Genetic Improvement Of Lecanicillium Attenuatum By RNA Interference And Its Control Effect On Diaphprina Citri(Hemiptera:Psyllidae)

Diaphorina citri is one of themost importanct pests in many citrus production regions around the world.It washarmed by piercing-sucking mouthparts,which mainly harms the citrus juices and causes tenderness.Tips withered and deformed.In addition,D.citri can also secrete a large amount of white honeydew,which adheres to the leaves,which are easy to induce Capnodium citri,affects the photosynthesis of the leaves,and weaken the tree vigor.More seriously,D.citri is one of the vector insects of Huanglongbingdisease.Therefore,controling of D.citri is of great significance to the prevention and control of HLB.At present,chemical control is stillmain method in the control of D.citri.Although chemical control can quickly reduce the population of D.citri,it is easy to cause pesticide residues,environmental pollution,pest resistance and other problems.Biological control has huge potential for development and abundant resources.L.attenuatum,is one of the important biocontrol fungus,has strong infectivity to Hemiptera pests such as D.citri.Itcan penetrate the insect wall with the help of mycelium,destroy the normal life activities of insects,compete for nutrition,and eventually cause insect death.On the basis of evaluating the fatal effect of progressive narrow L.attenuatum on D.citri,the RNAi vector of vitellogenin geneof D.citriwas constructed by molecular modification and introduced into the genome of Ceratopsis citrinus,the transgenic strains were further evaluated to provide a new method for the genetic transformation of L.attenuatum.The results are as follows:1.Infection effect of L.attenuatum on D.citriUsing spore suspensions of different scales of L.attenuatum TN002,the adult,egg and 1-5 instar nymphs were treated by immersion method and bag spray method respectively,and the mortality rate was counted to evaluate the infection effect of the strain on D.citri.As a result,it was found that the LC₅₀0 was 3.08×10⁵spore.m L^-1 after treatment of adult of D.citri with a concentration gradient spore suspension for 10 d.After treatment with 3.0×10⁸spore.m L^-1 suspension for 7 d,the corrected mortality of eggs and first-instar nymphs was 20.39%and 48.38%.The mortality of 2-5 instar nymphs declined significantly with age?P<0.05?.The results showed that L.attenuatum has a certain infectivity to D.citri of all ages,but the virulence is low.Therefore,it is necessary to transform the stains by improving the speed during infection of D.citri.2.Construction and transformation of filamentous fungal expression vectorTo more intuitively evaluate the expression of the vector in the transgenic strain,according to the principle of homologous recombination,the one-step cloning method was selected to connect the PtrpC promoter and EGFP gene fragments to the original filamentous fungalexpression vector in the laboratory,and obtained the filamentous fungal expression vector with the reported gene EGFP,named pRCy3.The transcriptome database of D.citri was screened,and the conserved sequences DcVg1a and DcVg1b of the vitellogenin gene of D.citri were found.According to the principle of homologous recombination,the forward/reverse fragments of the target gene were transferred into pRCy3 by one-step cloning Between the vector promoter and intron,and performing bacterial solution PCR,enzyme digestion and sequencing verification to obtain a filamentous fungal expression vector containing the PtrpC promoter+forward target fragment+intron+reverse target fragment+TtrpC terminator structure,Complete the construction of ihpRNA expression cassette.3.Agrobacterium-mediated ihpRNA expression cassette transformation of L.attenuatumThe freeze-thaw method was utilized to transform the successfully constructed ihpRNA expression cassette,and theAGL1 strain of Agrobacterium positive clone was verified by medium screening,primer amplification and enzyme digestion to obtain the strainof A.containing the ihpRNA expression cassette.Subsequently,using acetosyringone induced culture method.The AGL1 stain was cultured for 6 h and co-cultured with the spore suspension of L.attenuatum for about 60 h,and then transferred to hygromycin selective medium for continuous cultivation and separation and purification.The PCR detection and found that the ihpRNA expression cassette has been successfully incorporated into the genome of L.attenuatum.The ihpRNA expression cassette of the vitellogenin gene of citrus psyllid has been integrated into the genome of Ceratosporium elegans,and the genetic transformation of the strain of Cerebella esculentus has been completed.4.Evaluation of the effect of transgenic strainsOn the basis of genetic transformation,the transgenic strains were evaluated from biological characteristics,genetic stability,virulence and gene expression level.In terms of biological characteristics,the growth rate and sporulation per unit of transgenic strains La::DcVg1a and La::DcVg1b were not significantly different from those of wild-type strains,which indicated that the transformation of vitellogenin gene of D.citri had no significant effect on the biological characteristics of L.attenuatum.In terms of genetic stability,the transgenic strains can still amplify positive bands with hygromycin detection primers after continuous culture on PDA medium without antibiotics for 10generations,which shows that the Agrobacterium mediated transformation method has good stability.In the virulence evaluation,the LC₅₀ and LT₅₀ values were calculated to evaluate the virulence of the strains to the adult of D.citri after 10 d immersion.It was found that the LT₅₀0 of La::DcVg1a and La::DcVg1b were 4.67 and 4.33 d,respectively,significantly shorter than that of wild-type strains?P<0.05?;the LC₅₀ of La::DcVg1a and La::DcVg1b were 1.10×10⁴ and 5.60×10⁴ spore.m L^-1,respectively,significantly lower than that of wild-type strains and La::EGFP?P<0.05?.Compared with the wild strains,the virulence of La::DcVg1a and La::DcVg1b increased by 28.00 and 5.50,respectively.The results showed that the virulence of La::DcVg1a and La::DcVg1b was significantly increased.In the evaluation of gene expression level,the transgenic strains La::DcVg1a and La::DcVg1b were significantly reduced at 4-6 d?P<0.05?compared with La::EGFP strain,and the transgenic strains La::DcVg1a and La::DcVg1b were immersed in the spore suspension for 15 s,and then collected every 24 h for RT-qPCR.The results showed that siRNA was released in the process of infection.This study provides a new idea for control of D.citri by entomopathogenic fungi.