Biodiversity And Biogeography Of Bemisia Tabaci And Begomoviruses In Pakistan

The whitefly,Bemisia tabaci(Gennadius)(Hemiptera:Aleyrodidae),is a cryptic species complex distributed worldwide.Whitefly poses a serious threat to agriculture production not only because they damage host plants directly by sap sucking but also because they transmit one of the most threatening groups of plant viruses i.e.,begomoviruses(Geminiviridea),which comprise more than 322 species and cause various diseases of dicotyledonous crops in temperate and tropical regions of the world.In Pakistan,begomoviruses has caused huge economic losses.For example,the cotton leaf curl disease(CLCuD)caused by several begomoviruses destructed the cotton industry of Pakistan during 1990s.Even now,CLCuD is responsible for 30%yield loss of cotton every year.Despite the significant economic importance,nation-wide information on the population structure of whiteflies and begomoviruses is not available yet.In this study,whiteflies was sampled from 42 locations during 2016-2017 from all the four provinces of Pakistan,namely,Punjab,Sindh,Khaiber-Pakhtun-Khawa and Baluchistan,respectively.Mitochondrial cytochrome oxidase I(mtCOI)gene sequencing was performed to determine the whiteflies genetically,with at least two,mostly three,whiteflies from each location.Degenerative PCR was used to identify the begomoviruses carried by the whiteflies.After that,sequence and phylogenetic analysis was conducted to understand the population structure of whiteflies and begomoviruses in Pakistan.Regarding the species identification of B.tabaci,the 124 mtCOI sequences obtained in this study revealed the presence of five different cryptic species in Pakistan,namely Asia ?-1,Asia ?-5,Asia ?-7,Asia ?-8 and MEAM-1,respectively.Among them,Asia ?-1,which was previously reported from a few areas in the country,was now found to be prevalent all over the country covering 88.7%of all the sequenced samples.Punjab province had the greatest diversity of whiteflies with four cryptic species identified(Asia ?-1,Asia ?-5,Asia ?-7 and MEAM-1),while Sindh had two(Asia ?-1 and Asia ?-8)and the other two provinces had only one species(Asia ?-1).Asia ?-8,which was not previously identified in Pakistan,was detected in Sindh in this study.Phylogenetic analysis showed that the pairwise distance for the five cryptic species identified ranged between 0.35 and 19.54%.The maximum intera-specific nucleotide variation for mtCOI sequences was found for Asia ?-1 at 2.55%,followed by Asia ?-7 at 1.54%and MEAM-1 at 0.35%,respectively.The greatest pairwise mean distance for Pakistan B.tabaci mtCOI sequences was observed for Asia ?-1 clade(0.89%)followed by Asia ?-7 at 0.61%and MEAM-1 at 0.16%,respectively.Regarding the identification of begomoviruses carried by the whiteflies,BLASTn analysis using the 177 sequences(about 570-bp in length)obtained in this study revealed 14 distinct begomovirus species,including five which were not previously reported in the country namely Corchorus yellow vein virus(CoYVV),Tomato leaf curl Kerala virus(ToLCKeV),Tomato leaf curl Karnataka virus(ToLCKV),Tomato enation leaf curl virus(ToELCV)and Tobacco curly shoot virus(TbCSV),respectively.Putative novel strains of CoYVV and Chilli leaf curl virus(ChiLC V)showing less than 90%identity with the previously available taxa were also identified.Diverse species of tomato infecting begomoviruses including ToLCKeV,ToLCKV,ToELCV,Cherry tomato leaf curl virus(CToLCV),Tomato leaf curl Palampur virus(ToLCPMV)and Tomato yellow leaf curl virus(TYLCV)were found scattered across the country.The greatest number of begomoviruses per single site was detected in Sindh where up to five different begomovirus species were identified from the same cropping field.Cotton leaf curl Multan virus-Rajasthan(CLCuMuV-Ra)was found prevalent at all the cotton growing areas of the country.Alternanthera yellow vein virus(AYVV)was also identified in patches from various locations from all the four provinces of Pakistan.Some begomoviruses were only identified at one or a few sites in different provinces.These include TbCSV identified in southern Punjab,Hollyhock yellow vein mosaic virus(HoYVMV)and Pedilanthus leaf curl virus(PedLCV)identified in central Punjab and Papaya leaf curl virus(PLCV)which was identified from Papaya growing areas of Sindh.Management of whiteflies is challenging because of its high fecundity,polyphagus nature and high resistance to various insecticides.Likewise,management of begomoviruses is also a challenge due to their continuous evolution and emergence of new strains.The begomovirus-B.tabaci interactions lead to continuous research activities regarding management of both virus and its vector,thus opening exciting new horizons to formulate potential control strategies to ensure a disease-free cropping environment.This study is the first attempt to characterize the population structure of whiteflies and begomoviruses at a national level in Pakistan.The data reported here should be useful in the developing control measures against whitefly and begomoviruses in this country.