| Heliothis assulta（Guenée）,a Lepidoptera Noctuidae,is an important pest of tobacco,pepper and other solanaceae crops.The pest is a serious hazard and difficult to control.Its loss on tobacco accounts for about 15%of the total loss of tobacco production in the outbreak year.At present,chemical prevention and control is still the main means of control for H.assulta,and environmental pollution,natural enemy killing,drug resistance promotion and other problems caused by H.assulta are becoming more and more serious,so it is urgent to find new green prevention and control technology to replace traditional pharmaceutical control.Biological control method has the characteristics of strong specificity and environmental friendliness,which plays a great role in promoting the reduction of pesticide use.Under the background of reducing the application of drugs and fertilizers,biological control methods have been widely used in agricultural production.The exploration and application of new biological control resources has become a research hotspot in the field of plant protection.As important biocontrol resources,entomopathogenic fungi have the characteristics of strong infectivity and specialization,and can be used in collaboration with other natural enemies.In this study,Beauveria bassiana,a highly pathogenic fungus of entomopathogenic fungus B.bassiana,was screened by indoor virulence assay;On this basis,the cooperative control technology of B.bassiana and Trichogramma/matrine on H.assulta was systematically explored.The infection mechanism of B.bassiana to H.assulta was clarified by biochemical assay.In addition,the research of pathogenic gene mining and dynamic gene expression determination technology of B.bassiana were carried out,which provided theoretical basis and technical support for the research and development of new biological control technology of H.assulta.The main results are as follows:1.Two strains of B.bassiana with high virulence against the larvae of H.assulta were screened.The virulence of B.bassiana strain Bb062,which was newly isolated and identified from the natural pathogen of B.bassiana in the field,and B.bassiana Bbr72 strain preserved in the laboratory were measured together.It was found that the virulence of B.bassiana strain Bb062 to the third instar larvae of H.assulta was the highest,and the virulence of B.bassiana strain Bbr72 was second.LC50 were 1.82×10~7 and 3.16×10~7 spores/m L,respectively.The pathogenicity time of Bb062 and Bbr72 to H.assulta larvae was significantly shorter than that of other strains,with LT50 of 4.72 d and 6.86 d,respectively.The cumulative corrected mortality of larvae treated with Bb062 and Bbr72 solution for 10 days was 91.07%and 75.11%,respectively.These results indicated that the two strains of B.bassiana had great application potential in biological control of H.assulta.2.The safety of the highly virulent B.bassiana strain Bbr72 against Trichogramma dendrolimi Matsumura and the synergistic effect of their synergistic application on the eggs of H.assulta were clarified.The results of bioassay showed that the five strains,including the highly virulent strain Bbr72,had no infection to Trichogramma;It was found that among the different strains tested,strain Bbr72 had the strongest virulence to Trichogramma eggs.The parasitism rate experiment showed that there was a certain density limit on the parasitism of Trichogramma to the eggs of H.assulta.The higher the density of Trichogramma,the higher the parasitism rate of eggs,but when it exceeded the threshold,the parasitism rate tended to be stable;The collaborative application experiment shows that the application order of the two biological control methods will have a certain impact on the final control effect.After inoculating B.bassiana,the combined application of them made the egg death rate reach 79%;After releasing the red eyed bee first,the egg death rate of the two combined application was94%;The egg death rate of Trichogramma and B.bassiana was 98%.Whether inoculating B.bassiana first or releasing Trichogramma first,the control effects of both are better than that of the two alone.3.The synergistic effect of the synergistic application of matrine and B.bassiana on the pathogenicity of H.assulta larvae was clarified.The toxicity test showed that among the five plant-derived pesticides,0.5%matrine had the highest toxicity to the third instar larvae of H.assulta,and the cumulative mortality of the third instar larvae of H.assulta after treatment with 125 mg/L water agent was 100%.The plate confrontation experiment showed that 0.5%matrine and 1.0×10~8 spores/m L B.bassiana was compatible.The spores of B.bassiana strain Bb062 were inoculated in sday medium supplemented with 125 and 62.5 mg/L 0.5%matrine.The colony diameter of B.bassiana Bb062 was not different from the control without 0.5%matrine;The results of laboratory bioassay showed that matrine and B.bassiana had obvious synergistic effect on the control of tobacco green insect.The concentration is 1.0×10~8、1.0×10~7、1.0×10~6 spores/m L B.bassiana Bb062 emulsion suspension was mixed with 62.5 mg/L and 125 mg/L 0.5%matrine respectively,the toxicity of the mixture to the 3rd instar larvae of H.assulta was greater than that of single spray.The field control effect verification results were consistent with the indoor bioassay results.4.To clarify the changes of protective enzyme activity in different instar larvae of H.assulta infected by B.bassiana strain Bb062 with different inoculation methods and concentrations.B.bassiana infection can activate and then inhibit the protective enzyme in H.assulta,and the activity of protective enzyme in young larvae changes more rapidly.The activities of SOD and CAT in different inoculation methods were higher than those in the control,and there were significant differences.The order of their activities was feeding method>dipping method>spray method,and the performance of each insect age was the same;After the 2nd-5th instar larvae of H.assulta were fed with strain Bb062,the changes of SOD and CAT activities of each instar larvae had peaks.The corresponding time of the peaks of the 2nd and3rd instar larvae was the same,which was 32 h and 24 h respectively,while the corresponding time of the peaks of the 4th and 5th instar larvae was the same,which was 40 h and 32 h respectively.Within 72 hours after treatment,the overall change of SOD in the larvae of the control group decreased gradually,while the activity of CAT increased gradually,and there was no significant difference between the two activities at each observation time point.The effects of different concentrations of B.bassiana on the activities of SOD and cat in the 3rd instar larvae of H.assulta were different,1.0×10~8 spores/m L and 1.0×10~7 spores/m L of SOD and CAT activities of the 3rd instar larvae of H.assulta treated with 1×10~7 spores/m L appeared at 32 h and 24 h,respectively,while 1.0×10~6 spores/m L treatment appeared at 40 h and 32 h,respectively.After infecting the larvae of H.assulta,strain Bb062 can activate the protective enzyme activity first and then inhibit it,and the change trend is related to the development stage of the larvae.5.It was confirmed that the hydrophobic protein Bbrhyd1 gene of type I regulated the surface structure,growth rate,heat resistance,hydrophobicity and virulence of the spores of B.bassiana.The Double-joint PCR method was used to construct the knockout vector,which was transformed into B.bassiana by protoplast transformation.Three target converters were obtained:ΔBBRHYD1-417,ΔBBRhy D1-458,ΔBBRHYD1-520.The results showed that Bbrhyd1 could affect the hydrophobicity of the colony and participate in the regulation of the formation of strea-like ridges on the surface of the conidia.The results of conidial growth rate and heat resistance showed that the growth rate and heat resistance of Bbrhyd1 mutant strain were higher than those of wild-type（WT）.In addition,the pathogenicity of WT and Bbrhyd1mutant against the 3rd instar larvae of H.assulta was compared by bioassay.It was found that infection mode could affect the toxicity of B.bassiana to pests.The fatality rate of WT strain was significantly higher than that of mutant strain when the body wall was directly infected,while the fatality rate of WT strain and mutant strain was significantly different when hemolymph was directly injected.6.This study proposed a new time series data analysis method PA,which is expected to be an important supplement for the analysis of differential gene expression in time series transcriptome data and deepen the understanding of the molecular mechanism of B.bassiana infection.With PA method and analyses the influence of beauveria transcriptome data of target pests based on points,four time series prediction including 868 genes in the 14 kinds of patterns,than in the past study found:（1）the overall level of both in different time period,or in a pair of waveform symmetry pattern,gene expression levels are declining;（2）In the first 48 h of B.bassiana infection,0～24 h was the most active stage of gene expression;（3）Some patterns with significant downtrends are rich in“adjustment”related functions.In addition,some patterns that may be closely related to fungus-host interactions are discussed. |
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