Resistance Improvement And Effect Evaluation Of Lecanicillium Lecanii To Strobilurin Fungicides

Lecanicillium lecanii is a species of excellent entomogenous fungus,it has great control efficiency.However,it is viable organism preparations when it is applied,fungicides have fatal influence on it.So Lecanicillium lecanii can not use with chemicals such as fungicides meanwhile in the field.Aim at the problem of Lecanicillium lecanii is sensitive to fungicides,must improve strains of actual production which is applied to biological control.This study screened resistance isolates to strobilurins fungicides,and took advantage of resistance gene to construct resistance engineered strains of Lecanicillium lecanii,and increased resistance to strobilurins?kresoxim-methyl?.The significant results have been achieved as follows.1.Screening resistance isolates.F-2 isolate was selected from phytopathogen which separation and purification screened resistance isolates of high level to kresoxim-methyl by spore germination.The EC₅₀ of F-2 isolate to kresoxim-methyl far and away greater than 2500?g/ml.By sequencing and authenticating ITS sequence,as a result,the high level of resistance F-2 isolate is Cladosporium tenuissimum.2.Cloning resistance gene.To clone resistance gene Cytb from F-2 isolate which has high level resistance to kresoxim-methyl,and clone Cytb gene from wild type sensibility B-3 isolate,to make a comparative analysis of both Cytb gene.To amplification full cds sequence of Cytb gene by ordinary PCR and 3'RACE PCR.After sequence alignment of both,the result is basic group mutation at the 429site of nucleotide sequence of F-2 isolate,make glycine sport alanine at 143 site of amino acid sequence?G143A?.So F-2 has high level resistance to kresoxim-methyl,the consequence is correspond with resistance mechanism of strobilurins fungicides.3.Constructing expression vector.To construct fungus ATMT expression vector including resistance mutant Cytb gene.On the basis of pBHt2 carrier,to connect both fungus expression strong promoter PtrpC and resistance Cytb gene.Then,to confix pBHt2 and target fragment which both double digestd with KpnI and HindIII by T4 DNA ligase.So constructing pBHt2 recombinant plasmid contain resistance mutant Cytb gene.4.Transformation of Lecanicillium lecanii by ATMT.Agrobacterium-mediated transformation of pBHt2recombinant plasmid,then,transformation of Lecanicillium lecanii by ATMT with co-culture of AGL-1and conidium of Lecanicillium lecanii,to transfer exogenous Cytb gene into Lecanicillium lecanii,and get transformation strains contain Cytb gene and verify them.5.Verify transformations.Genome DNA of transformations as template,PCR amplification target fragment,prove that exogenous Cytb gene have been successfully imported into Lecanicillium lecanii.cDNA of transformations as template,RT-PCR amplification target fragment,prove that exogenous Cytb gene have been successfully conducted transcription reaction into Lecanicillium lecanii.To measure resistance to kresoxim-methyl of transformations,and the resistance increased significantly than CA19,the highest resistance is 7.7 times,it illustrated that exogenous Cytb gene have been successfully translated protein and expressed into Lecanicillium lecanii.This far,engineered strains of Lecanicillium lecanii have significant resistance to kresoxim-methyl were successfull,which could improve ecological adaptability and applicability of Lecanicillium lecanii,and could have sound applied prospect.