Molecular Identification And Pathogenicity Of Geminiviruses Infecting Euphorbia Pulcherrima In Fujian China

Geminiviruses are characterized by their geminate virion particles and circular single-stranded DNA(ssDNA) genome.They infect both monocots and dicots,and have caused significant losses in many economically important crops.It has become a serious threat to crops and weeds in South China since 1990s.This study was carried out to identify begomoviruses in Euphorbia pulcherrima and construct their infecting clones.From one plant of Euphorbia pulcherrima beside the schoolyard of Affiliated Middle School of Fujian Normal University,we have collected and studied samples during three consecutive years.According to the year of collection,the samples are named as FZ06,FZ07 and FZ08.Firstly,PCR detection was carried using universal primer pair PA/PB for begomoviruses to amplify partial sequence of virus genome after the totle DNA of the plant was extracted.The PCR fragment was cequenced.According to this cequence,primers were designed to amplify the full-length DNA-A,and then,PCR products were cloned and sequenced.At the same time,the universal primer pair ofβ01/β02 and universal primers of PCRc1,PBL1v2040 and PrBV1855 were used to amplify the satellite DNA-βand the DNA-B, respectively.The results of sequencing indicated that threre were FE01,FE02 and FE03 three begomoviruses mixed-infection in FZ06,and there were no detection of DNA-B and DNA-β. At the same time,there were only FE01 and FE03 mix-infection in FZ07 and FZ08 with a small circular molecule DNA of 899bp by DNA-βprimer.The complete nucleotide sequences of DNA-A of FE01,FE02 and FE03 were determined,and they contain 2751,2754 and 2733 nucleotides,respectively.Comparisons show that the total DNA-A of FE01,FE02 and FE03 have the highest sequence identity with that of Euphorbia leaf curl Guangxi virus-[China:Guangxi 35-1:2002] (EuLGxCV-[CN:Gx35-1:02]),Ageratum yellow vein virus-Taiwan[Taiwan:Tainan:1999] (AYVV-TW[TW:Tai:99]) and Papaya leaf curl Guangdong virus-[China:Guangdong2:2002] (PaLCuGuV-[CN:Gd2:02]),and the max identities were 89.9%,90%and 97%,respectively. There were no sequence that can match with E-Sat-DNA in NCBI.In order to investigate the role of FE01.in pathogenicity,infectious clone of FE01-DNA-A was constructed.Infectivity assay showed that FE01-DNA-A alone could systemically infect Nicotiana benthamiana,Nicotiana tabacum Xanthi nc to produce with typical symptoms including vein swelling,severe leaf curling in these host plants.Southern blot analysis indicated that the infectious clone of FE01-DNA-A could replicate and accumulate in host plant successfully.