| China is the first largest potato producer in the world. Between1980and2012, potato ranks fourth among the most important food crops in China, potato yields have a significant increase from9.7to15.2t/ha. However, potato yields in China are low relative to world levels, comparing with USA, Germany and Egypt average34,31and21t/ha, respectively. And potato viral diseases were considered as a major factor for potato production, for viruses infections have led to potato degeneration with yield reductions of from15%to90%. Regarding to potato carlaviruses, the incidence of PVS and PVH infecting potatoes is very high in China, as we know; mixed infections between PVS with other viruses can dramatically increase the severity of symptoms which further reduce potato yields. Most researchers only focus on development of appropriate diagnostic and control measures. However, studies on characterization of PVS strains, new host and construction of PVH infectious cDNA clone have not been reported.During2010-2014, incidence and distribution of potato viruses in some provinces of China was conducted. Virus disease-like symptoms in potato plants were sampled from different provinces of potato fields and were detected for seven viruses by RT-PCR and western blotting assays. Investigation of the incidence of the viral infections counts for51.7%in potato plants. Potato virus X, S and H are the most prevalent potato viruses sharing32.4,36.6and12.1%, respectively. Potato virus Y, Potato leaf roll virus, Potato virus A and Potato virus M were found in7.9,6.8,3.0and1.8%, respectively. In addition, PVS was widespread during survey in single, double and mixed infections sharing7.5,20.4and7.2%, respectively.Potato virus S (PVS, Carlavirus) has been divided into two strains:Chenopodium non-systemic (Ordinary:PVSo) and Chenopodium systemic (Andean:PVSA or PVSCS) according to the reaction on Chenopodium spp. In my study, we showed that there are three PVS isolates:PVS ordinary isolate collected from Yunnan (PVSo-YN), and two Andean isolates collected from Shandong (PVSCS-SD) and from Heilongjiang (PVSCS-HL) were identified by host reactions, RT-PCR and western blotting. In Chenopodium spp. phenotype screening of the Shandong (PVSCS-SD) and Heilongjiang (PVSCS-HL) isolates revealed localized infections followed by systemic infections, but the Yunnan isolate (PVSo-YN) only exhibited with localized infection. Of these isolates, only PVSCS-SD infected tomato plants systemically. Subsequently, complete genome sequence of PVSo-YN isolate and partial sequences of PVSCS-HL covering TGB1(ORF3) to the3’ poly A terminus (2573nts) were determined. The PVSo-YN genome contains six overlapping ORFs and consists of8,488nucleotides (nt), excluding the3’poly (A) tail. PVSo-YN shares nt sequence identities of93-97%in all the ORFs of three PVSo strain isolates, whereas this isolate has lower (77-90%) identities with the two PVScsstrains, except for ORF2where they share95-97%identity. On the other hand, PVSCS-HL partial sequence shared nt and aa sequence identities of83-97%and88-97%with PVSo-YN strain. Amino acid alignments showed that PVSCS/O strains with different positions could be characterized the relationship between systemic and localized infections on Chenopodium. Phylogenetic tree analyses based on nucleotide and amino acid sequences also showed that PVSO-YN clusters with the three Ordinary strain isolates, and that the PVSCS-HL isolate form a separated cluster and also belongs to Andean isolates.Potato virus H(PVH), a proposed new member in the genus Carlavirus in the family Betaflexiviridae, was first discovered in potatoes and occurred widely in Yunnan, Guangxi, Inner Mongolia, Hebei, Liaoning, Heilongjiang and Xinjiang. Herein, the full length infectious cDNA clones of PVH (pCa-PVH-3and pCa-PVH-2) have been generated successfully by sub-cloning three or two corresponding genomic cDNA fragments into pCass-Rz vector containing double35S promoter. The clones can be inoculated to N.benthamiana and Potato plants by agro-infiltration inoculation; the infectivity was confirmed by RT-PCR detection. The PVH infectious clone will be an important tool in the study of PVH by using reverse genetic approach, such as viral gene expression and their functions, virus replication and virus-host or vector interactions in the future.In addition, Potato virus H infecting pepino (Solanum muricaturn) was identified for the first time in China. During a study of serological relationships between PVH and PVM on potatoes, potato virus H (PVH) was detected serendipitously in symptomless pepino plantlets in Beijing, grown from tissue culture stocks. To confirm the presence of PVH on S. muricatum, surveys were conducted in2012and2013in Gansu, Yunnan and Guangxi provinces and Beijing. Fruits and leaves were collected randomly from pepino plants displaying no obvious symptoms. For PVH detection, a combination of RT-PCR, genome sequencing and serological assays were used. RNAs extracted from fruits and leaves were amplified using RT-PCR with primer pairs PVHCPF and PVHCPR, and extracted samples were reacted by Western blotting with the specific polyclonal antiserum against PVH. Among the fifty plants randomly collected, fruits and leaves of nine plants tested positive for PVH. Subsequently, an RT-PCR product of the expected size (2.6kb) encompassing the triple-gene block, the capsid protein gene, and the cysteine-rich protein gene, was amplified with a specific primer pair, and then cloned into pMD19-T and sequenced(PVH-Pepino with GenBank Accession no. KF546312). Further sequence comparison showed that PVH-Pepin shared91-98%nucleotide sequence identity in the genes mentioned above with those of the reported potato isolates PVH-Ho and PVH-YN.PVH-Pepino shared deduced amino acid identity of98-99%in CP gene with PVH-Ho and PVH-YN, respectively, but only shared57-67%amino acid identities with other reported carlaviruses. Thus, latent infection of PVH on S. muricaturm was confirmed. To our knowledge, this is the first report of S. muricaturm as a natural host of PVH. Our results suggest that PVH, as a new member of the genus Carlavirus, has a wider host range than originally expected. Potatoes and pepino are crops widely grown in China. The fact that no symptoms were expressed by PVH in pepino plants (symptomless carrier) and only mild symptoms expressed by PVH in diseased potatoes makes detection and remediation of this disease more difficult. |
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