First Report of Apple Latent Spherical Virus Infecting Kiwifruit in China

China is the center of origin of kiwifruit (Actinidia spp.) and the largest producing country in both terms of yield and acreage (Ferguson 2015). Kiwifruit virus diseases usually cause leaf yellowing, mosaic spots, fruit malformation, resulting in reduced yield and quality (Blouin et al. 2013). In August 2019, virus-like symptoms including chlorotic spots, crinkle, yellowing, and deformation were observed on leaves of kiwifruit grown in a 2,000-m2 commercial orchard in Chongqing, China, with disease incidence reaching approximately 20%. Twenty-eight leaf samples (CQ1-28) were randomly collected from symptomatic plants and mixed in equal amounts. Extraction of total RNA from above pooled sample, removal of ribosomal RNA and subsequent high-throughput sequencing (HTS) were conducted as previously reported (Wen et al. 2020). After filtering low-quality reads (Q30>94.16%), a total of 82,730,664 clean reads with an average length of 150 nucleotides (nt) were obtained and de novo assembled into contigs using CLC Genomics Workbench 11.0 (Qiagen) with k-mer values ranging from 31 to 121. BLASTx against the NCBI GenBank database showed that 10 contigs, 344 to 2,905 nt in length, were annotated as apple latent spherical virus (ALSV) (AB030940.1 and AB030941.1), which belongs to the Cheravirus genus and was originally identified in apple (Li et al. 2000). The nt identity between these contigs and the reference genome ranged from 75.2 to 85.7%. Primers CP-F/CP-R (5′-RTAGGHCARGGKGCTTGYTTGAGTA-3′/5′-CCAATAGGCTGTAGTRCTGGCC ATA-3′) targeting a 750 bp fragment CP gene of ALSV were designed based on the obtained contigs and the AB030941.1 sequence to verify the sequencing results in the individual samples. Total RNA was individually extracted from leaf samples CQ1-28 using a cetyltrimethylammonium bromide (CTAB) based method (Li et al., 2008) and subjected to reverse transcription polymerase chain reaction (RT-PCR). Target products were obtained from four samples (CQ2, CQ6, CQ8 and CQ15) and Sanger sequenced. Alignment of the four sequences showed that they shared 90.4 to 100% nt identity with each other and 75.7 to 77.3% nt identity with the corresponding AB030941.1 sequence. Furthermore, 49 symptomatic leaf samples were collected from Hubei (6), Jiangxi (6), Henan (31), Shanxi (4) provinces and Shanghai (2), China, and were tested for the presence of ALSV by RT-PCR with primers CP-F/CP-R. Three additional samples (Z2, ZZ19 and ZZ21) from Henan province were found ALSV positive. The detection rate of ALSV in Chongqing city and Henan province was 14.3% and 9.7%, respectively. To analyze the phylogenetic relationship of the ALSV isolates identified in this study, the seven CP sequences (GenBank accession nos. PV931796.1 to PV931802.1) of ALSV-positive kiwifruit samples together with the available sequences of ALSV from apple and Angelica sinensis (OP038546.1) (Jin et al. 2023) were aligned and a phylogenetic tree was constructed. The ALSV isolates obtained from kiwifruit clustered in the same branch with those infecting apple and A. sinensis. Since the mixed infections of ALSV with one or more of Actinidia virus 1, citrus leaf blotch virus, Actinidia virus A, Actinidia virus B and Actinidia seed borne latent virus, ALSV pathogenicity in kiwifruit remains challenging to confirm. To our knowledge, this is the first report of ALSV naturally infecting kiwifruit, expanding the host range of this virus and providing a crucial basis for viral disease monitoring.