| Xylotrechus rusticus(Coleoptera:Cerambycidae),a kind of borer pest that harms the poplars,has severely occurring in the northeast region of China since the beginning of this century,causing many poplars to wind down and windbreak,which directly damages the poplars economic value and ecological function.In order to solve the problem about no insect pathogenic microorganisms and natural enemies can be used to control the population of X.rusticus so far.This study takes 6 strains of Beauveria spp.and a natural enemy insect,Dastarcus helophoroides(Coleoptera:Bothrideridae),as the main research subjects that Beauveria spp.pathogenicity,the optimal culture conditions,environmental factors affecting the pathogenicity,the X,rusticus immune response to the Beauveria spp.infection and control effect of X.rusticus in the forest were studied.One of the parasitic natural enemies of Cerambycidae,D.helophoroides,the survival ability,the physiological changes and the expression of heat shock proteins(HSP)under cold stress were studied.The results listed below:(1)In this study,a strain of Beauveria bassiana isolated from adult X.rusticus,four strains of Beauveria bassiana isolated from other insects and a strain of Beauveria brongniartii were selected to compare their pathogenicity.The results showed that the strain Bb01 appeared the strongest pathogenicity.The cumulative mortality rate reached 96.96%,the corrected mortality rate reached 96.64%,the LT50 reached 3.28 d.Strain CFCC83486 and strain CFCC81428 also appeared strong pathogenicity.Therefore,the strain Bb01 should be selected as the best strain to control the larvae of X.rusticus.(2)It was selected by orthogonal test of 5-factor 3-level culture conditions.The growth condition of Bb01 strain was modified Martin medium,the most suitable culture temperature for colony growth and sporulation was 25?,and the most suitable medium pH was 5.The added carbon source,nitrogen source and microelements were sucrose,ammonium sulfate and iron sulfate(the amount added was the same as the original medium).(3)In this study,the pathogenicity of strain Bb01 was tested by 6 temperature gradients(24??25??26??27??28??29?),5 humidity gradients(95%?90%?85%?80%?75%),4 ultraviolet irradiation time gradients(10 min?15 min?20 min?25 min),5 kinds of culture mediums and Bb01 suspension added 3 induced enzyme fermented solution(chitinase fermented solution,lipase fermented solution,proteinase fermented solution).The results showed that when the ambient temperature was 25?,the environmental humidity was 95%,Bb01 appeared the strongest pathogenicity.UV ultraviolet irradiation could also affect the pathogenicity of Bb01.When the irradiation was 10 minutes,it can still ensure that Bb01 has strong pathogenicity,reached 73.33%.The Bb01 strain cultured by A1 medium has the strongest pathogenicity compared with the conidia cultured by other mediums,and its cumulative mortality rate reached 98.33%at the 10th day.The chitinase fermented solution,lipase fermented solution and proteinase fermented solution appeared synergistic effect on the lethality of the Bb01 strain.The cumulative mortality of the three fermented solution treatment groups reached 100%at the 10th day;the LT50 of the test group added lipase fermented solution was the shortest,2.36 d.Therefore,the chitinase fermented solution,lipase fermented solution and proteinase fermented solution have potential application value as the synergist of Bb01 strain.(4)The total blood cell concentration and phenol oxidase activity were measured at 13 time points.The results showed that the total blood cell concentration and phenol oxidase activity of the larvae and adults showed a trend of increasing first and then decreasing.The blood cell concentration of the larvae and adults reached the highest concentration at 36 h and 48 h after infection,which were 3.53×107 cells/mL and 3.63×107 cells/mL.The phenol oxidase activities of the larvae and adults reached the highest levels at 5.1 U and 4.4 U at 28 h and 40 h after infection,respectively.Therefore,the larvae and adults increased both the concentration of total blood cells and the activity of phenol oxidase to activate an immune response after infected by Bb01 strain.(5)The control effects of highly pathogenic Beauveria bassiana strains Bb01,CFCC83486,CFCC81428,the Bb01 strain with three kinds of induced enzyme fermented solution(lipase,chitinase,and protease),D.helophoroides adult and D.helophoroides egg,were tested in the forest.The results showed that Bb01 strain and D.helophoroides egg appeared the best control effects,reached 74.97%and 79.87%respectively.Therefore,Beauveria bassiana Bb01 strain and D.helophoroides eggs are recommended to control X.rusticus in the forest.Three kinds of induced enzyme fermented solution all appeared synergistic effects on the pathogenicity of the Bb01 strain.The group added lipase fermented solution appeared the best control effect,reached 83.33%.Therefore,the chitinase fermented solution,lipase fermented solution and proteinase fermented solution can be used as synergists and have potential application value in practical applications.(6)D.helophoroides adults induced by low temperature and 5 temperature gradients(0??-5??-10??-15??-20?)were set to measure the low-temperature survival ability of D.helophoroides adults and 6 temperature gradients(5??0??-5??-10??-15??-20?)were set to measure the physiological changes in D.helophoroides body.The results showed that the super cooling point of D.helophoroides adults induced by low temperature was lower than the super cooling point of D.helophoroides adults feeding under room temperature.The super cooling points were-25.1? and-16.9? respectively.With the decrease of the environmental temperature,the survival ability of the D.helophoroides adults became weaker.When the temperature was-20?,the survival rate was the lowest,but the survival ability became stronger after induced by low temperature.Therefore,low temperature induction can improve the cold tolerance of D.helophoroides adults.Superoxide dismutase(SOD)activity increased when the D.helophoroides under cold stress;the catalase(CAT)activity increased with the treatment temperature decreased.The activity of lactate dehydrogenase(LDH)decreased first and then increased with the extension of cold stress time.Both the fat content and the water content of the D.helophoroides decreased with the decrease of temperature.It showed that the D.helophoroides body activated the corresponding self-protection mechanism to maintain the metabolism.(7)The Beta-actin gene and the heat shock protein HSP90,HSP70 genes of the D.helophoroide were cloned by RACE technology,and the full-length of HSP90,HSP70,Beta-actin genes and the conservative sequences of HSP60 were obtained.At the same time,the expression levels of HSP90,HSP70,HSP60,and sHSP20.99 of the D.helophoroide under cold stress were measured by qRT-PCR.The results showed that HSP90,HSP70,and HSP60 showed a trend of increasing first and then decreasing under cold stress.sHSP20.99 showed a downward trend with the temperature decreasing.It showed that D.helophoroide maintained the normal structure of the protein in the body by regulating the expression of heat shock proteins,prevents its denaturation thereby helping to maintain the normal metabolism and growth. |
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