First Report of Papaya leaf curl China virus infecting Acalypha australis in China

Abstract

Papaya leaf curl China virus (PaLCCNV), a monopartite begomovirus (genus Begomovirus, family Geminiviridae), was originally reported on Carica papaya in Guangxi province of China (Wang et al. 2004). The virus has been reported to infect C. papaya, Solanum lycopersicum, Nicotiana tabacum, Corchoropsis tomentosa, Siegesbeckia orientalis, and Ageratum conyzoides. In January 2015, plants of Acalypha australis, a common and widespread weed species, were found exhibiting yellow vein symptoms in fields of Nanning city, Guangxi province. Five symptomatic leaf samples were collected from each of five diseased plants. Total DNA was extracted from the five samples with the EasyPure Plant DNA kit (TransGen Biotech, Beijing, China) and was used as a template for polymerase chain reaction with the degenerate begomovirus primers AV494 (5′-gccyatrtayagraagccmag-3′) and CoPR (5′-gangsatghgtrcadgccatata-3′). The expected approximately 570-bp fragment was detected from all five samples, indicating that A. australis plants showing yellow vein symptoms were infected by a begomovirus. Begomoviral genomes were amplified from the total DNA using rolling circle amplification (RCA) (TempliPhi kit; GE Healthcare, Buckinghamshire, UK), followed by digestion with endonucleases BamH I, EcoR I, Hind III or Pst I, respectively. Amplicons corresponding to full-length begomoviral genomic components, obtained from RCA products digested with Hind III, were gel purified, ligated into the plasmid pGEM-3Z (previously digested with the corresponding restriction enzyme mentioned above), and transformed into Escherichia coli DH5α. Three clones from each sample were selected for sequencing (Invitrogen, Shanghai, China). Sequencing results were assembled and analyzed using DNAStar software version 5.0 (DNAStar, Madison, WI). A similarity search for each sequence was carried out using BLASTn to identify related sequences in the GenBank database (https://www.ncbi.nlm.nih.gov/). Pairwise nucleotide (nt) sequence identities were determined using the Species Demarcation Tool (SDT1.0). Full-length begomoviral genomic sequences were 2,735 nt long and encoded six putative open reading frames. The cloned genomic sequences shared 99.5 to 100% nt identities with each other. One representative sequence was deposited in GenBank as the accession number KX273343, which shared 91.8 to 99.6% nt identities with isolates of PaLCCNV in the database, with the highest nt identities to PaLCCNV-G111 [CN:Gx:Age:14] (HG003651). In accordance with the threshold of 91% for begomovirus species demarcation (Brown et al. 2015), the virus associated with A. australis yellow vein disease is an isolate of PaLCCNV. According to the obtained sequences, the specific primer pairs W-F2 (5′-GTATACACGCCACTCTCGCATTG-3′) and W-R2 (5′-CTGGACAATCAAAAATCCCCTAT-3′) were designed to amplify the full-length genome and detect the presence of PaLCCNV in A. australis samples. The expected approximately 2.7-kb full-length genomic fragment was amplified from all five samples, indicating that A. australis plants showing yellow vein symptoms were infected by PaLCCNV. To our knowledge, this is the first report of natural occurrence of PaLCCNV on A. australis in China. A. australis may play an important role as a reservoir of PaLCCNV, especially during the non-crop-planting period, but additional studies analyzing a large number of plant samples from different geographical locations within China are necessary to establish the role of this host as a virus reservoir.