First Report of Apple Rubbery Wood Virus 2 Infecting Pear (Pyrus spp.) in China

Abstract

Apple rubbery wood virus 2 (ARWV-2) is a recently identified phenuivirus-like virus that possesses a tripartite negative-sense single-stranded RNA genome and is associated with apple rubbery wood disease (Rott et al. 2018). Another phenuivirus-like citrus virus A was recently reported to be able to infect Pyrus communis (Svanella-Dumas et al. 2019). In spring 2017, a leaf sample of a P. pyrifolia cv. Cuiguan tree (ID: S17-E2) showing a mosaic disease-like symptom was subjected to a high-throughput sequencing (HTS) assay. Library preparation from rRNA-deleted total RNA and sequencing were performed by Biomarker Biology Technology (Beijing, China) with the Illumina HiSeq 50 Cycle Single Read Sequencing version 4 platform. Analysis of the sequence data was performed with CLC Genomics Workbench version 10.1.1 (QIAGEN). BLASTn and BLASTx searches against the NCBI GenBank database allowed the identification of three assembled contigs sharing 96 to 100% nucleotide (nt) identities with the L, Ma, and Sa RNAs of the ARWV-2 genome (Rott et al. 2018). Additionally, two known pear-infecting viruses (apple chlorotic leaf spot virus [ACLSV] and apple stem pitting virus [ASPV]) were also detected in the sample. Then, a primer pair SF/SR (5′-ACAAGGCAGTAGTTATTATCAGCAA-3′/5′-TTCTGCAACTAACTTCAAGGCTG-3′) was designed based on the contig sequence matched to the viral Sa segment. A reverse transcription PCR (RT-PCR) product with a size of 486 bp was produced from the sample and shared 99% identity with the corresponding sequence of ARWV-2, confirming the presence of ARWV-2 in the sample. Furthermore, the partial L (6,740 nt, accession no. MN163133), Ma (1,593 nt, accession no. MN163134), and Sa (1,363 nt, accession no. MN163135) segments of the ARWV-2 were amplified from S17-E2. They shared about 97, 97, and 95% nt identities with the corresponding segments of ARWV-2 R12, respectively. To understand the infection status of the virus in pear trees, 65 pear leaf samples, including 49 P. pyrifolia samples and 16 P. bretschneideri samples, were randomly collected from commercial orchards in Fujian, Jiangxi, Hubei, and Shandong provinces and were subjected to RT-PCR detections for ARWV-2. In total, 24 (36.9%) samples, including 20 P. pyrifolia samples and four P. bretschneideri samples, were positive for the virus, suggesting the common presence of ARWV-2 in Chinese pear trees. The obtained amplicons shared about 99% nt identities, indicating a low sequence diversity among the viral isolates. In August 2018, five 1-year-old seedlings of P. betulifolia were individually double grafted with a bud from the ARWV-2-infected S17-E2 as an inoculum and a bud from virus-free P. communis A20 as an indicator. Five plants mock inoculated using healthy pear buds were used as negative controls. In June 2019, chlorotic spots and vein yellowing, which are indicative of ACLSV and ASPV infections in A20 (Nemeth 1986), were observed on indicator plants inoculated with S17-E2 buds. RT-PCR tests using primer pair SF/SR revealed that three out of the five S17-E2 bud-inoculated A20 plants were positive for ARWV-2. These RT-PCR products shared 99.4 to 100% identities with the corresponding ARWV-2 sequence from HTS. The results demonstrated the infectious nature of ARWV-2 in pear plants. Further surveys are needed to understand the effect of ARWV-2 on pear growth. To our knowledge, this is the first report of ARWV-2 infection in pear. The results extend our knowledge of the host ranges of the virus.