“First report of the hibiscus strain of citrus leprosis virus C2 infecting passionfruit (Passiflora edulis)”

Authors: OLMEDO-VELARDE, ALEJANDRO - University Of Hawaii; Roy, Avijit; LARREASARMIENTO, ADRIANA - University Of Hawaii; WANG, XUPENG - University Of Hawaii; Padmanabhan, Chellappan; NUNZIATA, SCHYLER - US Department Of Agriculture (USDA); NAKHLA, MARK - US Department Of Agriculture (USDA); HU, JOHN - University Of Hawaii; MELZER, MICHAEL - University Of Hawaii

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/25/2022
Publication Date: 3/6/2022
Citation: Olmedo-Velarde, A., Roy, A., Larreasarmiento, A., Wang, X., Padmanabhan, C., Nunziata, S., Nakhla, M.K., Hu, J., Melzer, M.J. 2022. “First report of the hibiscus strain of citrus leprosis virus C2 infecting passionfruit (Passiflora edulis)”. Plant Disease. https://doi.org/10.1094/PDIS-10-21-2314-PDN.
DOI: https://doi.org/10.1094/PDIS-10-21-2314-PDN

Interpretive Summary: An Hibiscus strain of Citrus leprosis virus C2 (CiLV-C2H) infecting Hibiscus-rosa-sinensis was reported from Hawaii in 2013 but there is no report of CiLV-C2H causing Citrus Leprosis (CiL) disease in Citrus. Recently, CiLV-C2H has been found in a sweet orange (Citrus sinensis) tree in Colombia in mixed infection with CiLV-C2. Both CiLV-C2 and CiLV-C2H are transmitted by the flat mite (Brevipalpus yothersii). In 2019, passionfruit plants displaying chlorotic spots on young leaves, and green spots and veins in senescing leaves were observed at two community gardens in Honolulu. Symptoms resembled Brevipalpus-transmitted viruses like CiLV-C2H infecting hibiscus and passionfruit green spot virus (PfGSV) infecting Passiflora Spp. To identify the virus associated with the passionfruit infection, multiple molecular assays were performed using total RNA from infected leaves and flat mites. Mites collected from symptomatic passionfruit leaves confirmed the presence of CiLV-C2H inside mite. Current findings of CiLV-C2H in passionfruit and presence of the known vector B. yothersii in Hawaii increases the emerging threat of CiL disease in the USA.

Technical Abstract: In Hawaii passionfruit (Passiflora edulis) is grown primarily in residential properties, community gardens (CG), and as naturalized plants. In 2019, passionfruit plants displaying chlorotic spots on young leaves, and green spots and veins in senescing leaves were observed at two CG in Honolulu. Symptoms are resembling to passionfruit green spot virus (PfGSV) infection in Passiflora Spp. in Brazil (Ramos-González et al. 2020) and hibiscus strain of citrus leprosis virus C2 (CiLV-C2H) infection in Hibiscus in Hawaii (Melzer et al. 2013). Both the viruses are belonging to the genus Cilevirus under the family Kitaviridae. Total RNA was extracted from two pools comprised of 40 symptomatic leaves collected from both the CG following a CTAB-based procedure (Li et al. 2008). To know the virus associated with the P. edulis infection, a RT-PCR assay was performed using generic CiLV-C2 (Olmedo-Velarde et al. 2021) and PfGSV primers (Ramos-González et al. 2020). RT-PCR assay amplified CiLV-C2 amplicon but failed to produce PfGSV amplicon from infected leaves. Amplicon sequencing followed by a BLAST search showed the nucleotide sequence had >99% identity with the CiLV-C2H-RNA1 (KC626783). Total RNA was used to prepare a Ribo-depleted RNA library before underwent high throughput sequencing (HTS) in a NextSeq550 Illumina platform (2x75 cycles). Out of 6.5 million raw reads only 14,640 were mapped to the viral database. After assembled the virus reads 12 CiLV-C2H and 3 Watermelon mosaic virus contigs were identified. RNA1 contigs covered 80.17% of the CiLV-C2H genome whereas RNA2 contigs covered 94.5% with average coverage depth of 31.660 and 57.121, respectively. To obtain the near complete genome of CiLV-C2H, HTS data was assembled, and gaps were filled up by overlapping RT-PCR followed by Sanger sequencing. RNA1 (8536 nt, Acc. No. MW413437) and RNA2 (4878 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, all 40 symptomatic leaf samples were individually tested by RT-PCR and thirty samples were positive to CiLV-C2H. Furthermore, 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, lesions were observed in recipient plants, and tested RT-PCR positive for CiLV-C2H. Total RNA from individual Brevipalpus mite was isolated, and cDNA was prepared to 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 28S ribosomal mite RNA sequence (MZ478051) shared 99-100% nucleotide identity with the 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 (hibiscus), Aracea (Dieffenbachia sp.), and Rutaceae (Citrus spp. and Swinglea glutinosa) (Roy et al. 2015). CiLV-C2H infects hibiscus alone and citrus in mixed infection with CiLV-C2 (Roy et al; 2018) which is responsible to cause citrus leprosis disease. Confirmatory detection of CiLV-C2H in passionfruit (Passifloraceae) increase the number of reservoir host of CiLV-C2H in Hawaii. To know the probability of citrus leprosis disease occurrence in Hawaii, role of mite vectors in CiLV-C2H transmission from infected hibiscus and passionfruit to citrus needs to be investigated.