First report of Gulupa baciliform virus A associated with passion fruit in China.

Passion fruit (Passiflora edulis) is a commercially important crop known for its nutritional value, high antioxidant content, and use in beverages and desserts. Gulupa baciliform virus A (GBVA), tentatively named Badnavirus in the family Caulimoviridae, is a cryptic circular double-stranded DNA (dsDNA, ≈6,951 bps) virus recently reported in Colombia with asymptomatic infection of passion fruit (Sepúlveda et al. 2022). In July 2024, viral-like symptoms, including vein clearing, mottling, leaf distortion, and yellowing, were observed on passion fruit leaves in Pingtang, Guizhou, China (Supplementary figure S1), with a 30% incidence rate, and 30 samples were collected. Total RNA was extracted from these samples using TRIzol reagent (Invitrogen, CA, USA). The intact RNA from the two pools (each consisting of 14 samples) was subjected to library construction using TruseqTM Small RNA sample prep Kit (Illumina) followed by Hiseq sequencing (Hiseq2000 Truseq SBS Kit v3-HS, Illumina platform) to complete the sRNA sequencing. Approximately 12,923,899 and 10,877,466 raw while 10,135,256 and 4,184,706 clean reads were obtained for the samples in pool A and pool B, respectively. Next, the de novo assembly was carried out using metaSPAdes (Nurk et al., 2017) which resulted in 4,770 virus related trimmed contigs with the maximum length of 883 bp. BLASTn searches of sequence contigs against the NCBI viral genome database confirmed the presence of telosma mosaic virus (TEMV, Hanoi isolate, accession No. NC_009742, >96.0% nucleotide identity), East Asian passiflora virus (EAPV, Taiwan isolate, accession No. KP114137, >97.5% nucleotide identity), and papaya leaf curl Guangdong virus (PaLCuGdV, Fujian isolate, accession No. FJ495184, >98.0% nucleotide identity). Interestingly, more than 70 sequence contigs revealed >98.7% nucleotide identity with the GBVA Colombia isolate (accession No. MW393828). Whereas, the overall similarity of newly identified GBVA Guizhou isolate was calculated to be more than 99% with corresponding sequences in NCBI database. Moreover, out of 4,770 trimmed contigs, the mapping of 241 contigs to the reference genome sequence (accession No. MW393828) using Geneious R9 further confirmed the presence of GBVA. To validate the Hiseq sequencing results, total RNA was extracted from the same samples used for high throughput sequence (HTS) analysis, and reverse-transcription polymerase chain reaction (RT-PCR) was performed using specific primers (GBVA-P2-F1: 5´-ATGAATTGGAAATCAACYGTAG-3´/GBVA-P2-R1: 5´-TTCTTCTCGAGGCGTCTCTT-3´) targeting the p2 protein region of the reference genome (accession No. MW393828) (Sepúlveda et al. 2022). Amplicon of expected size of 408bp was subjected to Sanger sequencing, and the resulted sequence (GBVA, Guizhou isolate) was submitted to GenBank (accession No. PQ240853). Multiple sequence alignment was executed based on the p2 protein sequence of GBVA in MEGA X with the ClustalW program. Subsequently, the phylogenetic tree was constructed employing the maximum likelihood method (Supplementary figure S1) (Kumar et al. 2018). Phylogenetic analysis showed that the newly identified isolate has highest similarity with the corresponding isolates from Colombia, indicating the close origin of Colombian and Chinese isolates. To our knowledge, this is the first report of GBVA associated with passion fruit in China, which indicates that further research into it's exact symptom expression may be warranted for global passion fruit production.