| Entomogenous fungi with its wide host, not easy to produce resistance, simple production process, environment friendly and other advantages becomes a research hotspot of biological control in domestic and foreign. Our country is rich in resources of entomogenous fungi, but acaricidal fungi is rarely reported. Two strains of pathogenic fungi were isolated fromPanonychus citri (McGregor) collected from the field, and were identified as Sarocladium implicatum (CQBBW8 ) and Cladosporium cladosporioides (CQBBW3 ), respectively. In order to strengthen the understanding of these two strains and lay a theoretical foundation for the development of creatures acaricide, the two strains were studied in terms of acaricidal activity, biological characteristics, insecticidal mechanism and genetic transformation in this paper. The results were as follows:1. The LC50 value of CQBBW8 strain against P. citri was 1.2×106 spores/mL, and the LT50 value was 5.1759 d, and the acaricidal activity was better than that of CQBBW3 strain (4.66×10g spores / mLand 6.2353 d). The study of biological characteristics found that the optimum culture medium for growing and sporulation of CQBBW8 strain was PDA and SEA medium, respectively, and the starch agar medium and Czapek's medium were best to CQBBW3 strain. Mannitol and Glycine were the best carbon and nitrogen sources for hyphal growth of CQBBW8 and CQBBW3 strain.The most suitable culture temperature for hyphal growth and spore germination was 25? and the most suitable temperatures for sporulation was 30? of CQBBW8 and 15? of CQBBW3. The suitable pH value of mycelial growth and sporulation of two strains was 6.0?8.0 and the spores germination rate was higher in the strong acid environment. UV had certain impact on the growth and sporulation of two strains,and had a strong killing effect to spores. In addition, two strains had a good compatibility with fungicides of cuprous oxide and five kinds of acaricides, they can be used interoperable in the fields.2. In this study, the pathogenicity of CQBBW8 and CQBBW3 strains against the 4th instar larvae of silkworm and silkworm pupa were determined by "dipping method" respectively. And the expression of extracellular protease and chitinase in the cicada slough culture medium was determined by the Folin reagent method and chitinase activity assay kit.The results showed that the CQBBW8 strain has better insecticidal effect, the mortality rate and the LT50 value of P. citri and silkworm larvae, and the LC50 value of silkworm pupa of CQBBW8 and CQBBW3 strains were 80.00%?4.3439d?9.4817×108 spore /mL and 69.99%?5.5697 d?1.26×1010 spore/mL,respectively. The changes of extracellular protease and chitinase of CQBBW8 and CQBBW3 strains in cicada culture medium showed that both protease and chitinase were enhanced, and the order of action was the same in the process of degradation of cicada, but protease was the main effect. And CQBBW8 strain was stronger than CQBBW3 strain to decompose protein and chitin of body wall by comparing the changes of protease and chitinase activity in these two strains?3. The activities of three kinds of detoxification enzymes in the P. citri were changed to different degrees after being infected by CQBBW8 and CQBBW3, respectively,the sequence of response was: glutathione-S-transferase (GSTs) , carboxylate enzyme (CarE) , acetylcholinesterase (AchE). The activity of GSTs in P. citri increased rapidly from the first day, and maximum enzyme activities caused by inoculation of CQBBW8 and CQBBW3 strains were third and fourth days respectively, and the enzyme activities were 8.13 and 6.53 U/mL prot, respectively.The CarE activity increased rapidly from the 2nd day, and the peak of enzyme activity caused by the infection of the two strains also appeared on the 3rd and 4th day respectively, the enzyme activities were 12.98?10.06 U/mLprot respectively.The AchE activity increased rapidly from the 3rd day, the highest activity were 0.1818 and 0.1120 U/mL prot respectively. The activities of the three kinds of detoxification enzymes began to decrease rapidly at the 4?5th day,and reached or below control at 6th and 7th day, indicating that the P. citri was close to death.4. The T-DN A random insert mutant library of CQBBW8 strain was constructed by Agrobacterium tume facie ns-mediated transformation (ATMT),and the functions of related genes of phenotypic mutant strains were analyzed. The results showed that the transformation efficiency of the genetic transformation system was 100?200 transformants/106 spores, and 41 mutants with large phenotypic changes were screened from the constructed mutant library containing 2800 transformants.The sequences of T-DNA insertion site flanking of 9 mutants were amplified by Hi-TAILPCR,then these sequences were compared with the genome database of CQBBW8 strain. The corresponding gene The corresponding genes were: peptide transporter (B11 transformant) , RNApolymerase ? transcription factor (B36 transformant),fumaric acid acetoacetic acid (FAA) hydrolase family (B54 transformant) , P450 (W27, W55 transformant) and functionally unknown gene(B77 transformant) and so on, their specific functions need to be further verified. |
PDF Full Text Request