sources and 6 combinations of carbon and nitrogen on strain stability,growth characters and virulence were observed.The results indicated that the variation of M.anisopliae was mainly controlled by its genetic materials,but also influenced by the composition nature of media.Among the six regular media,strain M₃₃₇grew most stably on PPDA,but those nutrition poor ones such as CMA and WBA would cause variation very frequently.Among nitrogen sources, those from animal were more stable than those from plant.Mean while,strain M₃₃₇grew most stably on media containing maltose and lactose as carbon source.The experiment results also showed that strain M₃₃₇grew most stably on medium containing C:N ratio of 2:1.7.The compatibility of 8 chemical insecticides(including 4.5%Beta-Cypermethrin EC, 2.5%Deltamethrin EC,21%Pesticide destroying EC,25%Benzoylphenal ureaâ…¢SC,20% Fenvalerate EC,40%Omethoate EC,40%Phoxim EC and 18%Dimehypo WA)with Metarhizium spp.(including M₃₃₇,M₁₀₃,M₁₀₄,M₁₁₅,M₃₃₅and M₃₃₆)were studied in this experiment.The results showed that all chemical insecticides inhibited the conidial germination to a certain degree,and the inhibition became stronger as concentration increased.But low sublethal dose of chemical insecticide had less inhibitory effect on conidial germination.The results of bioassay showed that it was obvious of the synergism against Masson's pine caterpillars(D. punctatus),when strain M₃₃₇of M.anisopliae(1.9×10¹⁰spores·L^-1)mixed with Fenvalerate (80000×),Deltamethrin(60000×),Phoxim(10000×),Pesticide destroying(25000×)and Benzoylphenal ureaâ…¢(15000×),and the LT₅₀of mixtures would be shortened about 9 days,7 days,6 days,5 days and 3 days respectively,comparing with that of using fungal insecticide of M. anisopliae(1.9×10¹⁰spores·L^-1)alone.8.According to the analysis of Co-toxicity coefficient,M.anisopliae could increase 1.78 times in its toxicity to larvae of D.punctatus when it was mixed with B.bassiana.The effect of control test in the field showed that the mixture insecticide was great superior in controlling D.punctatus to using fungal insecticide of M.anisopliae and B.bassiana alone.9.The density of Masson's pine caterpillars and arthropod communities in various layers of pine forest were investigated before and after spraying microbial insecticide of M.anisopliae in Shanghang county in Fujian province.T test of the density of Masson's pine caterpillars,vertical pattern of arthropod community and its parameters were discussed.The results showed that M. anisopliae had obvious biocontrol effect on Masson's pine caterpillars,but it had no impact on the non-target invertebrates apparently.The diversity and stability of arthropod community in pine stand was improved.