First Report of the Hibiscus Strain of Citrus Leprosis Virus C2 Infecting Passionfruit (Passiflora edulis)

Abstract

In Hawaii, passionfruit (Passiflora edulis; Passifloraceae) is grown primarily in residential properties and community gardens (CG). In 2019, passionfruit plants displaying chlorotic spots on young leaves, and green spots in senescing leaves were observed at two CG in Honolulu. Symptoms resembled those of passionfruit green spot virus (PfGSV) infection in Passiflora spp. (Ramos-González et al. 2020) and of the hibiscus strain of citrus leprosis virus C2 (CiLV-C2H) infection in hibiscus in Hawaii (Melzer et al. 2013). Both viruses belong to the genus Cilevirus, family Kitaviridae. Total RNA was extracted from two sample pools comprised of 40 symptomatic leaves collected from both the CG following a CTAB-based procedure (Li et al. 2008). To identify the virus associated with the P. edulis infection, reverse transcription (RT)-polymerase chain reaction (PCR) was performed using CiLV-C2 (Olmedo-Velarde et al. 2021) and PfGSV specific primers (Ramos-González et al. 2020). RT-PCR assay amplified the CiLV-C2 amplicon but failed to produce the PfGSV amplicon from infected leaves. Amplicon sequencing followed by a BLASTn search showed the nucleotide sequence had >99% identity with the CiLV-C2H-RNA1 (KC626783). A ribo-depleted RNA library created using the TruSeq Stranded Total RNA Library Prep kit (Illumina) underwent high throughput sequencing (HTS) on a NextSeq550 Illumina platform (2x75 cycles). The 6.5 million raw reads obtained were trimmed, filtered, and de novo assembled using Metaviral SPAdes v. 3.15.02 (Antipov et al. 2020). The resulting contigs were searched against an in-house database generated from GenBank virus and viroid sequences using BLASTn. This identified 12 and 3 contigs corresponding to CiLV-C2H and watermelon mosaic virus, respectively, with the latter being previously reported in passionfruit (Watanabe et al. 2016). RNA1 contigs covered 80.17% of the CiLV-C2H genome, whereas RNA2 contigs covered 94.5% with an average coverage depth of 31.660 and 57.121, respectively. To obtain the near complete genome of CiLV-C2H, gaps from the assembled HTS data were filled by overlapping RT-PCR followed by Sanger sequencing. RNA1 (8,536 nt, Acc. No. MW413437) and RNA2 (4,878 nt, MW413438) genome sequences shared 99.2% and 97.0% identity with CiLV-C2H-RNA1 (KC626783) and -RNA2 (KC626784). To further confirm the presence of CiLV-C2H in symptomatic P. edulis plants, 40 symptomatic leaf samples were individually tested by RT-PCR, and 30 samples were positive. Brevipalpus mites collected from CiLV-C2H-positive P. edulis leaves were transferred to common bean (Phaseolus vulgaris) seedlings (Garita et al. 2013). At 15-30 days post-transfer, RNA extracted from lesions observed in recipient plants tested positive for CiLV-C2H by RT-PCR. Total RNA from individual Brevipalpus mites was isolated, and cDNA was prepared to tentatively identify the mite species involved in CiLV-C2H transmission in passionfruit (Druciarek et al 2019, Olmedo-Velarde et al. 2021). CiLV-C2H was detected in individual mites, and the 28S ribosomal mite RNA sequence (MZ478051) shared 99-100% nucleotide identity with B. yothersi (MK293678 and MT812697), a vector of CiLV-C2 (Roy et al. 2013). CiLV-C2 currently has a host range limited to the families Malvaceae, Araceae, and Rutaceae (Roy et al. 2015). CiLV-C2H infects hibiscus alone and citrus in mixed infection with CiLV-C2 (Roy et al; 2018) which is responsible for causing citrus leprosis disease. Detection of CiLV-C2H in passionfruit expands the number of host families of CiLV-C2H.

High-throughput sequencing application in the detection and discovery of viruses associated with the regulated citrus leprosis disease complex

“…In this study, analysis of HTS data confirmed the presence of CiLV-C2 and CiLV-C2H either in single or in mixed infection in all three tested plant species but inconsistent with the real time RT-PCR results. The known natural host range of CiLV-C2H was expanded when HTS was applied successfully for detection of CiLV-C2H in passion fruit in Hawaii ( Olmedo-Velarde et al, 2022 ). The HTS protocol also successfully identified OFV orchid strain 2 (OFV-Orc2) ( Dichorhavirus ) in rough lemon and mandarin trees causing citrus leprosis symptoms ( Olmedo-Velarde et al., 2021 ) in Hawaii and in S. auriculata showing yellowing and mottling symptoms in Florida ( Dey et al., 2022 ).…”

Kitaviruses: A Window to Atypical Plant Viruses Causing Nonsystemic Diseases

“…This suggests the existence of phylogenetic gaps within and among genera, meaning that a diversity of unknown kitaviruses might exist. At the same time, although among isolates of some species different phylogenetic lineages and some uniqueness in their genomic organizations have been detected, intraspecies variability is low even between isolates collected from geographically distant places (25,73,78,90,93,101,102,107,120). Taken together, the data hint that although the spread of some kitaviruses likely could have been biased by anthropic actions possibly associated with the trade and exchange of their plant hosts, e.g., the cileviruses citrus leprosis virus C (CiLV-C; Cilevirus leprosis) and citrus leprosis virus C2 (CiLV-C2; Cilevirus colombiaense) in citrus and ToFBV in tomato (Solanum lycopersicum), there are regional diversity hot spots where kitaviruses of different genera have evolved from ancestors likely spread by continental drift.…”

“…In that region, isolate-specific arrays of ORFans have been found and the relationships of these ORFs with the wide natural host range of PfGSV have been speculated (93). Isolates of CiLV-C2 have been found infecting citrus and hibiscus (Hibiscus rosa-sinensis) in Colombia (103) and passion fruit (84) and hibiscus in the United States (73,107). Isolates collected in hibiscus and passion fruit show less than 5% nucleotide sequence variability, but they show a more accentuated divergence, i.e., 80-86% nucleotide sequence identity, with the RNA1-2 of the isolate infecting citrus in Colombia (73).…”