Study On The Diversity Of Fungi Symbiotes In White Backed Planthopper And Its Sensitivity To Different Fungicides

Endosymbiont fungi play an important role in growth,development and reproduction of insect and can be used as new targets to effectively prevent pests.To clarify the species,abundance and function of symbiotic fungi in the White-backed planthopper(WBPH)can lay a theoretical foundation for further research.Using ITS amplicon high-throughput sequencing technologies comprehensively analyzed the diversity of endosymbiont fungi in different growth period of WBPH.We compare the difference among three methods(ITS amplicon high-throughput sequencing,Denatured gradient gel electrophoresis and the metagenomics technology).The functional of symbiotic fungi in WBPH female adult was studied by metagenomic technique.The inhibition effect of five fungicides on WBPH and its symbiotic fungi was investigated.The sensitivity of different species of symbiotic fungi to different fungicides was determined by fluorescence quantitative PCR.The growth curve of a in vitro symbiotic fungus and its sensitivity to different fungicides were studied.The results are as follows.(1)The structure and diversity of the symbiotic fungi of the WBPH at different stages were identified by ITS amplicon high-throughput sequencing.A total of 5 phyla,15 classes,21 orders,31 families,37 genera and 47 species were identified.The species with the highest abundance in each taxonomic order were Ascomycota(93.23%-99.91%),Sordariomycetes(84.02%-99.50%),Hypocreales(84.00%-99.50%),Ophiocordycipitaceae(76.28%-99.18%),Hirsutella(75.75-98.69%)and Hirsutella Proturicola(75.75-95.76%).The diversity of female adult symbiotic fungi of WBPH was also detected by DGGE technique,and a total of 12 fungi were detected.Only the Cladosporium halotolerans was identified in the two methods.(2)Based on metagenomic techniques,the symbiotic fungi species annotation and functional gene analysis of WBPH female adult were obtained.The symbiotic fungi species consisted of 11 phyla,31 classes,65 orders,127 families,205 genera and 323 species.Compared with the annotation results of ITS amplicon,13 genera and 5 species were identified.Meanwhile,the difference in species abundance was compared.ITS amplification results showed that only Hirsutella Proturicola had a higher abundance.However,the results of metagenome showed that Hirsutella Proturicola was not the dominant species.Ophiocordyceps sinensis was 11.54%.(3)The results of KEGG database annotation showed that there were 3294 Unigens in total.The number of metabolic pathway of metabolism genes is most.It is mainly distributed in Carbohydrate metabolism,Carbohydrate metabolism,overall Overview and Amino acid metabolism.The annotation results of eggNOG database showed that the most annotated gene was Function unkown,followed by Posttranslational modification,protein turnover,Translation.CAZY database annotations revealed that the number of genes in the CAZY database was higher than that of Glycosyl Transferases,followed by Glycoside Hydrolases.-Cal:The least functional genes noted are Carbohydrate Esterases.(4)We then fed WBPH with rice plants treated with different fungicides[70%propineb wettable powder(WP)(PR),70%propamocarb hydrochloride aqueous solution(AS)(PH),25%trifloxystrobin and 50%tebuconazole water-dispersible granules(WG)(TT),40%pyrimethanil suspension concentrate(SC)(PY),and 50%iprodione SC(IP)]and evaluated their effects on YLS abundance and WBPH survival rate.The fungicide IP show the significant inhibitory effect on symbiotic fungi in WBPH.The abundance of two symbiont fungi(Ascomycetes symbiotes and Cla-like symbiotes)was significantly reduced upon exposure to 50%IP.The counts of Ascomycetes symbiotes,the most abundant YLS species,were also suppressed by the other fungicides tested.In conclusion,50%IP was the most effective fungicide,reducing YLS abundance and WBPH survival rate under controlled conditions,suggesting its potential use to control WBPH.(5)A strain of symbiotic fungi was obtained from in vitro culture.By the identification of 18s-its rDNA molecular sequence,the properties of ITS properties compared with those of Moesziomyces sequence were found to be 100%.So the in vitro symbiotc fungi was named as Moe-like Symbiotes.Meanwhile,the growth curve of Moe-like Symbiotes was measured,and the sensitivity of Moe-like Symbiotes to five fungicides was detected by antifungal loop experiment.