First Report of Actinidia virus A and Actinidia virus B on Kiwifruit in China.

Abstract

At present, two viruses affecting kiwifruit (Actinidia spp.), Actinidia virus A (AcVA) and Actinidia virus B (AcVB), both belonging to the genus Vitivirus in the family Betaflexiviridae, have been reported from New Zealand (2). The infected trees showed leaf vein chlorosis, flecking, and ringspots. China is the largest commercial kiwifruit producer. During field investigations in the growing season of 2013, symptoms of leaf chlorosis or ringspots, similar to those caused by AcVA and AcVB (1), were observed on some kiwifruit (Actinidia chinensis) plants in Hubei Province in the central China. Leaf samples were collected from three symptomatic and two symptomless plants of two A. chinensis cultivars. Total nucleic acids were extracted from the samples using a CTAB-based protocol described by Li et al. (3) and used as template in RT-PCR for the detection of AcVA and AcVB. Each virus was detected using two sets of primers reported by Blouin et al. (1). Primer sets AcVA 1F/1R and AcVA5F/5R were used for the AcVA detection, and AcVB1F/1R and AcVB5F/Viti3'R were used for the AcVB detection. AcVA was detected in three symptomatic plants (ID: Ac-HN-1, Ac-HN-3, and Ac-HN-5), and AcVB was detected in two symptomatic plants (ID: Ac-HN-1 and Ac-HN-3) and in one symptomless plant (ID: Ac-HN-2). Neither virus was detected in the second symptomless plant (ID: Ac-HN-4). Samples Ac-HN-1 and Ac-HN-3 had mixed infection of AcVA and AcVB, and sample Ac-HN-2 had the latent infection of AcVB. The sequenced 283-bp RT-PCR amplicons of the replicase-encoding gene from AcVA isolates AC-HN-3 and AC-HN-5 using AcVA1F/1R shared 90.8% nucleotide (nt) identity with the corresponding sequence of the New Zealand AcVA isolate (GenBank Accession No. JN427014.1). The 269-bp fragments of the RNA-binding protein-encoding gene obtained by using AcVA5F/5R shared 85.5 to 85.9% nt identities with the corresponding sequence of JN427014.1. The AcVB5F/Viti3'R products of 365 to 369 bp from three AcVB isolates shared 85.5 to 88.6% nt identities with the corresponding sequence of the New Zealand AcVB isolate. The representative sequences were submitted to GenBank with accession numbers KJ696776 and KJ696777 for the 269-bp fragments of AcVA-HN-1 and AcVA-HN-3, and KJ696778 and KJ696779 for the 365-bp and 369-bp fragments of AcVB-HN-1 and AcVB-HN-2, respectively. In addition, 12 and 14 out of 42 kiwi samples (excluding HN-1 to HN-5) collected randomly were positive for AcVA and AcVB as detected by RT-PCR. Meanwhile, the sample affected by AcVA-HN-5 was subjected to deep sequencing of the small RNAs (sRNAs) for complete survey of the infecting viruses. De novo assembly of sRNAs generated four sequence contigs, with lengths ranging from 161 to 285 nt, matching to ORFs 1 to 3 of the genome of the New Zealand AcVA isolate with significant nucleotide (91 to 95%) and amino acid (80 to 94%) similarities, and some other contigs from a new virus (unpublished). The result further confirmed AcVA infection in the kiwi plant. To our knowledge, this is the first report of both AcVA and AcVB outside of New Zealand. The Chinese isolates of the two viruses are distinct from those reported from New Zealand. The results provide valuable information for improving the viral sanitary status of the kiwifruit germplasm in China.