Screening And Preparation Of Highly Toxigenic Strains Of Beauveria Bassiana

Holotrichia diomphalia is a larva of the Coleoptera Scarabaeoidae.Because of its characteristics of eating habits,food intake,concealment and lag,it often inhabits the underground,causing severe damage to roots and the crop yield loss or even crop failure,especially in eastern coastal areas such as Anhui,Jiangsu,Shandong,and Henan.Therefore,it is recognized as a major soil-borne pest which is difficult to predict and control at home and abroad.In recent years,the use of a large number of chemical pesticides has caused pests to produce severe drug resistance,causing serious pollution to the ecological environment and a serious threat to human health.However,the production and development of new insecticides require much money and long time.Therefore,the selection of highly virulent strains as an industrial strain for the development of new insecticides is an important issue for the prevention and control of Holotrichia diomphalia.In this paper,the virulence assay of B.bassiana against Holotrichia diomphalia was used as a breakthrough to explore the possibility of using biocontrol fungus to control Holotrichia diomphalia.Firstly,five strains of potentially virulent strains with high virulence were screened out by collecting corpses from different regions.Bioassays were performed and the virulence of these five strains of B.bassiana to larvae was measured in the laboratory.The dominant strains were screened as production strains in the development of anti-bacterial agents.On the basis of single factor,granule preparations were made by using spore powders of high-yield strains such as spore powder,carriers,stabilizers to be optimized and selected.From the above studies,the possibility of the use of B.bassiana in the prevention and treatment of pests was initially explored,which provided a reliable experimental basis for improving the application value and actual control effect of B.bassiana granule preparations.The main findings are as follows:?1?For the sequence comparison of five strains combined with morphological characteristics,this results showed that all 5 strains belonged to the B.bassiana strain,and the DNA-ITS base sequence of the BbQLU1 strain was 572 bp.The database of B.bassiana has 99.21%in the homology.?2?The laboratory virulence test showed that the BbQLU1 strain was treated at a concentration of 1×10⁸ CFU ml^-1 suspension and cultured at room temperature.The corrected mortality rate of the tested larvae reached 98.1%on the 35th day,and it's LT₅₀is 22.15 d.For GC-MS analysis of the toxin components of the B.bassiana fermentation broth,the material with a toxic effect on fleas was determined.The relative retention time of 2-piperidone?peak III?was 11.698 min.The retention time of methyl m-methylbenzoate?peak VI?was 12.066 min.?3?The kaolin,beet powder,attapulgite,talcum powder,and calcium bentonite used in this study were used as carriers,it resulted in the GT₅₀0 of 5.97 d and the LT₅₀0 of29.20 d,which beet powder had significant differences among them.Secondly,in the selected trehalose,tyrosine and unequal ratio between them as activator,the trehalose and tyrosine ratio of 3:1 can have a significant difference in them,which are the GT₅₀0 of5.87 d and the LT₅₀0 of 33.26 d.Lignin,sucrose,nano-ZnO,ascorbic acid and nano-TiO₂were used as stabilizers,nano-ZnO had significant difference among them,which are the GT₅₀0 of 5.98 d and the LT₅₀0 of 28.76 d.On the basis of single factor,we tested the auxiliary agents through three factors and three levels in the Box-Behnken experiment.It was determined that the content of adjuvants of Beauveria bassiana granules preparation was:carrier is 82.0%,promoter is 1.9%and stablizer is 4.8%.?4?The B.bassiana preparations prepared in this article are dry and loose particles with a relatively uniform texture.And it safely has no special pungent odor and purple-red appearance.By variance analysis(F_?2,28?=190.1,P<0.01),the viability of the B.bassiana granules differed significantly under different temperature conditions,indicating that they exhibited better performance at low temperatures on the storage effect.