Study On Screening For Virulence Strains Of Metarhizium Anisopliae Against Hylobitelus Xiaoi Zhang And It's Pathogenesis

H.xiaoi is a new dangerous pest to pine,which gives rise to a serious problem to forestry production.M.anisopliae is one of main pathogenic fungi for biological control.In this thesis,the virulence strains Ma1291-2 and MaWP-01 of M.anisopliae against H.xiaoi had been screened.Accordingly their biological characteristics and pathogenic mechanism against H.xiaoi and the defense response of H.xiaoi to infection by M.anisopliae were systematically studied.Based on these,the biological controlling experiments with them in forest on H.xiaoi were studied.It may serve as a theoretical and application basis for biological control on H.xiaoi.1.The virulence of 10 M.anisopliae strains was tested to H.xiaoi larvae by spraying with (1.5±0.5)×10⁶ spore·mL^-1,the results showed that the highest corrected mortality of H.xiaoi larvae was 91.90%while the lowest was 15.28%,the highest cadaver rate was 91.32%,the lowest being 24.80%after seven days.The median lethal times(LT₅₀) were between 5.39 and 10.12 days,and the average individual spore yield of cadaver was from 10⁶ to 10⁷ spore. Among them,the Ma1291-2,MaWP-01 and Ma1775 strains were stronger virulence to H.xiaoi larvae,which cadaver rate were 89.56%,91.32%and 88.12%respectively,and the LT₅₀ were 5.49,5.72 and 5.53 days respectively,and their average individual spore yield of cadaver were higher than others too.By the time-dose-mortality modeling for estimating parameters to the dose and time effects of strain Ma1291-2 and MaWP-01 against H.xiaoi larvae,it found that the time of stronger lethal effect were eight and nine days respectively,and the peak time of death was more early with the spore concentration of treatment increasing.2.The virulence of 12 M.anisopliae strains against H.xiaoi adults was screened by dipping method with(1.5±0.5)×10⁸ spore·mL^-1,and a stronger virulence was used to infect trials in forest.After 30 days,the results showed that the strain Ma1291-2 was the most virulent among them,which was followed by MaWP-01.The correct mortality,muscardine cadaver rate and spore content individual of H.xiaoi treated with the strain Ma1291-2 were around 70.59%, 70.81%and(1.90±0.34)×10⁸ spore individual respectively,and the quickest killing speed with the LT₅₀ for 13.89 days.The correct mortality and muscardine cadaver rate of H.xiaoi treated with the strain MaWP-01 were 70.02%and 69.79%respectively,the LT₅₀ was 15.98 days.By the time-dose-mortality modeling for estimating parameters to the dose and time effects of strain Ma1291-2 against H.xiaoi adult,it found that the time of stronger lethal effect were 16-18 days,and the peak time of death was more early with the spore concentration of treatment increasing.By infect trials in forest with strain Ma1291-2,the correct mortality of H.xiaoi were (57.85±1.19)%and(44.35±2.91)%respectively.These results suggest that the Ma1291-2 and MaWP-01 strains could be incorporated into an integrated control programme for the biological control of H.xiaoi.3.The research on biological characteristics of the Ma1291-2 and MaWP-01 strain,the results showed that PPDA medium was the most suitable for their mycelia growth and conidia production among five kinds of commonly used medium,while glucose was the best carbon source,and the most ideal nitrogen source was peptone or yeast extraction.For the most spore germination rate of two strains,the best carbon source is glucose or soluble starch,and the most ideal nitrogen source was peptone or beef extraction.The optimum temperature for the spore production and germination of two stains were 25℃to 28℃,whilst the optimal humidity for spore germinating was more than 90%.In addition,the spore producing and germinating of two strains would be inhibited by the illumination.4.The infected position and process of the strain Ma1291-2 of M.anisopliae on H.xiaoi adult was studied using scanning electron microscope.The results showed that the infected position is abdomen.The spores had germinated after being inoculated for two days.It generated the oriented-growing spore tubes and the end of it presented enlargement after being inoculated for three days,but the appressorium have not been observed.From four to five days after being inoculated,the mycelium generated as well as the spore orienting growth.There were new myceliums and spores generate on just lethal H.xiaoi adults after being inoculated for eight days.For nine to ten days,non-moisturizing training muscardine cadaver was covered myceliums and produced lots of spores for the secondary infection.5.To explore cellular defense reaction of H.xiaoi adult to M.anisopliae isolate strains Ma1291-2,it was found that the concentration and behavioral of hemolymph cell of H.xiaoi were caused a strong immune response to infection of M.anisopliae.Follow the number of hemolymph cell were increase in the infection process,the hemolymph cells aggregated into nodule formation and participating in encapsulation and phagocytosis,finally,the hemolymph cells were been melanism and hardly injured to breakage.From five to eight days after being inoculated,the concentration of total hemolymph cell(THC) increased hardly and decreased follow it.And it reached the highest concentration with(4.71±0.02)×10⁴ cell·uL^-1 on the seventh day.The percentage of the plasmatocyte,granulocyte,sherulocyte,oenocytoide and prohemocyte are(4.46±0.16)%,(17.28±0.16)%,(8.54±0.40)%,(5.01±0.32)%and (4.90±0.05)%in normal hemolymph of H.xiaoi adult,respectively.However,the concentration of plasmatocyte,granulocyte and sherulocyte increased hardly and decreased follow it from six to ten days after being inoculated.However,the concentration of prohemocyte decreased hardly on the sixth days,and the concentration of oenocytoide was change slightly in the infection process.6.The phenoloxidase(PO) activity of hemocyte lysate solution of H.xiaoi larvae and adult were 2.04 and 1.91 times higher than those of plasma respectively.The PO activity of hemocyte lysate solution of H.xiaoi larvae was the highest,which it was 1.52 times higher than that of adult.After being activated by mycelial lysates solution of different virulence stains to H.xiaoi,the PO activity of the hemolymph of H.xiaoi larvae and adult were significantly increased.However,the virulence of stains to H.xiaoi was stronger;the activation effect on the prophenoxidase(proPO) was decrease.The PO activity was highest after being activated by mycelial lysates solution of inpathogenicity strains Ma20.In the infection process,the PO activity of H.xiaoi larvae was the highest from the third to fourth day,and it was the highest in adult from eighth day to tenth day after being inoculated.7.Not only are there more stable and higher spore germination rate and better adaptability to high temperature spore in oil formulation,but also it is favorable to adsorption and enhance virulence to insect.Infection test and control trial by ultra-low-volume spraying with the oil formulation of the strains Ma1291-2 and MaWP-01 were studied in forest.The results showed that the spore germination rate of two strains in oil formulation(Cooking oil V:Diesel oil V=3: 7) were over 80%after 48 hours.By infecting trials to H.xiaoi in forest with them,the mortality of H.xiaoi were extremely significant higher than the control.In the infection test,the mortality of H.xiaoi were extremely significant than the control in forest.After controlling for a year,the population densities of H.xiaoi were extremely significant lower than the control,and the control effect was(46.7±1.28)%and(37.57±1.26)%respectively.After two years,the population densities of H.xiaoi were still significantly lower than the control,of which the controlled effect was(45.47±1.70)%and(36.69±0.90)%respectively.It showed that the oil formulation of two strains had a certain controlling affection to H.xiaoi,and the strain Ma1291-2 has a higher application value in bio-control on H.xiaoi.8.By making of non-woven fabric band with the strain Ma1291-2 of M.anisopliae and control experiment on H.xiaoi in forest,the results showed that the correct mortality and cadaver rate of H.xiaoi were 64.25%and 62.25%respectively in indoor infecting test to H.xiaoi,and median lethal times(LT₅₀) was 14.43 days.By controlling trial in forest,the control effect on H.xiaoi was(53.80±1.74)%and(51.54±1.35)%respectively in two trial plots. And the spore on non-woven fabric bands kept 10⁷-10⁸ spore·cm^-2 in 15 days,but sharply decreased after then.The spore germination rate was decrease slightly in 30 days,it was over 80%.It showed that the non-woven fabric band of strain Ma1291-2 has a certain application value for controlling on H.xiaoi.9.The spore germination rate of Ma1291-2 and MaWP-01 strains was studied after compositing stabilizer with 7 kinds of chemical insecticides,the results showed that the spore germination rate of two strains was inhibited significantly by chemical insecticides,and the inhibitory effects were enhanced with the concentration of insecticides increasing.There were significant different inhibition rate on spore in different time,which the inhibition rate in 24 hours was higher than it in 48 hours.It showed that the spore of two strains were sensitive to chemical insecticide.Among them,the Phoxim has the strongest inhibitory effects to spores of two strains.