Identification Of Whitefly-transmitted Geminiviruses In Southern Region Of China And Analysis Of Recombinantion And Mutation

The whitefly-transmitted geminiviruses(WTGs)are widely distributed in tropical and subtropical regions,causing destructive diseases on many important crops.In China,southern provinces,such as Guangdong,Guangxi and Hainan,etc,are severely affected by the diseases caused by WTGs.The WTGs,composing many species and often emerging as mixed infection in the field,are prone to generate new species or strains through genetic recombination and mutation.These new species or strains,with higher pathogenicity and wider host range,may cause epidemic or outbreak of viral diseases.In this study,the species,molecular characteristics,genetic population,pathogenicity,and recombinantion and mutation induced mixed infection of WTGs from Guangdong,Guangxi,Hainan,Yunnan and Hunanprovinces were dissected.The main results are as follows:(1)The occurrence and geographical distribution of the disease caused by WTGs in southern provinces,China were clarified.Eight new diseases were discovered.The disease caused by WTGs has occurred on 21 species of plants in 30 cities or counties in Guangdong,Guangxi,Hainan,Yunnan,and Hunan provinces.The symptoms mainly include leaf yellowing and curling,leaf curling,dark green and swollen veins,yellow veins,and yellow mosaic leaves etc in field.The disease incidence in fields varied greatly difference depending on the geographical location,the type of crop and growth period of crop.The incidence was high in the later stage of the crop in general field.Among30 cities and counties surveyed,tomato yellow leaf curl disease,okra leaf curl disease,pumpkin leaf curl disease,and sweet potato leaf curl disease were the common,which disease incidence were 5～100%,3～100%,3～30% and 3～10% respectively.The diseases cauced by WTGs were first founded on Malvaviscus arboreus,Hibiscus cannabinus,Ipomoea triloba,Passiflora edulis,Pouzolzia zeylanica,Acalypha australis,Mikania micrantha,Lycianthes biflora plants.(2)The specific species and new hosts of WTGs were identified.Nineteen species of WTGs were identified on samples of 21 species plants,namely ToLCTwV,TYLCV,PaLCuCNV,AYVV,SiLCuV,CLCuMuV,SLCCNV,SPLCV,SPLCGV,AYVCNV,EYVV,SYVV,MLCuGd V,Ma YMV,RaMoV,SiYVVV,PouGMV,PouMGDV,LyYMV.PouGMV,PouMGDV and LyYMV were tentative new species.A novel betasatellite(MLCuGd B)was identified.Three new host of CLCuMuV,namely Malvaviscus arboreus,Hibiscus cannabinus,Passiflora edulis were discovered.One new host of SiLCuV,TYLCV,SPLCV,PaLCuCNV,namely Lycopersicon esculentum,Mikania micrantha,Ipomoea triloba,Acalypha australis was discovered.(3)The molecular characterization of three novel WTGs and a novel betasatellite were identified.PouGMV genome was 2 723 nucleotides(nt)in size and encoded seven ORFs.The nucleotide sequence shared the highest identity with AFSP6 d and AFSP8 d isolates of AYVV at 80.5%.No DNA-B component and satellite was detected.PouMGDV genome was 2 739 nt in size and encoded six ORFs.The nucleotide sequence had the highest identity with PouGMV-TY01 at 86.7%.No DNA-B component and satellite was detected.The DNA-A of LyYMV was 2 752 nt in size and encoded seven ORFs.DNA-B of LyYMV was 2 704 nt in size and encoded two ORFs.The common region(CR)between DNA-A and DNA-B was 191 nt long.The DNA-A sequence shared the highest identity with ToLCSuV-FI09No1 at 80.2%.The DNA-B sequence shared the highest identity with Pep LCIV-[Tomato2] at 76.3%.MLCuGd B was 1 344 nts in size,single-stranded circular,and contained A-rich region,SCR,and βC1 gene on the complementary strand.Its nucleotide sequence shared the highest identity with ToLCPB-Laguna1 at 71.9%.Based on the ICTV guidelines for Begomovirus and Betasatellite demarcation,PouGMV and PouMGDV were two novel monopartite begomoviruses.LyYMV was a novel bipartite begomovirus.MLCuGd B was a novel betasatellite.(4)The population genetic characteristics of five WTGs that harmed tomato in China were analyzed.The population genetic characteristics of ToLCTwV and TYLCV that harmed tomato in China were relatively conserved and stable.The nucleotide sequence identity was over98% during isolates from China.The population of PaLCuCNV had certain variations.The nucleotide sequence identity was 91.3～99.5% during all isolates of PaLCuCNV.The population of AYVV had large differences.The nucleotide sequence identity was87.0～99.5% during each isolate.AYVV-SY08 shared the highest identity with Sanya isolated from papaya in Hainan province at 99.5%.In this study,SiLCuV was first detected on tomato plants.The nucleotide sequence identity was 94.5～98.1% between Tomato isolate and other isolates of SiLCuV,with the highest identity at 98.1% to Hn57 isolate.(5)The infectious clones of four WTGs were constructed,the infectivity and symptoms on Nicotiana benthamiana and Lycopersicon esculentum of four viruses were examined.Infectious clones of ToLCTwV,TYLCV,ToLCGuV,and SiLCuV,were successfully constructed.The infectious clones were inoculated to N.benthamiana and L.esculentum by agro-inoculation.The infectious clones of ToLCTwV,TYLCV,ToLCGuV,SiLCuV could infect N.benthamiana plants.The inoculated N.benthamiana plants displayed shrinking,leaf curling etc.ToLCTwV,TYLCV,ToLCGuV could infect L.esculentum plants.The inoculated plants displayed shrinking,leaf yellowing,leaf curling etc.(6)The trends of recombination and mutation on three WTGs in mixed-infection tomato plants were initially explored.The genome sequence of TYLCV,ToLCGuV and ToLCTwV could be mutated or recombined in mixed infection tomato plants.With the extension of the mixed infection time,the variants and recombinants became more abundant.The missed mutants were found at 6 months of mixed infection,and recombinants were found at 30 months of mixed infection.One of the recombinants has reached the new species demarcation.This study indicated that geminiviruses could form a new strain or new species finally by mutations such as sequence deletion,mutation,and recombination in mixed infection plants.This study clarified the occurrence and geographical distribution of WTGs disease in Guangdong,Guangxi,Hainan and other provinces,identified specific WTGs species,discovered three novel WTGs,a new satellite,seven new hosts of WTGs,further cleared the genetic characteristics and pathogenicity of WTGs that infected tomato,explored the trends of recombination and mutation of geminiviruses in complex infection tomato plants.The results laid the foundation for comprehensive prevention and control of the WTGs diseases,and provided scientific basis for predicting the prevalence of tomato yellow leaf curl disease and controlling the outbreak of recombinants.