Incidence of Citrus leprosis virus C and Orchid fleck dichorhavirus Citrus Strain in Mites of the Genus Brevipalpus in Mexico.

The incidences of Citrus leprosis virus C (CiLV-C) and Orchid fleck dichorhavirus Citrus strain (OFV-citrus) were determined in field populations of Brevipalpus mites from 15 citrus-producing states in Mexico. Mites were collected from orange, grapefruit, mandarin, lime, and sweet lime orchards. Brevipalpus yothersi (Baker) (Trombidiformes: Tenuipalpidae) was the most abundant species followed by Brevipalpus californicus (Banks) (Trombidiformes: Tenuipalpidae), which confirmed previous reports. The viruses CiLV-C and OFV-citrus were found in both mite species. The incidence of CiLV-C, OFV-citrus and both viruses simultaneously (CiLV-C and OFV-citrus) was 17.2, 10.3, and 3.4% (n = 116) for B. yothersi, and 12.5, 20.8, and 4.1% (n = 24) for B. californicus, respectively. No significant difference was found when the incidence of these viruses was compared between both mite species. The importance of our results in relation to the epidemiology of leprosis is discussed.

Sublethal effects of spirodiclofen on biological and demographic parameters of the citrus leprosis mite Brevipalpus yothersi (Acari: Tenuipalpidae).

BACKGROUND: Brevipalpus yothersi Baker is one of the main vectors of citrus leprosis. Knowing the biology of this mite species when under chemical control is extremely important to understand its population dynamics, and then to solve problems of management of this pest. Therefore, we assessed the effects of one sublethal dose of spirodiclofen (0.48 ppm) on biological and demographic parameters of B. yothersi under laboratory and greenhouse conditions, comparing them with non-exposed mite population. RESULTS: Under laboratory conditions, where citrus fruits were used as a substrate, the duration of developmental stages (pre-egg-laying and egg-laying) and mite longevity showed no differences between treatments. However, the number of laid eggs increased in acaricide-treated fruits. In the greenhouse experiment, where citrus plants were used as a substrate, the instantaneous growth rate (ri) of mites was positive in all untreated plots. In contrast, in treated plants, ri was negative in 12 experimental units, and mite populations were suppressed in five of them. Moreover, mite population dynamics had a positive ri in three treated plants. CONCLUSIONS: Spirodiclofen sublethal dose had no negative effect on the offspring of B. yothersi females. However, it is not safe for citrus leprosis mite since it may increase egg number per female in some conditions. Biological and demographic differences could influence mite population dynamics in the field, requiring appropriate management strategies to improve citrus leprosis control. © 2019 Society of Chemical Industry.

Dissecting the Subcellular Localization, Intracellular Trafficking, Interactions, Membrane Association, and Topology of Citrus Leprosis Virus C Proteins.

Citrus leprosis (CL) is a re-emergent viral disease affecting citrus crops in the Americas, and citrus leprosis virus C (CiLV-C), belonging to the genus Cilevirus, is the main pathogen responsible for the disease. Despite the economic importance of CL to the citrus industry, very little is known about the performance of viral proteins. Here, we present a robust in vivo study around functionality of p29, p15, p61, MP, and p24 CiLV-C proteins in the host cells. The intracellular sub-localization of all those viral proteins in plant cells are shown, and their co-localization with the endoplasmic reticulum (ER), Golgi complex (GC) (p15, MP, p61 and p24), actin filaments (p29, p15 and p24), nucleus (p15), and plasmodesmata (MP) are described. Several features are disclosed, including i) ER remodeling and redistribution of GC apparatus, ii) trafficking of the p29 and MP along the ER network system, iii) self-interaction of the p29, p15, and p24 and hetero-association between p29-p15, p29-MP, p29-p24, and p15-MP proteins in vivo. We also showed that all proteins are associated with biological membranes; whilst p15 is peripherally associated, p29, p24, and MP are integrally bound to cell membranes. Furthermore, while p24 exposes an N-cytoplasm-C-lumen topology, p29, and p15 are oriented toward the cytoplasmic face of the biological membrane. Based on our findings, we discuss the possible performance of each protein in the context of infection and a hypothetical model encompassing the virus spread and sites for replication and particle assembly is suggested.

Production of mono- and polyclonal antibodies to Citrus leprosis virus C2 and their application in triple antibody sandwich ELISA and immunocapture RT-PCR diagnostic assays.

The newly discovered Citrus leprosis virus cytoplasmic type 2 (CiLV-C2) is one of the causal virus of citrus leprosis disease complex; which leads to substantial loss of citrus production in the states of Meta and Casanare of Colombia. Specific and sensitive detection methods are needed to monitor the dissemination of CiLV-C2 in Colombia, and to prevent introduction of CiLV-C2 to other citrus growing countries. Toward this end, putative coat protein gene (CPG) of CiLV-C2 was amplified from CiLV-C2 infected citrus tissues. The CPG was cloned, expressed and purified a recombinant coat protein of ∼31kDa which used to generate monoclonal antibodies and polyclonal antisera. Four monoclonal antibodies and two polyclonal antisera were selected as being specific following Western blotting. The monoclonal antibody MAb E5 and polyclonal antiserum PAb UF715 were selected testing with an extract of CiLV-C2 infected leaves using triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA). In addition, an immunocapture RT-PCR was standardized using MAb E5 for specific and sensitive detection of CiLV-C2. The standardized TAS-ELISA and IC-RT-PCR were able to detect CiLV-C2 in the extracts of symptomatic citrus leprosis tissues up to the dilutions of 1:160 and 1:2580, respectively. Result demonstrated that CiLV-C2 is present in citrus orchards in Meta and Casanare citrus growing areas of Colombia. TAS-ELISA could be used for routine detection of CiLV-C2, epidemiological studies, and for border inspections for quarantine purposes. IC-RT-PCR could be valuable for CiLV-C2 validation and viral genome analysis.

Detection of Citrus leprosis virus C using specific primers and TaqMan probe in one-step real-time reverse-transcription polymerase chain reaction assays.

Citrus leprosis virus C (CiLV-C), a causal agent of the leprosis disease in citrus, is mostly present in the South and Central America and spreading toward the North America. To enable better diagnosis and inhibit the further spread of this re-emerging virus a quantitative (q) real-time reverse transcription polymerase chain reaction (qRT-PCR) assay is needed for early detection of CiLV-C when the virus is present in low titer in citrus leprosis samples. Using the genomic sequence of CiLV-C, specific primers and probe were designed and synthesized to amplify a 73 nt amplicon from the movement protein (MP) gene. A standard curve of the 73 nt amplicon MP gene was developed using known 10(10)-10(1) copies of in vitro synthesized RNA transcript to estimate the copy number of RNA transcript in the citrus leprosis samples. The one-step qRT-PCR detection assays for CiLV-C were determined to be 1000 times more sensitive when compared to the one-step conventional reverse transcription polymerase chain reaction (RT-PCR) CiLV-C detection method. To evaluate the quality of the total RNA extracts, NADH dehydrogenase gene specific primers (nad5) and probe were included in reactions as an internal control. The one-step qRT-PCR specificity was successfully validated by testing for the presence of CiLV-C in the total RNA extracts of the citrus leprosis samples collected from Belize, Costa Rica, Mexico and Panama. Implementation of the one-step qRT-PCR assays for CiLV-C diagnosis should assist regulatory agencies in surveillance activities to monitor the distribution pattern of CiLV-C in countries where it is present and to prevent further dissemination into citrus growing countries where there is no report of CiLV-C presence.