Complete genomic sequence of turnip mosaic virus infecting passionfruit in Fujian province of China

Contigs with sequence identities to turnip mosaic virus (TuMV) were detected by deep sequencing of the small RNAs extracted from a passion fruit (Passiflora edulis) plant showing virus-like symptoms in Fujian Province, China. The complete genome of the TuMV, designated TuMV-FJ here, was determined based on the sequences of these contigs and Sanger sequencing of RT-PCR and 5′/3’ RACE amplicons. The TuMV-FJ genome consists of 9,833 nucleotides excluding the poly(A) tail. It contains an open reading frame encoding a polypeptide with 3164 amino acid residues. The polypeptide was predicted to be cleaved into ten functional proteins by viral proteases. Phylogenetic analysis indicated that TuMV-FJ is a member of a world-B phylogenetic group of TuMV. TuMV-FJ shared more than 94% nucleotide and amino acid sequence identities with its closest relative, namely BJ-B01, a TuMV isolate from Brassica oleracea. A RT-LAMP assay was developed to facilitate the molecular detection of TuMV-FJ. As far as we know, this is the first report of the complete genome sequence of TuMV infecting P. edulis. In addition, the RT-LAMP assay provides a rapid, robust and highly sensitive approach for the detection of TuMV in passion fruit.

As typical to other potyviruses, TuMV has a singlestranded positive-sense RNA genome ~ 10 kb in size (Lefkowitz et al. 2018). The viral genome has one major open reading frame (ORF) encoding a polyprotein that is autocatalytically hydrolyzed into 10 mature proteins. TuMV has a wide host range but mainly infects domestic brassica crops (Kawakubo et al. 2021). Like other potyviruses, TuMV is transmitted by aphids in a non-persistent manner (Shattuck 1992). TuMV can be divided into six phylogroups that differ in host range and geographical distribution, namely basal-Brassica (basal-B), basal-Brassica/Raphanus (basal-BR), Asian-Brassica/Raphanus (Asian-BR), world-Brassica (world-B), Iranian and Orchis, respectively (Kawakubo et al. 2021;Ohshima et al. 2002). Though TuMV on yellow passion fruit has recently been identified in Guangdong Province, China , the genome sequence of TuMV in passionfruit was, until recently, not available. Here, we report the first complete genome sequence of a TuMV isolate infecting passionfruit in Fujian Province China, which is referred to as TuMV-FJ in our study. During a survey of passion fruit viruses in 2018 in Fujian Province, samples were collected from 20 passion fruit plants from a commercial orchard showing mosaic, crinkle and yellow spot symptoms on the leaves. The presence of TuMV in one of the symptomatic passion fruit samples (TuMV-FJ, as shown in Fig. S1) was detected by ELISA (Agdia, Elkhart, USA). Total RNA was extracted from the symptomatic leaves of TuMV-FJ sample using TRIzol Reagent (Invitrogen, USA). A library of small interfering RNAs (siRNAs) was constructed and sequenced using a HiSeq X Ten sequencer (Illumina, USA) by Sangon Biotech Co., Ltd. (Shanghai, China), obtaining a total of 4,392,365 clean reads after trimming adapter sequence and low-quality reads. Eight contigs shared 98%-100% identity with the reported genomic sequences of TuMV. In addition, contigs with homologies to telosma mosaic virus (96%-100%) and cucumber mosaic virus (95%-100%) were also identified. TuMV infection was also confirmed by RT-PCR using a specific primer pair flanking a 641 bp region of the coat protein (CP) of TuMV (5ˊ-TGT GTT TATVAYC AYC ARG CAG GYGA-3´ and 5ˊ-CGC TGA AGA CCA TAT CGT GGC ATG -3´). To determine the complete nucleotide sequence of TuMV-FJ, the gap regions of the virus genome were amplified and sequenced by RT-PCR using four virus-specific primer sets (Table S1) designed based on the contigs. Terminal sequences of the viral genome were determined by rapid amplification of cDNA ends (RACE) using commercial kits (Invitrogen, USA). The RT-PCR amplicons of the expected sizes were cloned and sequenced in both directions by Sangon Biotech Co., Ltd. (Shanghai, China). The final sequences were assembled and analysed using the DNAMAN 9 program (Lynnon, Quebec, Canada). The complete genome sequence of TuMV-FJ has been deposited in GenBank under the accession number MK340758.
Viral sequences closely related to TuMV-FJ were obtained by a homology search against the GenBank database. The cleavage sites within the TuMV-FJ genome were determined utilizing an online website (http:// www. dpvweb. net/ potyc leava ge). Multiple sequence alignment was performed using the MAFFT algorithm (Katoh and Standley 2013) implemented in PhyloSuite 1.21 (Zhang et al. 2020), with ambiguously aligned regions trimmed using the program Gblock 0.91b (Talavera and Castresana 2007). Phylogenetic analysis was carried out with the maximumlikelihood method implemented in IQ-tree 1.6.8 (Nguyen et al. 2014) using the GTR + I + G4 + F substitution model, which was selected by ModelFinder (Kalyaanamoorthy et al. 2017). Support for the inferred tree was assessed by ultrafast bootstrapping with 10,000 replicates. The presence of putative recombination events were identified using RDP 5.3 (Martin et al. 2015) implementing seven different algorithms, including RDP, GENECONV, Bootscan, MaxChi, Chimaera, Siscan and 3Seq.
The phylogenetic relationship between TuMV-FJ and the other TuMV isolates is illustrated in the phylogenetic tree (Fig. 1). The maximum-likelihood tree showed that TuMV isolates could be separated into six phylogenetic groups (Kawakubo et al. 2021). TuMV-FJ was clustered into a clade formed by world-B TuMV isolates with high confidence (bootstrap value, BP = 100%, Fig. 1). Members of this clade have a considerable diversity in sampling locations and host origins. Within this clade, BJ-B01, an isolate from Brassica oleracea, was more closely related to TuMV-FJ.
Recombination analyses did not identify any recombination event in the TuMV-FJ genome. Taken together, these analyses suggest that TuMV-FJ is a novel isolate of TuMV from passionfruit. To our knowledge, this is the first report of the complete genome sequence of TuMV infecting P. edulis.
The results from the RT-LAMP assay showed that only the RNA of TuMV had an amplification curve, and the color of the reaction solution turned green (Fig. S2). Comparison of the sensitivity of the RT-LAMP assay with conventional RT-PCR was done by using a serially tenfold diluted cDNA from the RNA of TuMV. The lowest detection limit of RT-LAMP and conventional RT-PCR was 1,000-and tenfold, respectively (Fig. S3), indicating that the RT-LAMP assay was 100 times more sensitive than conventional RT-PCR. In conclusion, the developed RT-LAMP assay provides a rapid, robust and highly sensitive approach for the detection of TuMV in passion fruit plants. Out of the 80 passion fruit plants collected from eight different commercial orchards (10 samples per orchard) in Fujian Province, China, only 1.3% were found to be positive for TuMV using RT-LAMP (Fig. S4). The RT-LAMP results were subsequently confirmed by RT-PCR (Fig. S5). Despite the low incidence of TuMV in the passion fruit from the orchards, continued monitoring of the virus may be necessary given that it is a newly reported virus in this crop. Data availability Sequence data obtained in this study has been deposited in GenBank under the accession number MK340758.

Declarations
Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors.

Competing interest
The authors declare no completing interests.