Viruliferous Mites Research Articles

Brevipalpus yothersiBaker (Tenuipalpidae) development in sweet orange plants is influenced by previous mite infestation and the presence of shelters.

Citrus leprosis is the most important viral disease affecting citrus. The disease is caused predominantly by CiLV-C and is transmitted by Brevipalpus yothersi Baker mites. This study brings some insight into the colonization of B. yothersi in citrus [(Citrus × sinensis (L.) Osbeck (Rutaceae)] previously infested by viruliferous or non-viruliferous B. yothersi. It also assesses the putative role of shelters on the behavior of B. yothersi. Expression of PR1 and PR4 genes, markers of plant defense mechanisms, were evaluated by RT-qPCR to correlate the role of the plant hormonal changes during the tri-trophic virus-mite-plant interplay. A previous infestation with either non-viruliferous and viruliferous mites positively influenced oviposition and the number of adult individuals in the resulting populations. Mite populations were higher on branches that had received a previous mite infestation than branches that did not. There was an increase in the expression of PR4, a marker gene in the jasmonic acid (JA) pathway, in the treatment with non-viruliferous mites, indicating a response from the plant to their feeding. Conversely, an induced expression of PR1, a marker gene in the salicylic acid (SA) pathway, was observed mainly in the treatment with viruliferous mites, which suggests the activation of a plant response against the pathogen. The earlier mite infestation, as well as the presence of leprosis lesions and a gypsum mixture as artificial shelters, all fostered the growth of the B. yothersi populations after the second infestation, regardless of the presence or absence of CiLV-C. Furthermore, it is suggested that B. yothersi feeding actually induces the JA pathway in plants. At the same time, the CiLV-C represses the JA pathway and induces the SA pathway, which benefits the mite vector.

Pest categorisation of High Plains wheat mosaic virus.

The EFSA Panel on Plant Health conducted a pest categorisation of High Plains wheat mosaic virus (HPWMoV) for the EU territory. The identity of HPWMoV, a member of the genus Emaravirus (family Fimoviridae), is well established and reliable identification methods are available. The pathogen is not included in the EU Commission Implementing Regulation 2019/2072. HPWMoV has been reported from Argentina, Australia, Canada, Ukraine and USA, and it is not known to be present in the EU. HPWMoV infects plant species of the family Poaceae (i.e. wheat, maize and several other cultivated or wild Poaceae species). It is the causal agent of High Plains disease of wheat and maize, inducing symptoms ranging from mild to severe mosaic, chlorosis and necrosis in wheat, and chlorotic streaks in maize plants. The virus is transmitted by the wheat curl mite Aceria tosichella, which is present in the EU. HPWMoV transmission via seeds was reported to occur in sweet corn. Sweet corn seeds for sowing were identified as the most relevant pathway for entry of HPWMoV into the EU. Seeds from other hosts and viruliferous wheat curl mites were identified as entry pathways associated with uncertainties. Machinery not appropriately cleaned may move infected seeds and/or parts of cereals infested by viruliferous mites. Cultivated and wild hosts of HPWMoV are distributed across the EU. Would the pest enter and establish in the EU territory, economic impact on the production of cultivated hosts is expected. Phytosanitary measures are available to prevent entry and spread of the virus in the EU. HPWMoV fulfils the criteria that are within the remit of EFSA to assess for it to be regarded as a potential Union quarantine pest.

Circulative Transmission of Cileviruses in Brevipalpus Mites May Involve the Paracellular Movement of Virions.

Plant viruses transmitted by mites of the genus Brevipalpus are members of the genera Cilevirus, family Kitaviridae, or Dichorhavirus, family Rhabdoviridae. They produce non-systemic infections that typically display necrotic and/or chlorotic lesions around the inoculation loci. The cilevirus citrus leprosis virus C (CiLV-C) causes citrus leprosis, rated as one of the most destructive diseases affecting this crop in the Americas. CiLV-C is vectored in a persistent manner by the flat mite Brevipalpus yothersi. Upon the ingestion of viral particles with the content of the infected plant cell, virions must pass through the midgut epithelium and the anterior podocephalic gland of the mites. Following the duct from this gland, virions reach the salivary canal before their inoculation into a new plant cell through the stylet canal. It is still unclear whether CiLV-C multiplies in mite cells and what mechanisms contribute to its movement through mite tissues. In this study, based on direct observation of histological sections from viruliferous mites using the transmission electron microscope, we posit the hypothesis of the paracellular movement of CiLV-C in mites which may involve the manipulation of septate junctions. We detail the presence of viral particles aligned in the intercellular spaces between cells and the gastrovascular system of Brevipalpus mites. Accordingly, we propose putative genes that could control either active or passive paracellular circulation of viral particles inside the mites.

Management of the Wheat Curl Mite and Wheat Streak Mosaic Virus With Insecticides on Spring and Winter Wheat.

The wheat curl mite (WCM, Aceria tosichella, Keifer) is an eriophyid mite species complex that causes damage to cereal crops in the Northern Great Plains by feeding damage and through the transmission of plant viruses, such as wheat streak mosaic virus. Insecticide treatments were evaluated in the greenhouse and field for efficacy at managing the WCM complex on wheat. Treatments tested were carbamates, organophosphates, pyrethroids, a neonicotinoid seed treatment, mite growth inhibitors, and Organic Materials Review Institute–approved biocontrols, soaps, and oils. Treatment with carbamates, organophosphates, and pyrethroids decreased WCM in greenhouse trials compared with untreated controls 14 days after infestation. The seed treatment, mite growth inhibitors, and organic pesticides did not reduce WCM populations effectively and consistently. The timing of application was tested using a sulfur solution as the experimental treatment. Treating plants with sulfur seven days after mite infestation reduced mites compared with the untreated control. In contrast, prophylactically applied sulfur and sulfur applied 14 days after mite infestation were not effective. When tested under field conditions with plots infested with viruliferous mites, there was no yield difference detected between untreated control plots and plots sprayed with insecticides. Select carbamates, organophosphates, and pyrethroids have a potential for use in greenhouse mite management when appropriate.

Lack of transmission of Sugarcane yellow leaf virus in Florida from Columbus grass and sugarcane to sugarcane with aphids or mites.

Sugarcane yellow leaf virus (SCYLV), the causal agent of yellow leaf disease, naturally infects at least three plant species in Florida: sugarcane (Saccharum spp.), the weed Columbus grass (Sorghum almum) and cultivated sorghum (S. bicolor). All three hosts are also colonized by the sugarcane aphid (Melanaphis sacchari), the main vector of SCYLV worldwide. To understand the high incidence of SCYLV observed in sugarcane commercial fields and in germplasm collections, we investigated the transmission efficiency of SCYLV from sugarcane and Columbus grass to sugarcane using the sugarcane aphid and a spider mite (Oligonychus grypus) that also tested positive for SCYLV in Florida. Healthy and SCYLV-infected leaf pieces of sugarcane and Columbus grass carrying viruliferous aphids or spider mites were transferred to virus-free plants of the yellow leaf susceptible sugarcane cultivar CP96-1252. Three- and 6-months post inoculation, the 108 aphid-inoculated plants of Columbus grass and the 90 mite-inoculated plants of sugarcane tested negative for SCYLV by tissue blot immunoassay (TBIA) or reverse transcription polymerase chain reaction (RT-PCR). Similar results were obtained for 162 aphid-inoculated plants of sugarcane, except for two plants that tested positive for SCYLV by TBIA and RT-PCR. In two field experiments planted with SCYLV-free and virus-infected sugarcane (cultivar CP96-1252), only 18–28% of healthy plants became infected during a 24- to 28-month period. SCYLV prevalence in these field experiments did not differ between aphicide treated and untreated plots. Incidence of M. sacchari haplotypes in the Everglades agricultural area also indicated that the predominant haplotype that is currently colonizing sugarcane was not a vector of SCYLV in Florida. Lack of virus transmission by the spider mite suggested that this arthropod only acquired the virus when feeding on infected plants but was unable to transmit SCYLV. The current vector of SCYLV in Florida remains to be identified.

Wheat streak mosaic virus alters the transcriptome of its vector, wheat curl mite (Aceria tosichella Keifer), to enhance mite development and population expansion.

Wheat streak mosaic virus (WSMV; genus Tritimovirus; family Potyviridae) is an economically important wheat virus that is transmitted by the wheat curl mite (WCM; Aceria tosichella Keifer) in a persistent manner. Virus-vector coevolution may potentially influence vector gene expression to prolong viral association and thus increase virus transmission efficiency and spread. To understand the transcriptomic responses of WCM to WSMV, RNA sequencing was performed to assemble and analyse transcriptomes of WSMV viruliferous and aviruliferous mites. Among 7291 de novo-assembled unigenes, 1020 were differentially expressed between viruliferous and aviruliferous WCMs using edgeR at a false discovery rate ≤0.05. Differentially expressed unigenes were enriched for 108 gene ontology terms, with the majority of the unigenes showing downregulation in viruliferous mites in comparison to only a few unigenes that were upregulated. Protein family and metabolic pathway enrichment analyses revealed that most downregulated unigenes encoded enzymes and proteins linked to stress response, immunity and development. Mechanistically, these predicted changes in mite physiology induced by viral association could be suggestive of pathways needed for promoting virus-vector interactions. Overall, our data suggest that transcriptional changes in viruliferous mites facilitate prolonged viral association and alter WCM development to expedite population expansion, both of which could enhance viral transmission.

Parámetros de transmisión del virus de la leprosis de los cítricos por Brevipalpus yothersi (Acari: Tenuipalpidae)

La leprosis de los cítricos, causada por virus (Citrus Leprosis Virus, CiLV), es un problema fitosanitario de importancia económica y cuarentenaria para la citricultura de países productores. En Colombia su presencia fue confirmada en 2004 en los departamentos del Meta y Casanare; el ácaro Brevipalpus yothersi Baker (antes identificado como Brevipalpus phoenicis Geijskes) (Acari: Tenuipalpidae), se reconoce como el principal vector del virus en el país. Se determinaron los parámetros: período de acceso para adquisición, período de acceso para inoculación y porcentaje de ácaros infectados con CiLV, por medio de pruebas de transmisión y análisis moleculares en el Centro de Investigación “La Libertad” de CORPOICA, Meta, Colombia. Al probar períodos de adquisición entre 10 minutos y 72 horas, se encontró que B. yothersi adquiere el virus después de 30 minutos de alimentación sobre hojas de naranja Valencia (Citrus sinensis L.) Osbeck con lesiones de leprosis. El período mínimo requerido por los ácaros para transmitir el virus hacia plantas de C. sinensis fue de 10 minutos. Para períodos de transmisión desde 10 minutos hasta 24 horas, los porcentajes de infección variaron entre 25 y 68,75 % en las hojas receptoras. De acuerdo a pruebas de transmisión y análisis RT-PCR, después de tres días de alimentación sobre lesiones de leprosis, el 40 % de las poblaciones del ácaro evaluadas adquirió el virus CiLV-C2. Los resultados obtenidos amplían el conocimiento de las interacciones planta - virus - vector, lo cual es fundamental para el establecimiento de programas de prevención y manejo de la enfermedad.

Pest categorisation of Citrus leprosis viruses.

The EFSA Panel on Plant Health performed a pest categorisation of the Citrus leprosis viruses for the EU territory and identified five distinct viruses, Citrus leprosis virus C (CiLV‐C), Citrus leprosis virus C2 (CiLV‐C2), Hibiscus green spot virus 2 (HGSV‐2), the Citrus strain of Orchid fleck virus (OFV) and Citrus leprosis virus N sensu novo (CiLV‐N) as causing this severe disease, most significantly in sweet orange and mandarin. These viruses have in common that they do not cause systemic infections in their hosts and that they all are transmitted by Brevipalpus spp. mites (likely but not confirmed for HGSV‐2). Mites represent the most important means of virus spread, while plants for planting of Citrus are only considered of minor significance. These well characterised viruses occur in South and Central America. Leprosis is currently regulated in directive 2000/29 EC and, together with its associated viruses, has never been recorded in the EU. All five viruses have the potential to enter into, establish in and spread within the EU territory, with plants for planting of non‐regulated hosts, fruits of Citrus and hitch‐hiking of viruliferous mites identified as the most significant pathways. Given the severity of the leprosis disease, the introduction and spread of the various viruses would have negative consequences on the EU citrus industry, the magnitude of which is difficult to evaluate given the uncertainties affecting the Brevipalpus spp. vectors (identity, distribution, density, transmission specificity and efficiency). Overall, leprosis and its five associated viruses meet all the criteria evaluated by EFSA to qualify as Union quarantine pests, but do not fulfil those of being present in the EU or of plants for planting being the main spread mechanism to qualify as Union regulated non‐quarantine pests. The main uncertainties affecting this categorisation concern the Brevipalpus spp. mite vectors.

First Report of Brevipalpus papayensis as Vector of Coffee ringspot virus and Citrus leprosis virus C

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.

Arabidopsis thaliana as a model host for Brevipalpus mite-transmitted viruses

Brevipalpus-transmitted viruses (BTV) are a taxonomically diverse group of plant viruses which severely affect a number of major crops. Members of the group can be sub-classified into cytoplasmic (BTV-C) or nuclear type (BTV-N) according to the accumulation sites of virions in the infected plant cells. Both types of BTV produce only local infections near the point of inoculation by viruliferous mites. Features of BTV-plant interactions such as the failure of systemic spread in their natural hosts are poorly understood. In this study we evaluated Arabidopsis thaliana, a model plant commonly used for the study of plant-virus interactions, as an alternative host for BTV. Infection of Arabidopsis with the BTV-N Coffee ringspot virus and Clerodendrum chlorotic spot virus, and the BTV-C Solanum violaefolium ringspot virus, were mediated by viruliferous Brevipalpus mites collected in the wild. Upon infestation, local lesions appeared in 7 to 10 days on leaves of, at least, 80 % of the assayed plants. Presence of viral particles and characteristic cytopathic effects were detected by transmission electron microscopy (TEM) and the viral identities confirmed by specific reverse-transcriptase polymerase chain reaction (RT-PCR) and further amplicon sequencing. The high infection rate and reproducibility of symptoms of the three different viruses assayed validate A. thaliana as a feasible alternative experimental host for BTV.

Citrus leprosis virus C Infection Results in Hypersensitive-Like Response, Suppression of the JA/ET Plant Defense Pathway and Promotion of the Colonization of Its Mite Vector.

Leprosis is a serious disease of citrus caused by Citrus leprosis virus C (CiLV-C, genus Cilevirus) whose transmission is mediated by false spider mites of the genus Brevipalpus. CiLV-C infection does not systemically spread in any of its known host plants, thus remaining restricted to local lesions around the feeding sites of viruliferous mites. To get insight into this unusual pathosystem, we evaluated the expression profiles of genes involved in defense mechanisms of Arabidopsis thaliana and Citrus sinensis upon infestation with non-viruliferous and viruliferous mites by using reverse-transcription qPCR. These results were analyzed together with the production of reactive oxygen species (ROS) and the appearance of dead cells as assessed by histochemical assays. After interaction with non-viruliferous mites, plants locally accumulated ROS and triggered the salicylic acid (SA) and jasmonate/ethylene (JA/ET) pathways. ERF branch of the JA/ET pathways was highly activated. In contrast, JA pathway genes were markedly suppressed upon the CiLV-C infection mediated by viruliferous mites. Viral infection also intensified the ROS burst and cell death, and enhanced the expression of genes involved in the RNA silencing mechanism and SA pathway. After 13 days of infestation of two sets of Arabidopsis plants with non-viruliferous and viruliferous mites, the number of mites in the CiLV-C infected Arabidopsis plants was significantly higher than in those infested with the non-viruliferous ones. Oviposition of the viruliferous mites occurred preferentially in the CiLV-C infected leaves. Based on these results, we postulated the first model of plant/Brevipalpus mite/cilevirus interaction in which cells surrounding the feeding sites of viruliferous mites typify the outcome of a hypersensitive-like response, whereas viral infection induces changes in the behavior of its vector.

Winter Wheat Cultivars with Temperature-Sensitive Resistance to Wheat streak mosaic virus Do Not Recover from Early-Season Infections.

Wheat streak mosaic virus (WSMV), Triticum mosaic virus, and Wheat mosaic virus, all vectored by the wheat curl mite Aceria tosichella Keifer, frequently cause devastating losses to winter wheat production throughout the central and western Great Plains. Resistant 'Mace' and 'RonL are commercially available and contain the wsm1 and wsm2 genes, respectively, for resistance to WSMV. However, the resistance in these cultivars is temperature sensitive, ineffective above 27°C, and does not protect against the other common wheat viruses. The majority of winter wheat in the Southern Great Plains is planted in early fall as a dual-purpose crop for both grazing and grain production. Early planting exposes wheat plants to warmer temperatures above the threshold for effective resistance. Studies were conducted to determine whether the resistance found in these cultivars would give infected plants the ability to recover as temperatures cooled to a range conducive to effective genetic resistance. RonL, Mace, 'TAM 111', 'TAM 112', and 'Karl 92' wheat were infested with WSMV viruliferous mites at temperatures above the resistance threshold. After the initial 4-week infection period, plants were subjected to progressively cooler temperatures during the winter months, well below the resistance threshold. Throughout the study, plant samples were taken to quantify virus titer and mite populations. Resistant RonL and Mace, which became severely infected during the initial infection period, were not able to recover even when temperatures dropped below the resistance threshold. However, TAM 112 showed resistance to WSMV but, more importantly, it also showed resistance to the wheat curl mite, because the mite population in this cultivar was significantly lower than on all other cultivars. The results of this study are significant in that they represent the first evidence of quantitative resistance to both WSMV and the wheat curl mite in a single wheat cultivar. Resistance to the wheat curl mite has potential to reduce losses to all mite-vectored virus diseases of wheat and not just WSMV.

Experimental host range of Citrus leprosis virus C (CiLV-C)

Citrus leprosis (CL) is a serious threat to the citrus industry, especially for sweet oranges. For a long time, Citrus spp. were considered the only susceptible hosts. However, other plant species were also found either experimentally or naturally to be susceptible to Citrus leprosis virus C (CiLV-C). To assess the experimental host range of CiLV-C, a large number of plant species were inoculated with Brevipalpus phoenicis, viruliferous to CiLV-C, under experimental conditions. Out of the 140 tested species (43 families), 59 species (24 families) developed localized chlorotic and/or necrotic lesions upon inoculation of leaves with viruliferous mites, and 40 species (18 families) of them yielded positive results for CiLV-C detection in at least one of the following assays: ELISA, RT-PCR, transmission electron microscopy and immunfluorescence. For those that developed lesions and yielded negative results in CiLV-C detection assays, the results may be attributed to the small number of lesions and their necrotic state with very little viral material. The fact that a considerable number of plant species are susceptible to the virus after mite inoculation brings up implications for the epidemiology, quarantine and evolution of the citrus leprosis pathosystem.

Common Bean: Experimental Indicator Plant for Citrus leprosis virus C and Some Other Cytoplasmic-Type Brevipalpus-Transmitted Viruses.

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.

Citrus leprosis virus C infects Arabidopsis thaliana, the model for plant-pathogen interactions

Caused by Citrus leprosis virus C (CiLV-C) (genus Cilevirus) and vectored by Brevipalpus phoenicis Geijskes, leprosis is one of the main diseases of the Brazilian citrus industry. Although this disease was reported decades ago, the mechanisms involved in plant- virus interaction are still largely unknown. Since Arabidopsis thaliana has emerged as the major experimental model for essentially all aspects of plant biology, we tested the ability of CiLV-C to infect this model plant, evaluating the response of 12 ecotypes: Bur-0, Kas-1, Van- 0, Bay-0, C-24, Col-0, Ts-1, Tsu-1, Bla-2, Kon, Sha and Nfa-8. Four-week-old plants grown under 16 h photoperiod and 22°C were infested with two viruliferous or non viruliferous mites, or were not exposed to mites. Each treatment had twelve replicates of each ecotype. Localized necrotic lesions in green and senescent leaves and localized green lesions in senescent leaves appeared after 5-10 days only in plants infested with viruliferous mites. Thin-sectioned symptomatic tissues contained short bacilliform particles and viroplasm similar to those found in citrus. RT-PCR assays using primers that amplify part of the CiLV- C movement protein gene (Locali et al., 2003) yielded amplicons from all symptomatic plants, which were sequenced, proving identical to those obtained from the naturally infected CiLV-C source. CiLV-C was able to infect all of the tested A. thaliana ecotypes with reactions that identified three resistance level groups: more susceptible (Bur-0, Kas-1, Van-0, Bay-0, and C-24), intermediate (Col-0, Ts-1, and Tsu-1) and more resistant (Bla-2, Kon, Sha, and Nfa-8). Besides extending the range of CiLV-C hosts to a member of the family Brassicaceae, the use of A. thaliana in further studies will provide the opportunity for uncovering mechanisms involved in host-virus interaction.

Clinical signs of deformed wing virus infection are predictive markers for honey bee colony losses

The ectoparasitic mite Varroa destructor acting as a virus vector constitutes a central mechanism for losses of managed honey bee, Apis mellifera, colonies. This creates demand for an easy, accurate and cheap diagnostic tool to estimate the impact of viruliferous mites in the field. Here we evaluated whether the clinical signs of the ubiquitous and mite-transmitted deformed wing virus (DWV) can be predictive markers of winter losses. In fall and winter 2007/2008, A.m. carnica workers with apparent wing deformities were counted daily in traps installed on 29 queenright colonies. The data show that colonies which later died had a significantly higher proportion of workers with wing deformities than did those which survived. There was a significant positive correlation between V. destructor infestation levels and the number of workers displaying DWV clinical signs, further supporting the mite’s impact on virus infections at the colony level. A logistic regression model suggests that colony size, the number of workers with wing deformities and V. destructor infestation levels constitute predictive markers for winter colony losses in this order of importance and ease of evaluation.

DETECTION OF FIG MOSAIC VIRUS IN VIRULIFEROUS ERIOPHYID MITE ACERIA FICUS

Fig leaves showing typical fig mosaic symptoms on cv. Bursa siyahi (donor plant) were cut under a stereo microscope into small pieces each hosting 10 putatively viruliferous eriophyid mites (Aceria ficus Cotte) and placed directly on the top leaves of healthy Cucumis sativus, Chenopodium quinoa. C. amaranticolor, Nicotiana occidentalis, Catharanthus roseus, Fraxinus excelsior plants, and fig seedlings. Donor and test plants were analyzed by electron microscopy, RT-PCR and sequencing, whereas the mites (ErMs) underwent molecular assays using Fig mosaic virus (FMV)-specific primers. Mite-infested leaves of fig seedlings and C. roseus showed small yellowish spots after 10 days and 6 weeks, respectively, whereas no symptoms were observed in other test or control plants for three months. Electron microscopy observations showed the occurrence of double membrane bodies (DMBs) in the palisade cells of donor and mite-inoculated fig plants, but not in C. roseus. However, 302 bp RT-PCR products specific to FMV were amplified from donor and inoculated figs, C. roseus and ErMs. Nucleotide identity with the sequence of the FMV isolate in GenBank (accession No. AM941711.6) was 87%, 89% and 87% for donor plant (JQ708183), inoculated fig seedlings (JQ708184) and C. roseus (JQ408437, JQ408438), respectively. The sequences obtained from ErMs (JQ408432, JQ408436) showed 87% and 88% nucleotide identity with the reference FMV isolate, respectively. When dsRNA extracts were analyzed to confirm virus presence in inoculated periwinkles, a complex dsRNA profile was obtained, suggestive of infection by a multipartite virus or by multiple viruses. Sequence from RT-PCR amplicons of dsRNA (JX040436) showed 88% identity with those the reference FMV isolate (AM941716.1) and the donor plant (JQ708183). According to these results, Madagascar periwinkle (C. roseus) can be retained as a new experimental host for FMV and A. ficus appears to be able to transmit FMV from fig to periwinkle plants.

Citrus leprosis virus C Naturally Infecting Commelina benghalensis, a Prevalent Monocot Weed of Citrus Orchards in Brazil.

There are two bacilliform, rhabdo-like viruses that cause citrus leprosis: Citrus leprosis virus C (CiLV-C), which accumulates in the cytoplasm of infected cells, and Citrus leprosis virus nuclear type (CiLV-N), which accumulates in their nucleus. The first one, the prototype of the new genus Cilevirus, is prevalent and occurs in several countries of the American continent, from Argentina to Mexico (1). The second type, still a tentative member of the Rhabdoviridae family, is of rare occurrence, with a few reports in Brazil and one in Panama (1). Leprosis is particularly important to the Brazilian citrus industry because of the 60 to 80 million dollars spent yearly for the control of Brevipalpus phoenicis (Geijskes, 1939) (Acari: Tenuipalpidae), the vector of the virus (1). For decades, the disease was considered unique to citrus plants; however, greenhouse experiments conducted in the 1990s demonstrated the mechanical transmission of CiLV-C to noncitrus plants (1). Years later, researchers were able to transmit the virus to nonrutaceous hosts using viruliferous mites (1,4). Recently, León et al. (2) reported the occurrence of the first noncitrus plant naturally infected by CiLV-C, the rutaceous Swinglea glutinosa Blanco (Merr.). Tropical spiderworts (Commelina benghalensis L.; Commelinaceae) are monocot weeds commonly found in citrus orchards in Brazil. In a survey conducted in orchards with high incidences of leprosis in the municipalities of Borborema and São José do Rio Preto, State of Sao Paulo, Brazil, tropical spiderworts were found exhibiting necrotic spots with a yellow halo in green leaves and green spots with necrotic center in senescent leaves. Since these symptoms are similar to those caused by CiLV-C in citrus, symptomatic plants were collected and subjected to transmission electron microscopy analyses and reverse transcription-PCR using primers that specifically amplify a region within the putative movement protein gene of the virus (3). Bacilliform virus particles and typical inclusions were seen in the lesions. Bands of the expected 344 bp size were seen in agarose gels of symptomatic samples only. The analysis of the consensus sequence showed 100% identity with CiLV-C sequence available in the GenBank (Accession No. YP_654542.1). Experimental transmission of CiLV-C by B. phoenicis reproduced the lesions in inoculated tropical spiderwort. Also, the virus could be easily transmitted back from C. benghalensis to sweet orange plants. Our data show that this widespread weed is the first monocot as a natural host for CiLV-C. Since tropical spiderwort is a host for B. phoenicis and symptomatic plants were found in two municipalities 130 km apart from each other, it is possible that this weed may serve as reservoir for the virus and play a relevant role in the spread of the disease in the field, but this issue still needs to be addressed in further experiments.

Quantitative trait loci analysis of citrus leprosis resistance in an interspecific backcross family of (Citrus reticulata Blanco × C. sinensis L. Osbeck) × C. sinensis L. Osb

Leprosis, caused by citrus leprosis virus (CiLV) and transmitted by the tenuipalpid mite Brevipalpus phoenicis, is one of the most important viruses of citrus in the Americas. Sweet oranges (Citrus sinensis L. Osb.) are highly susceptible to CiLV, while mandarins (C. reticulata Blanco) and some of their hybrids have higher tolerance or resistance to this disease. The mechanisms involved in the resistance and its inheritance are still largely unknown. To study the quantitative trait loci (QTL; quantitative trait loci) associated with the resistance to CiLV, progeny analyses were established with 143 hybrid individuals of ‘Pera’ sweet orange (C. sinensis L. Osb.) and ‘Murcott’ tangor (C. reticulata Blanco × C. sinensis L. Osb.) from controlled crossings. Disease assessment of the hybrid individuals was conducted by infesting the plants with viruliferous mites in the field. The experiment consisted of a randomized completely block design with ten replicates. The evaluated phenotypic traits were incidence and severity of the disease on leaves and branches, for a period of 3 years. The MapQTL™ v.4.0 software was used for the identification and location of possible QTL associated with resistance to CiLV on a genetic map obtained from 260 AFLP and 5 RAPD markers. Only consistent QTLs from different phenotypic traits and years of evaluation, with the critical LOD scores to determine the presence or absence of each QTL calculated through the random permutation test, were considered. A QTL was observed and had a significant effect on the phenotypic variation, ranging from 79.4 to 84% depending on which trait (incidence or severity) was assessed. This suggests that few genes are involved in the genetic resistance of citrus to CiLV.

Detection of pigeonpea sterility mosaic virus, the causal agent of sterility mosaic disease of pigeonpea in viruliferous mite vector by DAS-ELISA and DIBA

The sterility mosaic virus in pigeonpea is transmitted by the eriophyid mite Aeria cajani. Serological diagnostic methods like DAS-ELISA and DIBA were developed to detect the presence of Pigeonpea Sterility Mosaic Virus (PPSMV) from their viruliferous vector using whole mite extract as an antigen. Both DAS-ELISA and DIBA were sensitive for virus detection in vector mites. Extract from 180 mites gave a strong positive reaction. A weak positive reaction was observed in a minimum of 10 viruliferous mites by DAS-ELISA. Whereas the virus was detected from the extract of a minimum of 5 mites by DIBA. This indicated that DIBA is more sensitive for virus detection in vector mites when compared to DAS-ELISA.