Reassortment of genome segments creates stable lineages among strains of Orchid fleck virus infecting citrus in Mexico

Authors: Roy, Avijit; Stone, Andrew; OTERO-COLINA, GABRIEL - COLEGIO DE POSTGRADUADOS; WEI, GANG; BRLANSKY, RONALD - UNIVERSITY OF FLORIDA; Ochoa, Ronald - Ron; Bauchan, Gary; SCHNEIDER, WILLIAM - FLORIDA STATE UNIVERSITY; NAKHLA, MARK - ANIMAL AND PLANT HEALTH INSPECTION SERVICE (APHIS); Hartung, John

Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/4/2019
Publication Date: 11/29/2019
Citation: Roy, A., Stone, A.L., Otero-Colina, G., Wei, G., Brlansky, R., Ochoa, R., Bauchan, G.R., Schneider, W.L., Nakhla, M., Hartung, J.S. 2019. Reassortment of genome segments creates stable lineages among strains of Orchid fleck virus infecting citrus in Mexico. Phytopathology. 110:106-120. https://doi.org/10.1094/PHYTO-07-19-0253-FI.
DOI: https://doi.org/10.1094/PHYTO-07-19-0253-FI

Interpretive Summary: Sweet orange and other citrus trees in the united States are attacked by many diseases and insects. Plant viruses are important because they are especially difficult to control. One group of plant viruses, referred to as the leprosis group, does not occur in the United States at this time, though does occur in Brazil and Mexico. It is unusual and unexpected that this group of viruses does not occur in the United States. In this study ARS researchers cooperated with scientists from USDA APHIS and a university in Mexico to study the leprosis disease that occurs in Mexico. We had previously described the virus pathogen in detail and developed a diagnostic test for it, but we noticed that samples from some diseased trees in Mexico did not react in our detection assay. We discovered that this was because there was a previously unknown variant of the virus in these samples. The new virus is unusual because it has evolved by naturally exchanging genetic information with related viruses, and this exchange is limited to a particular portion of the virus genome. We determined the nucleotide sequence of the virus and established its relationship to other members of the group in detail. We also developed a new diagnostic test that will detect this virus as well as the previous virus from Mexico and other related viruses that have been recently described in Brazil. The relationships among the many members of the leprosis group of plant viruses has been very confusing and our work clarifies these relationships. This group of viruses is transmitted by a group of mites. Mites are very small creatures that are closely related to spiders and not easily visible to the naked eye. Finally, we identified the species of mite that transmits this virus in citrus. The mites feed on the citrus and in doing so transmit the virus. Another common host of this group of viruses is horticultural orchids. The most likely reason this group of viruses does not occur in the United States is that the specific species of mite prefers cool climates and does not survive well under the environmental conditions present in Florida and California. Our work will be important to the research community as well as to government regulatory agencies such as USDA APHIS who have the job of identifying these viruses at when alert growers or border inspectors discover symptoms on plants or on fruit or flowers entering the United States.

Technical Abstract: The genus Dichorhavirus accommodates a group of viruses with bipartite, negative sense, single stranded RNA genomes that are transmitted by flat mites to hosts that include orchid, coffee and citrus. The virus replicates and forms particles in the nucleus of infected plant cells. In citrus, the symptoms are limited to chlorotic spots that become surrounded by necrotic borders. The localized lesions are found on fruit, twigs and leaves and the virus is not systemic. The virus and disease in citrus in Mexico were referred to as citrus leprosis nuclear type (Cil-N) to distinguish it from a superficially similar disease caused by an unrelated cilevirus, but CiL-N has been reclassified as a strain of the orchid fleck virus (OFV-Cit). Dichorhavirus in citrus is widespread in Mexico and occurs in Central and South America. We have previously used RNA sequencing technologies on OFV-Cit samples from a site in Mexico to characterize and develop RT-PCR assays for OFV-Cit. In the process of assay validation, we obtained samples with clear symptoms of OFV-Cit that failed to produce an amplicon with RT-PCR. Further RNA sequencing and electron microscopy revealed the presence of a novel virus strain, OFV-Cit2 in these samples. Sequence comparisons with other members of the genus showed variation in the protein products encoded by RNA1. The strains of OFV clustered together, whether from orchid or citrus, and were clearly separated from other dichorhaviruses recently described from citrus in Brazil. Further analysis of the sequence data showed that variation in RNA1 between the original (OFV-Cit1) and the new (OFV-Cit2) strains was not observed with RNA2, but instead a common RNA2 molecule was shared among strains of OFV-Cit1 and OFV-Cit2, a situation strikingly similar to OFV infecting orchids. We also collected and identified the mite that transmits OFV-Cit1 and OFV-Cit2 as Brevipalpus californicus sensu stricto. OFV-Cit1 and OFV-Cit2 have coexisted at a site in Mexico prior to 2012 through the present time. Dichorhavirus has not been reported in citrus in the United States, and we used the sequence data to design diagnostic tests to detect and differentiate all strains of dichorhavirus from citrus. These tests were validated with plant samples obtained from Mexico and will be available to be used in survey programs for the early detection of any introduction of these viruses into the United States.