First Report of Prunus necrotic ringspot virus Infection of Apple in China.

China is one of the main apple-producing countries in the world with a planting area of 2.3 million ha and 41 million tons of fruit produced in 2014. During 2014 to 2015, a survey was conducted in apple orchards in Gansu, Hebei, Henan, Liaoning, and Shangdong provinces to assess the incidence of Apple chlorotic leaf spot virus (ACLSV), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV), Apple mosaic virus (ApMV), and Apple scar skin viroid (ASSVd). Shoot or leaf samples from a total of 216 trees in seven orchards were collected randomly and total RNA was extracted and tested using a reverse transcription (RT)-PCR as described by Hu et al. (2015). In RT-PCR, primer pair ILAR1/ILAR2 amplified a fragment of 204 bp corresponding to the partial coat protein (CP) gene of ApMV and Prunus necrotic ringspot virus (PNRSV) from 39 apple trees. The PCR products of six samples (FS06, FS07, FS08, HY, LD, and ZW) were cloned into the pMD18-T easy vector (Takara, Dalian, China) and sequenced. Nucleotide sequence was determined for one clone of each sample (GenBank Accession Nos. KU144872 to KU144878). BLASTn results showed that all sequences had the highest identities to CP genes of PNRSV, with 78% identity to one isolate from the United States (L38823.1) (Hammond and Crosslin 1995). To confirm the PNRSV infection in apple, a 1,487 bp fragment from RNA3 was amplified using primer pair PNRSV-RNA3-F1 (5′-AGTGTTCTATGGACGAAATGAGCCAGAT-3′) and PNRSV-RNA3-R1 (5′-TCACTTACCACTACGTACAAATCCCTAAC-3′), cloned, and sequenced. The sequence (KU144878) from one infected tree (FS06) showed identities of 86.3 to 99.1% to 10 previously reported PNRSV (FJ846090-92, FJ610344, JN416773, JN416776, L38823, S78312, U57046, and Y07568) and identities of 58.2 to 58.6% to three ApMV (AM490197, HE574164, and U15608) genome sequences, respectively. Furthermore, two PNRSV-infected (LD and ZW) samples and one PNRSV-free sample were inoculated to cucumber plants and maintained under the same conditions. Stunting symptom were observed after 15 days on cucumber plants that tested positive for PNRSV using RT-PCR, while no other virus was found in the PNRSV-positive cucumber. The leaves of seven PNRSV-positive apple trees showed mosaic and yellow vein banding symptoms, so we speculate that PNRSV could also be associated with the mosaic disease of apple. PNRSV, belonging to the genus Ilarvirus, typically causes fruit yield loss and affects fruit maturity and tree growth in Prunus species such as almond, apricot, peach, plum, and sweet cherry (Mink and Aichele 1984). Despite PNRSV having worldwide distribution in stone fruits, this virus has only been reported previously in apple in India (Chandel et al. 2008). To our knowledge, this is the first report of PNRSV in domestic apples in China. Further research is needed to evaluate the effect of PNRSV on performance and quality of apple. This work was supported by CAAS-ASTIP. © 2016, American Phytopathological Society. All rights reserved.