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First Report of Orchid Fleck Virus Associated with Citrus Leprosis Symptoms in Rough Lemon ( Citrus jambhiri ) and Mandarin ( C. reticulata ) the United States

2021; American Phytopathological Society; Volume: 105; Issue: 8 Linguagem: Inglês

10.1094/pdis-12-20-2736-pdn

1943-7692

Alejandro Olmedo-Velarde, Avijit Roy, Chellappan Padmanabhan, Schyler O. Nunziata, Mark K. Nakhla, Michael J. Melzer,

Phytoplasmas and Hemiptera pathogens

HomePlant DiseaseVol. 105, No. 8First Report of Orchid Fleck Virus Associated with Citrus Leprosis Symptoms in Rough Lemon (Citrus jambhiri) and Mandarin (C. reticulata) the United States PreviousNext DISEASE NOTE OPENOpen Access licenseFirst Report of Orchid Fleck Virus Associated with Citrus Leprosis Symptoms in Rough Lemon (Citrus jambhiri) and Mandarin (C. reticulata) the United StatesAlejandro Olmedo-Velarde, Avijit Roy, Chellappan Padmanabhan, Schyler Nunziata, Mark K. Nakhla, and Michael J. MelzerAlejandro Olmedo-Velardehttps://orcid.org/0000-0003-1688-0455Department of Plant and Environmental Protection Sciences, University of Hawaii at Manoa, Honolulu, HI 96822, Avijit Royhttps://orcid.org/0000-0002-2268-9713USDA, Animal Plant Health Inspection Service, Plant Protection and Quarantine, Science and Technology, Beltsville Laboratory, Laurel, MD 20708, Chellappan PadmanabhanUSDA, Animal Plant Health Inspection Service, Plant Protection and Quarantine, Science and Technology, Beltsville Laboratory, Laurel, MD 20708, Schyler NunziataUSDA, Animal Plant Health Inspection Service, Plant Protection and Quarantine, Science and Technology, Beltsville Laboratory, Laurel, MD 20708, Mark K. NakhlaUSDA, Animal Plant Health Inspection Service, Plant Protection and Quarantine, Science and Technology, Beltsville Laboratory, Laurel, MD 20708, and Michael J. Melzer†Corresponding author: M. J. Melzer; E-mail Address: melzer@hawaii.eduhttps://orcid.org/0000-0003-0390-3857Department of Plant and Environmental Protection Sciences, University of Hawaii at Manoa, Honolulu, HI 96822 AffiliationsAuthors and Affiliations Alejandro Olmedo-Velarde1 Avijit Roy2 Chellappan Padmanabhan2 Schyler Nunziata2 Mark K. Nakhla2 Michael J. Melzer1 † 1Department of Plant and Environmental Protection Sciences, University of Hawaii at Manoa, Honolulu, HI 96822 2USDA, Animal Plant Health Inspection Service, Plant Protection and Quarantine, Science and Technology, Beltsville Laboratory, Laurel, MD 20708 Published Online:22 Sep 2021https://doi.org/10.1094/PDIS-12-20-2736-PDNAboutSectionsView articlePDFSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat View articleCitrus leprosis is an economically important disease of citrus in South and Central America. The disease can be caused by several nonsystemic viruses belonging to the genera Cilevirus (family Kitaviridae) and Dichorhavirus (family Rhabdoviridae) (Freitas-Astúa et al. 2018; Roy et al. 2015). In February 2020, lesions consistent with citrus leprosis were observed on the leaves and stems of rough lemon (Citrus jambhiri) and mandarin (C. reticulata) trees in Hilo, Hawaii. Brevipalpus mites, vector of orchid fleck virus (OFV), were also present on these trees (Freitas-Astúa et al. 2018). To identify the virus associated with the symptoms, total RNA was isolated using a NucleoSpin RNA Plus kit (Macherey-Nagel) and underwent reverse transcription (RT)-PCR with two newly designed universal primers specific for dichorhaviruses (Dichora-R1-F1, 5′-CAYCACTGYGCBRTNGCWGATGA / Dichora-R1-R1, 5′-AGKATRTSWGCCATCCKGGCTATBAG). The expected ∼350-bp amplicon was obtained and directly sequenced in both directions. BLASTn and BLASTx searches revealed that the primer-trimmed consensus sequence (MT232917) shared 99.3% nucleotide (nt) and 100% amino acid (aa) identity with an OFV isolate from Germany (AF321775). OFV has two orchid- (OFV-Orc1 and OFV-Orc2) and two citrus- (OFV-Cit1 and OFV-Cit2) infecting strains (Roy et al. 2020). However, an isolate of OFV-Orc1 has recently been associated with citrus leprosis in South Africa (Cook et al. 2019). To confirm the presence of OFV in Hawaiian citrus and identify the strain, symptomatic tissue was submitted to USDA-APHIS-PPQ-S&T, where total RNA was extracted from the symptomatic tissue using an RNeasy Plant Mini kit (Qiagen). The RNA samples were tested with OFV-Orc and OFV-Cit generic and specific primers in a conventional RT-PCR assay following optimized RT-PCR protocols (Roy et al. 2020). Two additional sets of generic primers (OFV-Orc-GPF, 5′-AGCGATAACGACCTTGATATGACACC / OFV-Orc-GPR, 5′-TGAGTGGTAGTCAATGCTCCATCAT; and OFV-R2-GF1, 5′-CARTGTCAGGAGGATGCATGGAA / OFV-R2-GR, 5′-GACCTGCTTGATGTAATTGCTTCCTTC′) were designed based on available OFV phospho (P) and large (L) polyprotein gene sequences in GenBank. These assays detected OFV-Orc2 in the symptomatic citrus samples, with the nucleocapsid (1,353-bp), P (626-bp), and L (831-bp) gene sequences sharing 97 to 98% identity with published OFV-Orc2 sequences (AB244417 and AB516441). Ribo-depleted RNA (Ribo-Zero, Illumina) was prepared using a TruSeq Stranded Total RNA Library Prep kit (Illumina) and underwent high-throughput sequencing on a MiSeq platform (Illumina). The resulting 19.6 million 2 × 75-bp reads were de novo assembled using SPAdes version 3.10.0 (Bankevitch et al. 2012). In addition to sequences corresponding to citrus tristeza virus and citrus vein enation virus, two contigs of 6,412 nt (average depth, 18,821; MW021482) and 5,986 nt (average depth, 19,278; MW021483) were found to have ≥98% identity to RNA1 (AB244417) and RNA2 (AB244418) of OFV isolate So (Japan), respectively. This is the first report of OFV in Hawaii and the first time leprosis has been observed in the United States since it was eradicated from Florida in the 1960s, although that outbreak was attributed to infection by citrus leprosis virus-N0, a distant relative of OFV (Hartung et al. 2015). The recent detection of citrus leprosis associated with OFV infection in South Africa (Cook et al. 2019) and now Hawaii underscores the threat this pathogen poses to the global citrus industry.The author(s) declare no conflict of interest.References:Bankevich, A., et al. 2012. J. Comput. Biol. 19:455. https://doi.org/10.1089/cmb.2012.0021 Crossref, ISI, Google ScholarCook, G., et al. 2019. Eur. J. Plant Pathol. 155:1373. https://doi.org/10.1007/s10658-019-01854-4 Crossref, ISI, Google ScholarFreitas-Astúa, J., et al. 2018. Curr. Opin. Virol. 33:66. https://doi.org/10.1016/j.coviro.2018.07.010 Crossref, ISI, Google ScholarHartung, J. S., et al. 2015. Phytopathology 105:1277. https://doi.org/10.1094/PHYTO-03-15-0064-R Link, ISI, Google ScholarRoy, A., et al. 2015. Phytopathology 105:1013. https://doi.org/10.1094/PHYTO-12-14-0375-FI Link, ISI, Google ScholarRoy, A., et al. 2020. Phytopathology 110:106. https://doi.org/10.1094/PHYTO-07-19-0253-FI Link, ISI, Google ScholarThe author(s) declare no conflict of interest.Funding: Funding was provided by National Institute of Food and Agriculture (9050H).DetailsFiguresLiterature CitedRelated Vol. 105, No. 8 August 2021SubscribeISSN:0191-2917e-ISSN:1943-7692 DownloadCaptionComparison of banana plants in silicon-deficient soil amended or nonamended with calcium silicate and infected by Fusarium oxysporum f. sp. cubense (W. Zellner et al.). Photo credit: A. A. Fortunato and F. A. Rodrigues. Infected spicebush tree outer bark showing black vascular discoloration of the sapwood typical of laurel wilt (R. Olatinwo et al.). Photo credit: R. Olatinwo. Maize plants naturally infected by Bipolaris zeicola (S. S. Liu et al.). Photo credit: S. S. Liu. Metrics Downloaded 918 times Article History Issue Date: 1 Dec 2021Published: 22 Sep 2021First Look: 3 Mar 2021Accepted: 25 Feb 2021 Page: 2258 Information© 2021 The American Phytopathological SocietyFundingNational Institute of Food and AgricultureGrant/Award Number: 9050HKeywordsviruses and viroidsfruittree fruitspathogen detectionThe author(s) declare no conflict of interest.PDF downloadCited BySmilax auriculata: A New Host for Orchid Fleck Dichorhavirus Identified in Florida, USAK. K. Dey, M. 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