Analyses Of Mixed Viral Communities In Nectarine Trees With Different Disease Symptoms

Nectarine(Prunus persica var.nectarina)is an important fruit crop.However,nectarine can be affected by a number of viruses/viroids.In this study,we studied co-infecting viral communities using NGS technology in five individual trees of two nectarine cultivars associated with different disease phenotypes and performed comparative analyses of the potential contribution of the pathogens to disease symptoms.We identified new virus,amplified the full length of the genome and performed phylogenetic analysis of the detected virus.This study provides insight into the role of the mix viral communities infecting nectarine in host symptom development and will be seminal for further studing epidemiological features of host-pathogen interactions in nectarines.Then,we developed a mRT-PCR assay can simultaneously detect the PaLV,PeVD and NSPaV in a single reaction.The mRT-PCR protocol developed in this study is sensitive,reliable,convenient and economical.The mRT-PCR is helpful for large-scale surveys of viruses that infect peach.This study provides a theoretical basis for the prevention and treatment of nectarine virus disease.1.We analyzed viral communities in five individual trees of two nectarine cultivars with different disease phenotypes using NGS technology.Different viral communities were found in different cultivars and individual trees.A total of nine viruses/viroids in five families were identified in a single tree.To our knowledge,this is the first report showing that the most-frequently identified viral species co-infect a single individual peach tree,and is also the first report of PeVD infecting Prunus in China.Combining analyses of genetic variation and sRNA data for co-infecting viruses/viroids in individual trees revealed for the first time that viral synergisms involving a few virus genera in the Betaflexiviridae,Closteroviridae,and Luteoviridae families play a role in determining disease symptoms.Evolutionary analysis of one of the most dominant peach pathogens,PLMVd,shows that the PLMVd sequences recovered from symptomatic and asymptomatic nectarine leaves did not all cluster together,and intra-isolate divergent sequence variants co-infecting individual trees could be associated with disease symptoms.Our study provides insight into the role that mixed viral/viroid communities infecting nectarine play in host symptom development.2.We developed a multiplex RT-PCR(mRT-PCR)technique for simultaneous detection of three peach viruses: PaLV,PeVD and NSPaV using plant nad5 mRNA as an internal control.Using field samples of peach infected by these three viruses,we determined that the three primer pairs were suitable for detecting these viruses and that the optimal concentration of each primer pair was used in mRT-PCR.We also evaluated and optimized mRT-PCR parameters such as annealing temperature,detection limit using serial dilutions of RNA and cDNA et al.The newly developed mRT-PCR assay can simultaneously detect the PaLV,PeVD and NSPaV in a single reaction.To validate the reliability of mRT-PCR for virus detection,mRT-PCR was used to detect viruses in the leaves of 21 peach plants collected in Liaoning province,China,revealing single and mixed virus populations.The mRT-PCR assay developed in this study requires relatively little reagent use and is simple,reliable,convenient and cost-effective for largescale surveys of viruses that infect peach.3.We amplified complete nucleotide sequence of NSPaV isolate(named NSPaV-T04)by RT-PCR and RACE.The complete genome was 4,991 nt in length and encoded four open reading frames(ORFs).It had the same genome structures with several isolates deposite in GenBank.The complete nucleotide sequence of NSPaV-T04 had the highest similarity with the isolate NSPaV/12P42(KT273410)from America.Nucleotide and amino acid sequence analysis showed that the the domain of RdRp P1 was more variable and the domain of CP was more homologous compared with other domains.Phylogenetic analysis showed that the isolate NSPaV-T04 was most closely related to the isolate NSPaV/12P42(KT273410)from America.This is the first report of complete genome sequence of NSPaV in China.