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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Molecular Plant Pathology Laboratory » Research » Publications at this Location » Publication #398338

Research Project: Discovery, Characterization, and Diagnostics of Endemic and Exotic Citrus Pathogens Using High Throughput Sequencing (HTS)

Location: Molecular Plant Pathology Laboratory

Title: First report of Passion fruit green spot virus in yellow Passion fruit (Passiflora edulis f. flavicarpa) in Casanare in Colombia

item Roy, Avijit
item GUILLERMO, LEON - Colombian Corporation Of Agriculture And Livestock- Agrosavia
item NUNZIATA, SCHYLER - Animal And Plant Health Inspection Service (APHIS)
item PADMANABHAN, CHELLAPPAN - Animal And Plant Health Inspection Service (APHIS)
item RIVERA, YAZMIN - Animal And Plant Health Inspection Service (APHIS)
item BRLANSKY, RONALD - University Of Florida
item HARTUNG, JOHN - Retired ARS Employee

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/28/2022
Publication Date: 7/1/2023
Citation: Roy, A., Guillermo, L.M., Nunziata, S., Padmanabhan, C., Rivera, Y., Brlansky, R.H., Hartung, J.S. 2023. First report of Passion fruit green spot virus in yellow Passion fruit (Passiflora edulis f. flavicarpa) in Casanare in Colombia. Plant Disease. 107:2270.

Interpretive Summary: Passion fruit green spot cilevirus (PfGSV) is transmitted by the flat mite, Brevipalpus yothersi, and causes disease in passion fruit in Brazil. PfGSV was first identified in the 1990s, but molecular characterization was accomplished in 2020. Usually, yellowish foliar lesions arising during the initial stages which gradually turn to green island-like spots on the senescent leaves, are the typical symptoms of PfGSV infection. The genus Passiflora comprises around 520 species of which 170 are present in Colombia, with yellow passion fruit the most important in terms of net production and internal consumption followed, by purple passion fruit grown mainly for export. In 2022, PfGSV was reported in Hibiscus in Brazil and Paraguay. In addition, citrus leprosis is an economically virus disease caused by three cileviruses (CiLV-C, CiLV-C2 and CiLV-C2H) and all of them are present in Colombia. Common between these two pathogens are (1) both infect hibiscus and (2) are transmitted by same the vector B. yothersi. The recent report of CiLV-C2H in passion fruit in Hawaii and PfGSV in hibiscus in Brazil and Paraguay motivated us to investigate the possibility of cilevirus infection in passionfruit in Colombia. To determine the presence of PfGSV in citrus growing regions in Meta and Casanare, two surveys were conducted and eight yellow passionfruit suspected leaf samples were collected. Conventional RT-PCR specific for PfGSV followed by Sanger Sequencing confirmed the presence of PfGSV in yellow passion fruit in Colombia. High throughput sequencing was utilized for further confirmation and to determine the complete genome sequence of Colombian PfGSV isolates. Both PfGSV-RNA1 and -RNA2 segments of Casanare isolate shared 99% nt identity with PfGSV isolate infecting passion fruit in Brazil. We believe that our discovery identified PfGSV for the first time in Colombia. The findings from the study are critical for local growers and the possible movement of PfGSV from passionfruit to citrus by the same insect vector warrants further investigation.

Technical Abstract: Passiflora edulis, commonly known as passion fruit, is a vine species of passionflower native to South America. In Colombia, yellow passion fruit (P. edulis f. flavicarpa) is the most important in terms of net production and local consumption. Recently, two Brevipalpus-transmitted cileviruses, passion fruit green spot virus (PfGSV) and hibiscus strain of citrus leprosis virus C2 (CiLV-C2H), were detected in passion fruit in Brazil and Hawaii, respectively (Olmedo-Velarde et al. 2022; Ramos-González et al. 2020). CiLV-C2H infects both citrus and hibiscus in Colombia (Roy et al. 2015, 2018), but there was no report of PfGSV elsewhere apart from Brazil and Paraguay (da Costa-Rodrigues et al. 2022). Apart from emerging begomovirus diseases, five major viruses are known to infect passion fruit in Colombia: soybean mosaic virus (SMV), cowpea aphid-borne mosaic virus, passion fruit yellow mosaic virus, cucumber mosaic virus, and a tentative Gulupa bacilliform badnavirus A (Cardona et al. 2022). Current findings of CiLV-C2H in passion fruit and PfGSV in hibiscus motivated us to investigate the possibility of cilevirus infection in passion fruit in Colombia. During surveys, along with healthy yellow passion fruit leaves, five symptomatic plant samples from Meta and three from Casanare were collected and sent to the Molecular Plant Pathology Laboratory in Beltsville, MD, under APHIS permit. Samples from Meta showed leaf mottling, rugose mosaic, and leaf distortion, whereas leaf variegation, chlorotic spots, yellowing, green spots in senescent leaves and green vein banding were observed in the Casanare samples. Total RNA was extracted using RNeasy Plant Mini Kit (Qiagen, U.S.A.). To determine the potential cilevirus infection in these samples, three PfGSV specific primer pairs (Ramos-González et al. 2020) and one CiLV-C2 generic primer pair (Olmedo-Velarde et al. 2021) were used in RT-PCR assays. All five samples from Meta failed to produce either PfGSV or generic CiLV-C2 amplicons, but all three Casanare samples amplified 321, 244, and 299 nts of PfGSV-RNA1 and -RNA2 amplicons using C13F/C13R, C6F/C6R, and C8F/C8R primers, respectively. Bidirectional amplicon sequencing followed by BLASTn analysis revealed =99% nt identity with the PfGSV-RNA1 (MK804173) and -RNA2 (MK804174) genome sequences. An optimized Ribo-depleted library preparation protocol was used to prepare two cDNA libraries using the RNA extracts of PfGSV suspected positive (Casanare) and negative (Meta) samples (Padmanabhan et al. 2023). HTS libraries of Casanare and Meta samples resulted in 22.7 to 29.5 million raw reads, respectively. After adapter trimming and filtering, clean reads were mapped to the Arabidopsis thaliana reference genome and unmapped reads were de novo assembled (Padmanabhan et al. 2023). BLASTn analysis of the assembled contigs identified one to three contigs corresponding to PfGSV-RNA1 and -RNA2, respectively, from the Casanare sample and three contigs of SMV were identified in the Meta sample. No other virus sequence was obtained from either library. Assembled contigs of the Casanare sample covered 99.33% of the RNA1 and 94.42% of the RNA2 genome, with read depths of 64,474 and 119,549, respectively. Both RNA-1 (OP564895) and -2 (OP564896) segments of the Casanare isolate shared 99% nt identity with PfGSV isolate (MK804173 to 74). Meta sample contigs (OP564897) covered >99% of the SMV genome, which shared >99% nt identity with the Colombian SMV isolates (KY249378, MW655827). Our discovery identified PfGSV in Colombia; the first-time outside Brazil and Paraguay. The findings of PfGSV in passion fruit increase the potential threat and possibility of PfGSV movement via Brevipalpus sp. from passion fruit to other hosts including Citrus spp.