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Citrus leprosis (CL) caused by Citrus leprosis virus C (CiLV-C) is present in Latin America from Mexico to Argentina, where citrus plants are grown. CiLV-C is transmitted by the tenuipalpid mite, Brevipalpus phoenicis, causing localized lesions on citrus leaves, fruit, and stems. One limitation to study of the virus–vector–host relationship in this pathosystem is the lack of a suitable assay plant. On Citrus spp. used as susceptible hosts, symptoms may take weeks or months to appear after experimental inoculation by viruliferous mites. Common bean (Phaseolus vulgaris) was found to respond with localized necrotic lesions after inoculation with viruliferous B. phoenicis in 5 days. Thus far, 113 tested common bean varieties and lines and some recent accessions of varied genetic background behaved in a similar way. Black bean ‘IAC Una’ was adopted as a standard test variety. When inoculated leaves were left at 28 to 30°C, the period for the lesion appearance was reduced to only 2 days. Confirmation that the lesions on common bean leaves are caused by CiLV-C were made by transmission electron microscopy, immunofluorescence, enzyme-linked immunosorbent assay, and reverse-transcription polymerase chain reaction specific for CiLV-C. Common bean plants mite-inoculated with some other cytoplasmic-type Brevipalpus-transmitted viruses (BrTVs) (Passion fruit green spot virus, Solanum violaefolium ringspot virus, Ligustrum ringspot virus, and Hibiscus green spot virus) also responded with necrotic local lesions and may serve as test plants for these viruses. Two nuclear types of BrTV (Coffee ringspot virus and Clerodendrum chlorotic spot virus) were unable to produce symptoms on common bean.

Made available in DSpace on 2013-08-12T18:45:47Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-07-01 Made available in DSpace on 2013-09-30T17:53:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-07-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:14:50Z No. of bitstreams: 0 Made available in DSpace on 2014-05-20T13:14:50Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-07-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) USDA-ARS Fundecitrus The equivalent of US$75 million is spent each year in Brazil to control Brevipalpus phoenicis, a mite vector of Citrus leprosis virus C (CiLV-C). In this study, we investigated the possibility that hedgerows and windbreaks normally found in citrus orchards could host CiLV-C. Mites confined by an adhesive barrier were reared on sweet orange fruit with leprosis symptoms then were transferred to leaves of Hibiscus rosa-sinensis, Malvaviscus arboreus, Grevilea robusta, Bixa orellana, and Citrus sinensis. Ninety days post infestation, the descendant mites were transferred to Pera sweet orange plants to verify the transmissibility of the virus back to citrus. Nonviruliferous mites which had no feeding access to diseased tissue were used as controls. Local chlorotic or necrotic spots and ringspots, symptoms of leprosis disease, appeared in most plants tested. Results generated by reverse-transcription polymerase chain reaction with primers specific for CiLV-C and by electron microscope analyses confirmed the susceptibility of these plants to CiLV-C. Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) Cassava & Fruits Ctr APTA Citros Sylvio M, BR-13490970 Cordeiropolis, SP, Brazil FCAV UNESP, Depto Fitossanidade, BR-14884900 Jaboticabal, SP, Brazil ESALQ, Depto Fitopatol & Nematol, BR-13418900 Piracicaba, SP, Brazil ARS, USDA, USHRL, Ft Pierce, FL USA FCAV UNESP, Depto Fitossanidade, BR-14884900 Jaboticabal, SP, Brazil

Coffee ringspot virus (CoRSV) and Citrus leprosis virus C (CiLV-C) are two emergent pathogens that can cause significant losses to coffee and citrus, respectively. Recently, it was verified that Brevipalpus phoenicis, originally reported as vector of these viruses, is indeed a complex group reclassified into eight mite species (Beard et al. 2015). Among those, B. papayensis Baker (Acari: Tenuipalpidae) occurs in citrus, but seems to be prevalent in coffee plantations. However, it is still unclear the relationship (if any) of B. papayensis with CiLV-C or CoRSV. In order to determine whether or not this mite is capable of transmitting these viruses, one single female specimen was collected from Coffea arabica cv. Catuai in Atibaia, SP-Brazil, and multiplied in laboratory as an isoline. Specimens were mounted in Hoyer’s medium and morphologically confirmed by phase contrast microscopy as B. papayensis, according to the classification of Beard et al. (2015). This isoline population was reared onto healthy coffee leaves, in 25 ± 1°C, 14 h light/10 h dark and 60 ± 10% humidity. RT-PCR assays using primers for the detection of each of the viruses were performed in order to confirm the virus-free colony status. B. papayensis mites were transferred to either coffee leaves exhibiting ringspots or sweet orange fruits symptomatic for leprosis, which served as sources of inocula for 7 days. Viral acquisition was confirmed by RT-PCR of viruliferous mites using specific primers for the detection of CoRSV (Kitajima et al. 2011) or CiLV-C (Locali et al. 2003), according to Kubo et al. (2011). After the acquisition period, 60 mites from each source of inoculum were transferred to 20 Arabidopsis thaliana plants (6 mites per plant), a plant species that can host both. Fifteen days after infestation, typical pinpoint lesions were observed and virus presence was confirmed by RT-PCR in 60% of the CiLV-C- and 30% of the CoRSV-inoculated plants. Additionally, common bean plants (Phaseolus vulgaris cv. Una) were infested with 15 B. papayensis mites from the isoline population reared onto CiLV-C inoculum (one mite per leaf, isolated by entomological glue). Perhaps due to the low efficiency in single mite transmission, only 27% of the leaves became symptomatic. The presence of CiLV-C was confirmed by RT-PCR and sequencing (98% of similarity to GenBank accession no. KP3367461). Fifteen females were collected directly from coffee leaves with CoRSV symptoms in Cordeiropolis, SP-Brazil, and transferred to isolated leaves of common bean plants. Symptoms of necrotic local lesions appeared 10 days after inoculation in 50% of the plants. The presence of CoRSV was confirmed by RT-PCR in both coffee and bean leaves. Amplicons obtained from the latter were sequenced (QG979998) and found 98% identical to CoRSV sequences available in GenBank. This is the first report of common bean as an experimental host of CoRSV. B. yothersi has been shown to transmit cileviruses. B. californicus seems to be the only vector of the dichorhavirus Orchid fleck virus, while B. phoenicis sensu stricto is the vector of the tentative dichorhavirus Citrus leprosis virus N. This is the first confirmation that B. papayensis can transmit CiLV-C and CoRSV. This is also the first time, following the reclassification of the B. phoenicis group, that a transmission was demonstrated by a single species of Brevipalpus for both a dichorha- and a cilevirus. Since this mite species is prevalent in coffee plantations in Brazil, it is likely the main vector of CoRSV under natural conditions.

Citrus leprosis virus (CiLV), a tentative member of the Rhabdoviridae family, affects citrus trees in Brazil, where it is transmitted by mites Brevipalpus spp. It also occurs in other South American countries and was recently identified in Central America. This northbound spread of CiLV is being considered a serious threat to the citrus industry of the United States. However, despite its importance, difficulties related to the biology of CiLV have hindered much of the progress regarding its accurate detection, leaving both the analyses of symptoms and electron microscopy as the only tools available. An attempt to overcome this problem was made by constructing a cDNA library from double-stranded RNA extracted from leprosis lesions of infected Citrus sinensis (sweet orange) leaves. After cloning and sequencing, specific primers were designed to amplify putative CiLV genome regions with similarity to genes encoding the movement protein and replicase of other plant viruses. RNA from infected citrus plants corresponding to different varieties and locations were amplified by reverse transcription-polymerase chain reaction (RT-PCR) using the two pairs of primers. Amplified products were purified, cloned in pGEM-T, and sequenced. The sequences confirmed the genomic regions previously associated with CiLV. The results demonstrate that RT-PCR was specific, accurate, rapid, and reliable for the detection of CiLV.

In Colombia, citrus is cultivated in mostly small plantings that total 55,000 ha by approximately 25,000 farmers. Production includes 1,200 tons of fresh fruits and 60 tons of juice for domestic consumption, resulting in a net worth of US$650,000 per year. Most of the production comes from areas located between the Cordillera Occidental and Cordillera Central mountain ranges (departments of Antioquia, Caldas, Quindio, and Risaralda) near coffee plantations. The departments of Meta and Casanare, located at the east plains (Llanos Orientales), include a zone parallel (4 to 5°N, 72 to 74°W) to the east mountain range and generate approximately 10% of the total Colombian citrus production. Suspected citrus leprosis symptoms on leaves and fruits of sweet oranges (Citrus sinensis (L.) Osb.) were first observed by plant pathologists for CORPOICA (Colombian National Agricultural Research Organization) in citrus orchards in Casanare in 2003, and later in 2004, in Meta. To confirm the visual identification, leaves and fruits from Valencia sweet orange exhibiting typical lesions of leprosis were collected from several locations in the departments of Casanare (Yopal, Aguazul) and Meta (Guamal, Villavicencio, and Cumaral). Samples were fixed in cacodylate-buffered paraformaldehyde/glutaraldehyde solution and subsequently processed for examination in thin sections using electron microscopy. Samples were processed and examined at the Citrus Research and Educational Center (CREC) of the University of Florida, Lake Alfred, and the Agricultural College (ESALQ) of the Universidade de São Paulo at Piracicaba, SP, Brazil. Some leaf samples collected in Meta were also dried and used for detection of Citrus leprosis virus, cytoplasmic type (CiLV-C) by reverse transcription-polymerase chain reaction (RT-PCR) at the Centro APTA Citros Sylvio Moreira at Cordeirópolis (CAPTACSM). The RT-PCR was performed with primers that specifically amplify a fragment of the viral genome that codes for the putative cell-to-cell movement protein (1). Locations at CREC and ESALQ each observed, using electron microscopy, cell changes characteristic of CiLV-C that include short bacilliform particles in the endoplasmic reticulum and dense, vacuolated, and irregularly shaped viroplasm in the cytoplasm (2) in samples from Casanare and Meta. RT-PCR amplified cDNA fragments of the expected size for samples collected in Meta and one of the amplicons was sequenced (GenBank Accession No. DQ272491). The sequence obtained was found to have 98% nucleotide sequence identity to the Brazilian CiLV-C isolate (GenBank Accession No. AY289190.1). Mites collected from affected plants from the department of Meta were identified at ESALQ as Brevipalpus phoenicis (Geijskes), a known principal vector of CiLV-C (2). These several lines of evidence confirmed that the symptoms observed in sweet oranges at Meta and Casanare are due to the infection by CiLV-C. To our knowledge, this is the first report of this virus in Colombia. References:(1) E. C. Locali et al. Plant Dis. 87:1317, 2003, (2) J. C. V. Rodrigues et al. Exp. Appl. Acarol. 30:161, 2003.

Citrus leprosis virus (CiLV) constitutes one of the most important viruses in citrus in the areas where it occurs. Two morphological types of virus particles have been described from associated leprosis symptoms, nuclear (CiLV-N) and cytoplasmic (CiLV-C) (4). The CiLV-C is more common, representing more than 99% of samples collected from South and Central America (E. W. Kitajima and J. C. V. Rodrigues, unpublished). Both virus types are associated with the mite vector, Brevipalpus phoenicis (Geijskes) (Acari: Tenuipalpidae). So far, CiLV-C has only been naturally transmitted by these mites to citrus (3). Plants of Solanum violaefolium Schott (Solanaceae) and ornamental and sweet orange seedlings were infested with viruliferous adult female mites, colony no. 61 (GenBank Accession No. AY320027) that were previously maintained on citrus seedlings infected with CiLV-C according to Rodrigues et al. (3). Fifteen days after the mites were transferred, spotted yellowish symptoms were observed on leaves of plants of S. violaefolium and similar symptoms were observed after 25 days on citrus leaves. The symptomatic tissues were studied using transmission electron microscopy. Particles typical of CiLV-C were observed in samples from both plant species. To our knowledge, this is the first reported case of mites transmitting CiLV-C to a noncitrus host. Subsequent experiments showed that mites were able to transmit the virus between plants of S. violaefolium. Attempts to transmit the virus by mites from S. violaefolium to citrus were unsuccessful. The dsRNA viral electrophoresis profile showed differences between the two host plants. Reverse transcription-polymerase chain reaction (RT-PCR) and sequencing assays with primers designed to detect CiLV-C (2) amplified DNA fragments of the expected size and base composition. These data suggest the loss or alteration of some viral components from the Solanum sp. host that might be essential for the transmission or infection in citrus. Such a mechanism may explain why, despite the ever-increasing number of Brevipalpus-transmitted viruses in a large number of different host plant species (1), cross transmission is not common. This information shows one of the potential routes for CiLV to invade citrus orchards, and suggests one alternative host plant that allows rapid multiplication of the virus for characterization. References: (1) E. W. Kitajima et al. Exp. Appl. Acarol. 30:135, 2003. (2) E. C. Locali et al. Plant Dis. 87:1317, 2003. (3) J. C. V. Rodrigues et al. Proc. Int. Org. Citrus Virol. 174, 2000 (4) J. C. V. Rodrigues et al. Exp. Appl. Acarol. 30:161, 2003.

Coffee ringspot virus (CoRSV) (family Rhabdoviridae) is transmitted by Brevipalpus phoenicis (Geijskes) (Acari: Tenuipalpidae). Coffee ringspot disease was first reported in coffee plants from Brazil in 1939 (1). In August 2000, severe symptoms of concentric ringspots and “oak leaf” patterns on coffee leaves (Coffea arabica L. cv. Catuai) were observed during field inspections conducted in two areas of San Gabriel de Desamparados, Costa Rica. The disease caused premature fruit and leaf drop in the affected plants. Some areas within the ringspot lesions remained green on senescent leaves. Because CoRSV particles remain restricted to lesion areas (1), this virus has not been purified, and antiserum for virus detection is not available. Therefore, leaves with symptoms were collected and examined by transmission electron microscopy. In ultrathin sections of symptomatic leaves, arrays of rhabdovirus-like particles were associated with the nucleus as described for CoRSV (2). Healthy tissues did not contain similar arrays of bacilliform and bullet-shaped particles. Twenty mites collected from the infected plants at the same locations and time were slide-mounted and identified as B. phoenicis. High populations of this mite were also observed infesting plants of Cajanus cajan L. that were intercropped with coffee at the same location. Sweet orange trees growing in the same fields as shade for the coffee did not show symptoms of citrus leprosis, a disease caused by another Brevipalpus-transmitted virus that was recently reported in Panama (3). To our knowledge, this is the first report of a virus similar to CoRSV in Costa Rica. The spread of this virus, presumably CoRSV, could seriously affect the coffee industry throughout Central America by increasing production costs. It may be necessary to apply one or more foliar acaricides to effectively control the mite vector. References: (1) A. Bitancourt. O. Biol. 5:33, 1939. (2) C. M. Chagas et al. Phytopathol. Z. 102:100, 1981. (3) F. S. Dominguez et al. Plant Dis. 85:228, 2001.