Aline Daniele Tassi

Confirmation of the presence of the Citrus leprosis virus C (CiLV-C) in Southern Mexico

Citrus leprosis was detected in sweet oranges in Chiapas, Mexico, in 2005 based on symptoms. The disease was soon after observed in sweet orange orchards at Huimanguillo and Cunduacan, state of Tabasco. Leaf samples of leprosis-affected Valencia or Hamlin sweet oranges were collected and subjected to ultrastructural examination and molecular detection of CiLV-C by RT-PCR. Cytopathic effects typical of CiLV-C infection as presence of short, baciliform particles in the endoplasmic reticulum cisternae and electron dense, vacuolated viroplasma in the cytoplasm, as well as hypertrophy of some spongy parenchyma cells were observed in the tissues of the leaf lesions. RT-PCR using specific primers for CiLV-C produced DNA fragments of the expected size. Brevipalpus mites were present in these orchards and identified as B. phoenicis, the known vector for CiLV-C. Transmission tests with these mites confirmed their role as leprosis vectors to sweet orange but not to lemon, Volkamerian lemon, C35 citrange and Carrizo citrange. These data confirm the presence of CiLV-C in Tabasco, Mexico and that the citrus leprosis found in Chiapas was caused by CiLV-C.

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.

Citrus leprosis virus N: A New Dichorhavirus Causing Citrus Leprosis Disease

Citrus leprosis (CL) is a viral disease endemic to the Western Hemisphere that produces local necrotic and chlorotic lesions on leaves, branches, and fruit and causes serious yield reduction in citrus orchards. Samples of sweet orange (Citrus × sinensis) trees showing CL symptoms were collected during a survey in noncommercial citrus areas in the southeast region of Brazil in 2013 to 2016. Transmission electron microscopy analyses of foliar lesions confirmed the presence of rod-like viral particles commonly associated with CL in the nucleus and cytoplasm of infected cells. However, every attempt to identify these particles by reverse-transcription polymerase chain reaction tests failed, even though all described primers for the detection of known CL-causing cileviruses and dichorhaviruses were used. Next-generation sequencing of total RNA extracts from three symptomatic samples revealed the genome of distinct, although highly related (>92% nucleotide sequence identity), viruses whose genetic organization is similar to that of dichorhaviruses. The genome sequence of these viruses showed <62% nucleotide sequence identity with those of orchid fleck virus and coffee ringspot virus. Globally, the deduced amino acid sequences of the open reading frames they encode share 32.7 to 63.8% identity with the proteins of the dichorhavirids. Mites collected from both the naturally infected citrus trees and those used for the transmission of one of the characterized isolates to Arabidopsis plants were anatomically recognized as Brevipalpus phoenicis sensu stricto. Molecular and biological features indicate that the identified viruses belong to a new species of CL-associated dichorhavirus, which we propose to call Citrus leprosis N dichorhavirus. Our results, while emphasizing the increasing diversity of viruses causing CL disease, lead to a reevaluation of the nomenclature of those viruses assigned to the genus Dichorhavirus. In this regard, a comprehensive discussion is presented.

Present status of citrus leprosis in Argentina and Paraguay

Citrus leprosis (CL) was first described in South America in the 1920s. It is considered similar to a disease first observed back to 1860 in Florida. It is a destructive disease characterized by localized lesions on the leaves, fruits and stems, which may lead to the death of the affected plant if left untreated. Around 1940, CL was demonstrated to be transmitted by Brevipalpus mites in Argentina. Because little information exists on the status of CL pathosystem in Argentina and Paraguay, a survey was made in several citrus growing areas of these countries from 2009 to 2011, to evaluate its presence and relevance as well as the identification of the virus and the mite vector. CL was found in most of the sweet orange and/or mandarin orchards in Paraguay (Departamentos de Boqueron, Concepcion, San Pedro, Cordillera, Alto Parana, Itapua) and Argentina (Provincias de Misiones, Corrientes, Entre Rios). Incidence was usually low. The causal virus was identified as Citrus leprosis virus C (CiLV-C) by RT-PCR, electron microscopy and immunofluorescence. In all the visited regions in Paraguay and the region of Montecarlo, Argentina, the mites collected in plants infected by CiLV-C were identified as B. phoenicis.

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.

First report of Brevipalpus papayensis Baker (Acari: Tenuipalpidae) as vector of Coffee ringspot virusand 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.

Unveiling the complete genome sequence of clerodendrum chlorotic spot virus, a putative dichorhavirus infecting ornamental plants

The genus Dichorhavirus includes plant-infecting rhabdoviruses with bisegmented genomes that are horizontally transmitted by false spider mites of the genus Brevipalpus. The complete genome sequences of three isolates of the putative dichorhavirus clerodendrum chlorotic spot virus were determined using next-generation sequencing (Illumina) and traditional RT-PCR. Their genome organization, sequence similarity and phylogenetic relationship to other viruses, and transmissibility by Brevipalpus yothersi mites support the assignment of these viruses to a new species of dichorhavirus, as suggested previously. New data are discussed stressing the reliability of the current rules for species demarcation and taxonomic status criteria within the genus Dichorhavirus.

Dichorhaviruses in Their Host Plants and Mite Vectors

A group of related bacilliform, nuclear viruses with a bisegmented negative-sense RNA genome that are transmitted by Brevipalpus mites likely in a circulative-propagative manner were recently classified in the new genus Dichorhavirus, family Rhabdoviridae. These viruses cause localized lesions on leaves, stems, and fruits of economically significant horticultural and ornamental plant species. Among its members, orchid fleck virus, citrus leprosis virus N, and coffee ringspot virus are most prominent. This chapter summarizes the current knowledge about these viruses, available detection techniques, and their interactions with their plant hosts and mite vectors.

Brevipalpus-transmitted viruses: parallelism beyond a common vector or convergent evolution of distantly related pathogens?

Although diseases caused by Brevipalpus-transmitted viruses (BTV) became relevant for agriculture a century ago, their causal agents have been only recently characterized and classified in two new genera of plant-infecting viruses: Cilevirus and Dichorhavirus. In this review, we highlight both similarities and differences between these viruses emphasizing their current taxonomy and historical classification, phylogeny, genomic organization, gene expression, and the latest research developments on BTVs. Additionally, we stress particular features of interactions with their mite vectors and plant hosts that support, from an evolutionary perspective, the potential convergence of both viral groups.

First report of orchid fleck virus and its mite vector on green cordyline

Orchid fleck virus (OFV) and its mite vector Brevipalpus californicus were for the first time identified on green cordyline plants showing distinctive chlorotic and necrotic ringspots. Thin section electron microscopy revealed bacilliform, dichorhavirus-like particles within nuclear viroplasms. RT-PCR using OFV degenerate primers yielded a single amplicon, the nucleotide sequence of which closely matched the nucleoprotein gene of OFV.