Introduction

Crab apples belong to the genus Malus in the family Rosaceae and are popular ornamental trees in the commercial and residential landscape for their beautiful flowers or fruit [6]. They exhibit excellent ornamental characteristics and stress resistance and can thus be used as rootstocks for apples and potential pollinizers in commercial orchards. Crab apples are subject to the same pathogens as apples, including several viruses such as apple chlorotic leaf spot virus (ACLSV), apple stem grooving virus (ASGV) and apple stem pitting virus (ASPV). There are several viruses or virus strains that are latent in apple varieties but which can cause serious alterations in the leaves, fruit, and sterns of some crab apple varieties that can be used as indicators for detection of latent virus infections of apples [8].

Over the last decade, advances in DNA sequencing technology have led to the development of new approaches for the identification and detection of viruses and viroids [1, 20]. These new approaches involve sequencing of the total nucleic acid or small RNAs in samples from diseased plants by next-generation sequencing (NGS) and allow identification of pathogens by downstream analysis using bioinformatic tools. In the past few years, NGS using various technologies and templates (RNAs, DNA, short interfering RNAs, double-stranded RNAs) has contributed tremendously to unraveling the nature of microbial agents associated with disease and allowed the rapid identification and characterization of a number of novel or known plant viruses with an RNA or DNA genome [2, 3, 10].

Ornamental crab apples showing virus-like symptoms such as necrosis and chlorosis of leaves and decline of tree vigor were observed in a landscaped park in Beijing. To identify the potential viruses involved in the disease, symptomatic leaves were collected, and total RNA was extracted from the samples using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Total RNA was then quantified and assessed for quality using a NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, DE) and integrity using a Bio-Analyzer 2100 (Agilent Technologies, Waldbronn, Germany) and used for construction of a small RNA library with subsequent sequencing on a HiSeq2000 platform (Illumina). Sequence reads were trimmed to remove low-quality and adaptor sequences and assembled using the de novo assembly algorithm of Velvet [22]. The assembled contigs were analyzed by BLASTn and BLASTx searches against the GenBank database.

A total of 23,140,206 high-quality reads were obtained using the Illumina Hiseq 2000 Solexa platform after quality trimming, 12,700,358 of which were virus-derived. 5727 contigs were assembled using the Velvet program with a k-mer value of 17, with the size ranging from 33 to 364 nt. A BLASTn and BLASTx analysis of assembled contigs against the NCBI database, using a high-homology sequence search, identified 79 contigs showing high sequence similarity to ASGV (61 contigs, with 46% sequence coverage), ACLSV (nine contigs, with 7% sequence coverage) and PNRSV (nine contigs, with 7% sequence coverage), while all the remaining contigs were of host origin. RT-PCR was conducted with primers designed based on the assembled contigs, and this confirmed the presence of these three viruses. PNRSV, a member of the genus Ilarvirus in the family Bromoviridae, has been documented in peach, apple, plum, cherry and apricot in China [5] and was only partially sequenced in this study (GenBank accession number KX371574). BLAST analysis showed that this sequence shared 75% nucleotide sequence identity (the highest) with the corresponding region of the isolate ChrYL (GenBank accession number KT444702).

The complete genome sequence of ACLSV-BJ was obtained by Sanger sequencing RT-PCR and rapid amplification of cDNA ends (RACE) PCR for the 5′ and 3′ ends of the virus genome (Supplemental Table 1) and submitted to the GenBank database with the accession number KU960942. The genomic RNA of ACLSV-BJ was 7554 nucleotides in length excluding the 3′ polyA tail, and the 5′ and 3′ ends were 154 and 210 nt, respectively. Sequence analysis showed that the genome nucleotide sequence identities between ACLSV-BJ and other ACLSV isolates available in the GenBank database ranged from 67.0% (TaTao5) to 83.0% (SY01). The genomic organization of ACLSV-BJ was the same as that of previously described ACLSV isolates and consisted of three ORFs. ORF 1 (nt 155-5788) putatively encoded a 1877-aa long viral replicase polyprotein sharing 91% amino acid sequence identity with the Chinese pear isolates KMS, JB and YH [23] and recently described hawthorn isolate SY01 [18]. ORF2 (nt 5700-6983) encoded a 47-kDa movement protein and shared 93% amino acid sequence identity with the isolates KMS, JB, YH and SY01. BLAST analysis showed that the coat protein encoded by ORF3 (nt 6766-7344) shared 89-96% aa sequence identity with other isolates. A previous analysis based on the amino acid sequences of CP showed that the ACLSV isolates were separated into two major clusters in which the combinations of the five amino acids at positions 40, 59, 75, 130 and 184 (S40-L59-Y75-T130-L184or A40-V59-F75-S130-M184) were highly conserved within each cluster [21]. In the CP of ACLSV-BJ, the motif S40-M59-Y75-A130-S184 was identified. Moreover, the specific aa combination S73-D82-L83-G98 reported for isolates JB, KMS, YH, KRL (a kuerle isolate) and MO5, which clustered together in the phylogenetic tree, was also conserved in the CP of ACLSV-BJ (S71-D81-L82-G97). A phylogenetic tree (Fig. 1A) based on the complete genome sequences of ACLSV isolates aligned using Clustal W [17] was constructed using the neighbor-joining (NJ) method implemented in the MEGA6.06 program [17] with the best-fit model (Tamura-Nei model) recommended by a model test. This analysis showed that ACLSV-BJ clustered with the isolates SY01 from hawthorn, MO5 from apple, and JB, KMS and YH from pear, which was in accordance with the nt and aa sequence analyses.

Table 1 Possible recombination events in apple stem grooving virus populations identified using RDP3
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Fig. 1
figure 1

Neighbor-joining tree generated based on the complete genome nucleotide sequences of isolates of apple chlorotic leaf spot virus (A) and apple stem grooving virus (B). Data on geographic provenance and hosts are shown next to the GenBank accession number for each isolate used in the analysis. The number of bootstrap replicates was 1000. Branches with bootstrap values ≥70% are shown. The scale bar represents genetic distance (substitutions per nucleotide)

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The complete genome sequence of ASGV-BJ was also determined in this work and deposited in GenBank under the accession number KU947036. The complete nucleotide sequence of ASGV-BJ was 6509 nucleotides (nt) long, excluding the poly (A) tail. The genomic RNA had two overlapping ORFs. ORF1 (nt 42–6351) encoded a 2,105-amino-acid (aa) polypeptide containing methyltransferase-like, papain-like protease, helicase-like, and RdRp-like domains, and the coat protein located at the carboxy-terminal end of the polyprotein. This ORF was preceded by a 41-nucleotide-long non-coding region at the 5′ end of the genome and followed by a 150-nucleotide-long intergenic region. ORF2 (nt 4793-5755) encoded a 320-amino-acid polypeptide, a protein with conserved motifs for both movement proteins (MPs), and viral proteases. Sequence analysis showed that ASGV-BJ shared 78.2-80.7% nucleotide sequence identity with other isolates. In a previous study, the transcription start sites of both the CP and the movement protein (MP) sgRNAs of ASGV were mapped to a conserved hexanucleotide motif, UUAGGU, upstream of each sgRNA [9]. This conserved hexanucleotide motif was also found at nt 4647, upstream of the movement protein gene, and similarly, at nt 5607, upstream of the CP gene in the ASGV-BJ isolate. To elucidate the phylogenetic relationship of ASGV-BJ to other isolates, a phylogenetic tree was constructed using the available complete ASGV genome sequences. ASGV-BJ clustered together with the isolate ASGV_kfp (KR106996) from pear (Fig. 1B).

Recombination plays a significant role in the evolution of many viruses [13, 14, 19] and is well documented among plant-infecting single-stranded RNA viruses [4, 15]. Recombination analysis of ASGV isolates was carried out using the software RDP3 [11], which includes RDP, GENECONV, BOOTSCAN, MAXCHI, CHIMAERA, SISCAN, and 3SEQ, performed with the default configuration, except that the options of linear sequence and of disentangling overlapping signals were selected. An event detected by at least five different methods and with p-values < 10−6 was considered to be a positive recombination event. Of the 20 genomes analyzed, ten of them showed evidence of recombination (Table 1), indicating a relatively high recombination frequency within the population of fully sequenced ASGV isolates. Crossover sites were identified at different locations for these recombinants, suggesting that there were no hotspots. ASGV-BJ was found to be a recombinant originating from isolate ASGV_kfp (KR106996) and an isolate from South Korea associated with pear black necrotic leaf spot disease (AY596172). Recombination in ACLSV has been reported previously [7]. Our analyses confirmed previous reports, with no additional events detected in this study.