First report of Hibiscus infecting Cilevirus in Citrus sinensis in Meta and Casanare, Colombia

Authors: Roy, Avijit; Stone, Andrew; LEON, M - Corpoica; Hartung, John; Wei, Gang; MAVRODIEVA, V - Animal And Plant Health Inspection Service (APHIS); NAKHLA, M - Animal And Plant Health Inspection Service (APHIS); Schneider, William; BRLANSKY, R - University Of Florida

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/15/2018
Publication Date: 6/15/2018
Citation: Roy, A., Stone, A.L., Leon, M.G., Hartung, J.S., Wei, G., Mavrodieva, V., Nakhla, M.K., Schneider, W.L., Brlansky, R.H. 2018. Sweet orange showing leprosis symptoms in Colombia are naturally infected with Hibiscus infecting cilevirus and citrus leprosis virus C2. Plant Disease. https://doi.org/10.1094/PDIS-01-18-0150-PDN.
DOI: https://doi.org/10.1094/PDIS-01-18-0150-PDN

Interpretive Summary: Citrus leprosis is the name given to symptoms on citrus caused by any of several viruses. All are transmitted by a related group of very tiny spider-like creatures called mites that feed on citrus leaves and fruit by sucking juice from the plants. If the mites are not controlled the symptoms can become very severe and even kill the trees. Because the symptoms caused by the different viruses are essentially the same, advanced laboratory based detection methods are needed to identify the different viruses. Citrus leprosis was originally described in Florida one hundred years ago, but disappeared from Florida in the 1960’s. Constant vigilance is required to prevent the reintroduction of the virus and mites into the United States. We therefore work in cooperation with scientists in Colombia where citrus leprosis and a closely related virus disease of ornamental hibiscus are found. We have developed advanced molecular diagnostic methods to differentiate among this mixture of viruses. We have found for the first time, infections by two of these viruses within single symptomatic citrus leaves. These symptomatic leaves are infected with both a very severe form of leprosis virus previously associated with citrus and a second virus previously associated with ornamental hibiscus. Our results add to existing evidence that hibiscus may serve as an alternate host for the virus and mite vectors associated with citrus leprosis disease. Our work has also demonstrated extremely sensitive methods to identify these viruses and distinguish among them. Our results will be used immediately by collaborators at the US Department of Agriculture who are responsible for preventing the reestablishment of leprosis virus out of the United States.

Technical Abstract: In February 2015, two sweet orange samples with severe citrus leprosis (CiL) symptoms were collected from Meta and Casanare states in Colombia and assayed for Cilevirus, Dichorhavirus and Higrevirus associated with CiL disease. PCR primers specific for CiLV-C2, but not primers specific for CiLV-C, CiLV-N and Hibiscus green spot virus-2, amplified expected species specific products. Cilevirus infection in ornamental Hibiscus was previously reported from Hawaii and Florida. Hibiscus-infecting Cilevirus (HiCV) is a hibiscus strain of Citrus leprosis virus C2 (CiLV-C2H) with 92% amino acid identity with CiLV-C2. Natural infection of Hibiscus by CiLV-C2 was reported in Colombia but there was no report of CiLV-C2H infection in citrus. The incidence and distribution of CiLV-C2H in Colombia was determined with a new primer set specific to the putative coat protein gene (CPG) in a RT-PCR assay. This primer pair successfully amplified the 626 nt of CPG sequence from the samples tested previously above. In addition, 14 sweet orange samples that were archived in 2012 from Meta and Casanare were tested. The species-specific RT-PCR assays yielded the CiLV-C2 specific 795 nt amplicon from all 14 samples and the 626 nt amplicon specific to CiLV-C2H sequences from 12 samples. The RT-PCR products were purified, cloned and sequenced. BLAST analysis of CiLV-C2 and -C2H amplicon sequences (NCBI MG637132-MG637133) showed 99 or 86% nucleotide identities with CiLV-C2 isolate L147V1 (JX000024) and 85 or 98% with CiLV-C2H isolate from Hawaii (KC626783), respectively. These results confirmed the presence of CiLV-C2H in mixed infection of citrus with CiLV-C2 prior to 2012. This is the first report of CiLV-C2H in Colombia and is also the first report of CiLV-C2H infection in citrus. These RT-PCR based results were confirmed by Next Generation Sequencing of total RNA extracted from the samples with an Illumina HiSeq2000 sequencer. Small RNA libraries generated 34,407,866 and 36,485,310 single-end reads from healthy and CiL infected samples, respectively. The assembled contigs were analyzed using BLASTn against the CiLV-C2 and -C2H reference genomes in GenBank. The NGS results showed the sequences were related to CiLV-C2 and -C2H and confirmed by analysis of the species-specific RT-PCR amplicons described earlier. It is not yet clear if CiLV-C2H alone can produce symptoms in citrus or if symptoms are due to the synergistic effects of the viruses in dual infection. Further studies are also necessary to confirm Brevipalpus mite involvement in CiLV-C2H transmission and leprosis -like symptom production in citrus.