Evaluation Of Entomopathogenic Fungi (EPF) For The Control Of Citrus Red Mite,Panonychus Citri (Acari: Tetranychidae) And The Effects Of Lecanicillium Attenuatum–Bbzwn9

The citrus red mite（Panonychus citri）is an agricultural mite that causes severe economic damage.It has a short generation time,a high reproductive rate,and evolved resistance to several acaricides.alternative strategies including:chemical,biological,and physical.The routine method was chemical control of P.citri,for example:acequinocyl,fenbutatin oxide,hexythiazox,essential oil,pyridaben,spirodiclofen,or fenpyroximate,those ways have been triggered chemical resistance in the mites and caused some negative impact in environmental.Therefore,current research efforts have focused on this field.Predatory mites and entomopathogenic fungi（EPF）are commonly biocontrol methods.However,the application of predatory mites faces a series of issues including species and variety screening,impact of pesticides such as emamectin benzoate,lepimectin,and spirotetramat or other organism as fungi,and large-scale production and related technologies of field release.In contrast,EPF may have more advantages in controlling P.citri,and the previous studies about investigated EPF to manage P.citri are rare.Thus,this research project had selected EPF for the study.The current study investigated the ecological and molecular interactions between P.citri and EPF,focusing on systematic evaluation of EPF for the control of P.citri.In part one,the contents included bioassay of 15 fungi strains on the mites,fungal efficacy confirmation,infection observation and identification of the highest toxicity strain;in part two,the gene expression difference and immunity-related genes in citrus red mite during fungal infection were analyzed.briefly,the results provide a reference for the interaction between P.citri and EPF.1 Evaluation of EPF for control of the citrus red mite1.1 Screening and identification of fungiThe fungus that exhibited the highest bioactivity against P.citri among 15 fungal isolates tested was strain Bbzwn9,which caused a mortality rate>80%in mites in 8 d with exposure to 10⁷ and 10⁸ conidia/m L.In addition,this strain was shown LC₅₀ was 0.53×10⁶ conidia/m L,at 8 d.The fungus required high relative humidity（RH）of>70%.The mite mortality was not related to fungal hypha growth;however,it may have been related to the colony characteristics.Finally,morphological and molecular methods revealed that Bbzwn9 was similar to Leccanicillum attenuatum,a fungus that had previously been evaluated for the control of insects,especially aphid species.1.2 Evaluation and confirmation of fungal efficacyUsing Koch’s postulates,the evaluation of mite developmental stages,and steamed-mashed maize and plant pot assays,the results indicated that Bbzwn9 was a fungus that effectively controlled P.citri and showed the highest activity compared to other separated fungi（the fungi that were separated from the mite samples）.In the developmental stages test,the highest mortality rate occurred in adults（females）and the lowest in eggs.At 26°C,mortality rates of all stages including egg,larval,and protonymph stages were higher than at 22°C.LT₅₀ was 4.8 d,after expose to 10⁷conidia/m L.In addition,it was found that Bbzwn9 survived at low temperatures（about4°C）on steamed-mashed maize,especially in the dark-mode,for one month or more.Different species of tested plants（citrus and soybean）showed that plant species can affect the level of fungal control of P.citri as a result of different physical structures.1.3 Observation of fungal infectionUsing three methods,including fluorescein diacetate（FDA）,frozen cross section and lactophenol cotton blue（LPCB）,the infection processes of Bbzwn9 against P.citri in two developmental stages（eggs and adult females）were comparatively observed for successive 5 d after treatment.Meanwhile,the fungal content was detected with the specific primers of Bbzwn9 after the Bbzwn9 spray on the mite.It was concluded that Bbzwn9 germinated 2 d after conidia were sprayed on the mites and penetrated into the mite bodies within 3 d,except for on the eggs.This was because their physical structures,such as the cuticle layer,were different from those of the adult female mites.In addition,the fungal DNA concentration detection with the specific primers showed that sprayed adult female mites exhibited gradually higher DNA content with the prolong of spray treatment.This phenomenon was not found in the egg stage.2 Analysis of gene expression changes and immunity-related genes in citrus red mite during fungal infection of Lecanicillium attenuatum–Bbzwn92.1 Transcriptomic analysisDifferentially expressed genes（DEGs）were analyzed with the Illumina method by using the samples from five different treatments（control（WT）,1 d,2 d,3 d,and 4 d of spraying）.In total,11,041 unigenes had been annotated.In addition,a total of 412 new genes were annotated among total 983 new genes by the COG,GO,KEGG,eu Karyotic Ortholog Groups of proteins（KOG）,Pfams,Swiss-Prot,egg NOG,and Nr annotation databases.GO database analysis revealed that binding and catalytic activity gene numbers were the highest.A total of 12.5%of genes were annotated in the carbohydrate transport and metabolism group in GOG classification.In KEGG analysis,most expressed gene changes were found in the carbon mechanisms of the mechanism category.Among the differentially expressed genes（DEGs）,fewer up-regulated genes were observed than down-regulated genes at all treatment times.Moreover,the DEGs at the 4-d treatment had the largest number of both up-and down-regulated changes,which were expressed 2-fold more than at the 1 d treatment,and the smallest number was found at 2 d of treatment.2.2 Immunity-related genes in citrus red miteGenes annotated as hypothetical or unknown proteins in Nr annotation and with relative expression levels of FPKM<1 were filtered out.Nr annotation analysis of up-regulated genes revealed that uncharacterized protein genes were the most highly expressed genes in 1–4 d shared DEG analysis（41.67%）and analysis of DEGs for each day（1 d,2 d,3 d,and 4 d treatments）.Signal transducer,receptor activity and signaling pathways related to insect immunity system were the criteria to screen immunity-related genes in the mite.Finally,these genes with IDs of EVM0010587（NPY6R）,EVM0008503（Glu Cls）, EVMOOOO112（IGFALS）,EVM0004680（MYO313）,EVM0009298（HR3）,EVM0010387（CPG-2）,EVM0002854（NGB）,EVM0008239（Apo-D）,EVM0007645（CHPT1）,EVM0009824（CDK5）,EVM0007006（AGMO）,and New gene 1359（Cytochromb）in 2 d expression were suggested.Moreover,there were also a large number of binding function genes,and several gene showed significant expression when compared with normal treatment（WT）in hydrolase activity function.In addition to signal transducer,receptor activity and signaling pathways,binding and hydrolase activity functions may be also related to the immunity-related genes of citrus red mite.All of these findings on above could be more support the strategy of further developing several aspects of biological control（EPF）for use on P.citri in the platform of basic knowledge.