| Frankliniella occidentalis,a very important invasive pest,is becoming more and more serious because of its strong adaptability.Lecanicillium spp.are the most important pathogenic fungus and play a important role in bilogical control.To further understand the role of Lecanicillium against F.occidentalis in infection process and endogeneity in plant,the pathogenicity of Lecanicillium fungi to F.occidentalis was comprehensively described from the following aspects:classification of Lecanicillium,direct and indirect pathogenicity of Lecanicillium to F.occidentalis,defense response of F.occidentalis to Lecanicillium,genomics of Lecanicillium,genetic transformation of Lecanicillium and colonization of Lecanicillium in plants.The main results were as follows:1.Isolation,identification and evolutionary origin analysis of LecanicilliumThe phylogenetic analysis of all species of Lecanicillium was carried out by multiple genetic locus,and the disproportionation time was estimated by phylogeny.It was found that there were 8branches of the current Lecanicillium that were parallel derived and evolved with other genera of Clavicipitaceae.To solve the problem of attribution of Lecanicillium,it is obvious that more strains need to be discovered.In addition,two new species,Lecanicillium cauligalbarum and L.huhutii were identified based on phylogenetic analysis and morphological identification.Based on the comparison of the difference among single gene barcode and the combinations of barcodes,the combination of ITS and RPB1 gene barcodes could be used to identify all species in the Lecanicillium genus,which could be as quickly identify method.2.The pathogenicity of different strains of Lecanicillium to Frankliniella occidentalisThe pathogenicity of 7 strains of Lecanicillium to F.occidentalis was tested.It was verified that L.attenuatum,L.cauligalbarum,L.araneogenum and L.aphanocladii had certain pathogenicity to F.occidentalis for first time reports,but their pathogenicity were different.Among them,L.cauligalbarum was not only be pathogenic to the adult and nymph of F.occidentalis,but also significantly inhibited the oviposition of female adults of F.occidentalis.On this basis,the pathogenicity and sublethal effects of L.cauligalbarum and L.attenuatum on F.occidentalis were systematically compared.In general,the order of pathogenicity of L.cauligalbarum and L.attenuatum to each insect stage of F.occidentalis was as follows:adult>pupa>nymph.In terms of pathogenicity to adult and pupa of F.occidentalis,L.cauligalbarum showed a lower effective concentration than L.attenuatum.At higher concentrations,the lethal effect was faster than that of L.cauligalbarum.Compared with the control group,the development time of F.occidenalis treated by L.cauligalbarum and L.attenuatum were significantly shorter,and the intrinsic growth rate（rm）,limited growth rate（λ）,net reproductive rate（R0）,generation time（T）and total reproductive rate（GRR）were significantly lower than the control group,showing strong sublethal effect.The culture conditions between L.cauligalbarum and L.attenuatum were differences.On the basal culture medium,the conidium produced by L.cauligalbarum was significantly less than that produced by L.attenuatum.The sporulation of L.cauligalbarum was significantly affected by nitrogen sources,and can not use inorganic nitrogen basically.The optimal culture conditions for L.cauligalbarum were as follows:Starch 1%,yeast powder 3%,silkworm pupa powder 1.5%,potassium chloride 0.05%,dipotassium hydrogen phosphate 0.1%,magnesium sulfate heptahydrate 0.05%,ferrous sulfate heptahydrate 0.001%,agar 1.5%,distilled water 1000 m L,culture at 25°C.The sporulation yield of optimized medium was 4.5 times that of PDA medium and 15.6 times that of Shahler medium.4.Defense response of Frankliniella occidentalis to infection by L.cauligalbarum and L.attenuatumThere were significant changes of the activities of protective enzymes（superoxide dismutase,catalase,peroxidase）and detoxification enzymes（polyphenol oxidase,glutathione-s transferase,carboxylic acid lipase）,hormone contents（ecdysone and juvenile hormone）,the compositions of microorganisms（fungi,bacteria）after thrips were infected by L.cauligalbarum and L.attenuatum.The activities of SOD,CAT and POD of F.occidentalis increased to different degrees after the treatment of L.cauligalbarum and L.attenuatum.However,the changes of activities caused by two Lecanicillium were different,which showed that thrips had significantly different timing and degree of defense against these two Lecanicillium.With the infection of L.cauligalbarum and L.attenuatum,the abundance and community of fungi and bacteria in thrips changed in varying degrees,and the effects were different between two Lecanicillium.In the constructed microbe-enzyme/hormone network,12 strong positive correlations and 3 strong negative correlations were identified among 33microbe groups,4 enzyme activities and 1 insect hormone.Thus showed correlations between enzymes and microorganisms.5.The functions of surface protein of L.cauligalbarum and L.attenuatumThe sequencing and data assembly of the whole genome of L.cauligalbarum and L.attenuatum were completed for the first time.It was predicted that there were 9,596 protein-coding genes in L.cauligalbarum and 10,623 in L.attenuatum.It was found there were 330 cell surface-associated proteins of L.cauligalbarum,and L.attenuatum with 388 surface proteins by bioinformatics screening.These surface proteins were annotated to the PHI protein database,and 50 proteins were annotated to the PHI protein database for L.cauligalbarum and,while they were 53 proteins for L.attenuatum.38 kinds of same proteins were noted both in L.cauligalbarum and L.attenuatum.Among them,there were 5 kinds of proteins that could possibly improve the host virulence of L.cauligalbarum and L.attenuatum.6.The construction of genetic transformation system of Lecanicillium and function of important protein genesThe genetic transformation system was successfully constructed,and the transformation conditions were optimized:the initial agrobacterium OD660 was 0.15,the concentration of the spores of the cocci was 1×104/m L,the co-culture temperature was 22°C,the co-culture time was 48 h,and the concentration of AS was 200μmol/m L.Then,we obtained the converters that could stabilize the heredity and stimulate green fluorescence.Six surface proteins that might improve virulence were overexpressed in L.attenuatum.The bioassay results showed that the pathogenicity of all the six gene overexpressed strains was better than that of the wild strains,among which OE1 was the best one.The protein was with a chitinase structure.7.The colonize of Lecanicillium in kidney bean plant and its effect on Frankliniella occidentalisIt was found that L.attenuatum could colonize in kidney bean plant and it was speculated that it entered the plant through root hair.In addition,the plant height,fresh weight,taproot biomass and taproot length of kidney bean plant were significantly increased after the seeds treated and planted by L.cauligalbarum and L.attenuatum.Frankliniella occidentalis showed obvious repellency to the kidney bean plant treated by L.cauligalbarum and L.attenuatum.The life expectancy of F.occidentalis fed on plants colonized by L.cauligalbarum and L.attenuatum was significantly shorter than that fed on normal beans.In terms of the reproductive value of Frankliniella occidentalis,the peak values of F.occidenalis fed on plants colonized by L.cauligalbarum and L.attenuatum were also significantly lower than fed on normal beans,and the peak values of also appeared earlier than the control group. |
