Abstract The citrus leprosis is responsible for causing major damage in fruit production. The disease, in the stems, is characterized by caused “scaly bark” lesions of reddish tone, depending on the stage they can be called blisters and make ruptures in the longitudinal direction. The aim of this study was to analyze the evolution of leprosis lesions of type CiLV-C and the anatomical alterations in the twigs of field-collected plants in order to compare to the evolution of the foliar and twigs lesions of inoculated plants already described in literature. For anatomical analysis, the samples were fixed in Karnovsky solution, dehydrated in a graded ethylic series, embedded in hidroxy-ethyl methacrylate resin (Leica Historesin), sectioned (5-7 μm thick), and stained with Toluidine blue for usual histological analysis. Histochemical tests were also carried out. The digital images were capture in a microscope with video camera. The analyzed lesion followed the same pattern as described to foliar lesions, i.e., there was a necrotic center surrounded by a halo also necrotic. In the necrotic region there was the accumulation of lipid compounds. The cortical and, or phloematic parenchyma exhibited hyperplasia. In both lesions it was verified the decrease of starch amount and the increase of quantity of prismatic calcium oxalate crystals, and the presence of gummosis traumatic ducts. production disease stems scaly bark tone direction CiLVC CiLV C fieldcollected field collected literature analysis solution series hidroxyethyl hidroxy ethyl Leica Historesin, Historesin , Historesin) 57 5 7 (5- thick, thick thick) out camera ie i e i.e. compounds hyperplasia crystals ducts (5 i.e (

Resumo A leprose dos citros é responsável por causar grandes danos à citricultura. Nos troncos e ramos a doença caracteriza-se por causar lesões corticosas de tonalidade avermelhada. Dependendo do estágio da doença, as lesões podem ser denominadas pústulas e apresentar fendas no sentido longitudinal. O objetivo desse estudo foi analisar a evolução das lesões da leprose do tipo CiLV - C e as alterações anatômicas nos ramos de plantas provenientes do campo a fim de compará-las com a evolução das lesões foliares e com as lesões de ramos de plantas inoculadas descritas na literatura. Para tal, amostras de lesões em diferentes estágios de desenvolvimento foram coletadas, fotografadas e fixadas em solução de Karnovsky. Posteriormente, as amostras foram submetidas à desidratação em série etílica e infiltradas em resina hidroxi-etil-metacrilato (Leica Historesin). As amostras foram seccionadas em micrótomo rotativo a 5-7 μm de espessura e coradas com azul de toluidina para as análises histológicas usuais. Também foram realizados testes histoquímicos. A captura de imagens digitais dos materiais preparados em lâminas foi realizada em microscópio equipado com câmera de vídeo. A evolução das lesões nos ramos seguiu o mesmo padrão descrito para as lesões foliares, ou seja, houve a formação de uma pontuação necrótica central e de um halo necrótico circundante. Na região da necrose havia o acúmulo de compostos lipídicos. O parênquima cortical e floemático apresentava hiperplasia. Em ambas as lesões houve a diminuição da quantidade de amido, aumento de cristais de oxalato de cálcio e a presença de ductos traumáticos gomosos. citricultura caracterizase caracteriza se avermelhada longitudinal comparálas compará las literatura tal coletadas Karnovsky Posteriormente hidroxietilmetacrilato hidroxi etil metacrilato Leica Historesin. Historesin . Historesin) 57 5 7 5- usuais histoquímicos vídeo seja circundante lipídicos hiperplasia amido gomosos

ABSTRACT Zinnia sp. and hairy beggartick (Bidens pilosa) plants exhibiting symptoms of possible virus infection were found in the municipality of Santa Bárbara d’Oeste, São Paulo State, Brazil. Flexuous filamentous particles and cytoplasmatic inclusions typical of potyvirus infection were observed by transmission electron microscopy, respectively, in leaf extracts and cells of symptomatic leaves. Infection of both plants with bidens mottle virus (BiMoV) was confirmed by RT-PCR using potyvirus universal primers, followed by nucleotide sequencing of the amplicons. The nearly complete genome sequence of the Brazilian isolate, named BiMoV-BR, is 9700 nucleotides long and shares 95.6 % identity with the corresponding nucleotide sequence of a BiMoV isolate from the United States. BiMoV-BR was mechanically transmitted and caused systemic infection on plants of Zinnia sp., hairy beggarstick, sunflower (Helianthus annuus), and lettuce (Lactuca sativa). Myzus persicae transmitted the virus to Zinnia sp. plants with efficacy of 8 % and 42 %, using one and ten aphids per plant, respectively. This is the first detection of BiMoV in Brazil. Further studies are necessary to evaluate the distribution of this potyvirus in the country. sp Bidens pilosa dOeste, dOeste d Oeste, Oeste d’Oeste State Brazil microscopy respectively leaves (BiMoV RTPCR RT PCR primers amplicons BiMoVBR, BiMoVBR BR, BR 970 956 95 6 95. States beggarstick Helianthus annuus, annuus , annuus) Lactuca sativa. sativa . sativa) 4 plant country 97 9

ABSTRACT Sunflower (Helianthus annuus) plants showing symptoms of chlorosis, mosaic, chlorotic ringspot, and necrosis on younger leaves were found in a small experimental plot in Piracicaba, in the state of São Paulo, Brazil. Preliminary examinations by transmission electron microscopy of symptomatic leaf tissue revealed flexuous filamentous particles 13-15 nm wide and 700-750 nm long, and cytoplasmatic cylindrical inclusions typical of those found in plant cells infected by members of the Potyvirus genus. Total RNA extracted from symptomatic leaves and subjected to RT-PCR followed by partial nucleotide sequencing confirmed the presence of a potyvirus in the affected plants, which was identified as sunflower chlorotic mottle virus (SuCMoV), a member of the Sunflower chlorotic mottle virus (genus Potyvirus, family Potyviridae) species. Mechanical transmission assays with extracts of symptomatic sunflower leaves reproduced the original symptoms in sunflowers, mosaic symptoms in Zinnia elegans, and chlorotic local lesions in Chenopodium amaranticolor and C. quinoa. Sunflower and zinnia plants became infected after aphid transmission experiments with Myzus persicae. RT-PCR tests using specific primers for SuCMoV confirmed the presence of this virus in experimentally infected plants, meeting the criteria of Koch’s postulate. This is the first report of SuCMoV infecting sunflower plants in Brazil.

ABSTRACT Tomato fruit blotch virus (ToFBV) is a blunervirus that causes blotches on mature tomato (Solanum lycopersicon L.) fruits in Italy and Australia in 2020, and was newly detected in Brazil. A cytological study on pericarp tissues from the blotched areas of infected fruits collected in Brasília, Brazil, revealed characteristic cell alterations. Small and slender bacilliform particles (ca. 25 nm wide × 100 nm long) were found accumulating in the perinuclear space and the lumen of the endoplasmic reticulum of the epidermis, peri- and mesocarp cells. No viroplasm-like inclusion was observed either in the nuclei or in the cytoplasm. Such cell alterations are reminiscent of those described in cultured mosquito cells infected by negeviruses, an unofficial group of insect viruses. Negeviruses and some other arthropod-borne viruses shared a common ancestor in the RdRp gene with kitavirids, including blunerviruses. Although additional detailed studies are required, we show evidence that ToFBV particles are enveloped and bacilliform, and that such similarity in cytopathology seems to support the evolutionary relationship between plant kitavirids and insect negeviruses.

ABSTRACT: Physalis is an herbaceous species native to the Andes region. Currently, it is cultivated in various Brazilian states due to the economic interest of growers for this new fruit. Physalis plants grown in the field showed symptoms of shoot proliferation, leaf malformation, and chlorosis. Since these symptoms are commonly induced by phytoplasmas, this study investigated to confirm the presence of these prokaryotes in symptomatic plants. After DNA extraction from symptomatic and asymptomatic plants, phytoplasmas were found in all affected plants through the nested PCR. Examination by transmission electron microscopy (TEM) using appropriately prepared segments of leaf veins allowed the visualization of typical pleomorphic cells of phytoplasmas in the phloem of symptomatic plants. The computer-simulated RFLP patterns and the phylogenetic analysis allowed identifying the detected phytoplasmas as a ‘Candidatus Phytoplasma fraxini’-related strain belonging to the 16SrVII-B subgroup. Moreover, physalis was identified as an additional host species for phytoplasmas in the 16SrVII group, expanding the current knowledge on the host range of phytoplasmas in this group.

ABSTRACT Lima bean plants showing mosaic and leaf distortion symptoms, similar to those induced by viruses, were observed in several production areas in the states of Ceará and Piauí, Northeastern Brazil. The aim of this study was to identify RNA genome viruses that infect lima bean. Field research was conducted from 2017 to 2018. Fifty-five symptomatic samples were randomly collected from seven properties in five municipalities and tested by RT-PCR and DNA sequencing with specific or universal primers for two viruses and two virus groups. Four virus species were identified: cucumber mosaic virus (CMV), cowpea mild mottle virus (CPMMV), cowpea aphid-borne mosaic virus (CABMV) and cowpea severe mosaic virus (CPSMV), which had infection levels of 21.8, 52.7, 47.2, and 1.8%, respectively, as well as double and triple infections. The CMV isolates belonged to subgroup IA. The CPMMV isolates had high nucleotide identity with CPMMV isolates from Brazil, USA and Mexico. The CABMV isolates showed moderate nucleotide identity with Brazilian isolates. Only one sample was infected with CPSMV. This is the first record of CPMMV and CPSMV naturally infecting lima bean. Approaches to virus control are discussed.

ABSTRACT: Sesame (Sesamum indicum L.) plants exhibiting symptoms of phyllody disease were observed in commercial fields in Paraguay. The symptoms were indicative of infection by phytoplasmas. Thus, the present study investigated the association between affected plants and phytoplasma, which was later analyzed using molecular and phylogenetic methods. Total DNA was extracted from symptomatic and asymptomatic plants and used in nested PCR assays using primers R16SN910601/R16SN011119 and R16F2n/16R2. Amplified products of 1.2 Kb revealed the presence of phytoplasma in all diseased plants, and electron microscopy confirmed the presence of phytoplasmas within phloem vessels. Nucleotide sequences from sesame phytoplasma shared 99 % similarity with phytoplasmas belonging to group 16SrI. Computer-simulated RFLP indicated that the detected phytoplasma is representative of the 16SrI-B, therefore, a ‘Candidatus Phytoplasma asteris’-related strain. Phylogenetic analysis was in agreement with virtual RFLP. Our findings expand the current knowledge regarding distribution of representatives of the aster yellows group in a new agroecosystem and implicate sesame as a new host of 16SrI-B phytoplasma in Latin America.

ABSTRACT: 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.

Catharanthus roseus is a perennial, evergreen herb in the family Apocynaceae, which is used as ornamental and for popular medicine to treat a wide assortment of human diseases. This paper describes a new potyvirus found causing mosaic symptom, foliar malformation and flower variegation in C. roseus. Of 28 test-plants inoculated mechanically with this potyvirus, only C. roseus and Nicotiana benthamiana developed systemic mosaic, whereas Chenopodium amaranticolor and C. quinoa exhibited chlorotic local lesions. The virus was transmitted by Aphis gossypii and Myzus nicotianae. When the nucleotide sequence of the CP gene (768nt) was compared with other members of the Potyviridae family, the highest identities varied from 67 to 76 %. For the 3' UTR (286nt), identities varied from 16.8 to 28.6 %. The name Catharanthus mosaic virus (CatMV) is proposed for this new potyvirus.

Coffee ringspot is a minor coffee disease caused by the nuclear type of Brevipalpus mite-transmitted virus, Coffee ringspot virus (CoRSV). Recently outbreaks of the disease in some growing regions of the state of Minas Gerais, Brazil, were registered with qualitative and quantitative yield losses. Coffea arabica was the only species registered as natural host. A survey was made on a germplasm collection of Coffea and related species kept at the Centro de Café "Alcides Carvalho", Instituto Agronômico, Campinas, state of São Paulo (SP), Brazil, to assess natural susceptibility of Coffee species, other than C. arabica and some interspecific hybrids of Coffea as well as other non-Coffea plant species to the Coffee ringspot virus (CoRSV). The following plants were found with ringspot symptoms on their leaves and/or fruits besides C. arabica L.: C. kapakata (IAC 4511), C. dewevrei cv. Excelsa, C. canephora cv. Robusta, hybrid derivative of the C. arabica × C. racemosa (IAC1195-5-6-2), C. arabica × C. dewerei (Piatã IAC 387), Híbrido de Timor CIFC 832/1 (derivative from a natural crossing between C. arabica × C. canephora) and C. racemosa. Also Psilanthus ebracteolatus, a species close to the genus Coffee was also found with ringspot lesions on their leaves. All these plants were also found infested by Brevipalpus mites identified as B. phoenicis. Infection of these plants by CoRSV was confirmed by the observation of characteristic cytopathic effects in the tissues of the lesion and by RT-PCR using a pair of primer specific for CoRSV. Only with C. racemosa RT-PCR failed to amplify the CoRSV genome. The susceptibility of P. ebracteolatus to CoRSV adds new dimension regarding its controversial taxonomic position.

O gênero botânico Clerodendrum pertence à família Lamiaceae e compreende várias espécies ornamentais, Manchas cloróticas e necróticas em folhas de coração-sangrento foram observadas pela primeira vez em um jardim de Piracicaba, SP, associadas à infestação com Brevipalpus phoenicis (Acari: Tenuipalpidae). Exames de secções de tecidos das lesões foliares ao microscópio eletrônico revelaram ocorrência de efeitos citopáticos do tipo nuclear e concluiu-se que os sintomas eram causados por um vírus transmitido por Brevipalpus (VTB), o qual foi designado de mancha clorótica de Clerodendrum (Clerodendrum Chlorotic Spot Virus- ClCSV). O ClCSV é transmitido mecanicamente de coração-sangrento para coração-sangrento. Em ensaios preliminares foi transmitido por B. phoenicis e mecanicamente para várias outras plantas, além da ocorrência de sua disseminação natural por este ácaro para outras espécies. Visando complementar a caracterização do ClCSV foram feitos estudos sobre alterações anatômicas em folhas de plantas infectadas pelo ClCSV. Foram examinadas secções histológicas de folhas sadias e infectadas pelo ClCSV de C. x speciosum e de outras hospedeiras como Hibiscus schizopetalus, Salvia leucantha, Malvaviscus arboreus e Annona muricata. Constatou-se que o ClCSV causa alterações celulares semelhantes nas diferentes hospedeiras e os sintomas causados por este vírus são similares aos causados por outros vírus transmitidos por Brevipalpus como o vírus da leprose dos citros citoplasmático (Citrus Lepros Virus Cytoplasmic- CiLV-C) e nuclear (Citrus Leprosis Virus Nuclear- CiLV-N), mancha anular do cafeeiro (Coffee Ringspot Virus- CoRSV), mancha anular de Solanum violaefolium (Solanum violaefolium Ringspot Virus- SvRSV) e "Orchid Fleck Vírus" (OFV), representadas por hipertrofia e hiperplasia frequentemente acompanhadas de necrose nos tecidos do parênquima paliçádico e lacunoso.

The botanical genus Clerodendrum belongs to the family Lamiaceae and includes several ornamental species. Necrotic and chlorotic spots on leaves of bleeding heart were observed for the first time in a residential garden from Piracicaba Municipality, São Paulo State, Brazil, associated with an infestation by the flat mite Brevipalpus phoenicis (Acari: Tenuipalpidae). Tissue section analysis of the leaf lesions under transmission electron microscope showed characteristic cytopathic effects of the nuclear type, indicating that the symptoms were caused by a Brevipalpus-transmitted virus (BTV), named Clerodendrum Chlorotic Spot Virus (ClCSV). This virus was shown to be transmitted mechanically and by B. phoenicis to several plant species. In order to complete ClCSV characterization, anatomical changes in the leaf lesions of ClCSV-infected leaves were evaluated. Histological sections of healthy, control leaves and leaf lesions of ClCSV-infected plants of C. x speciosum, Hibiscus schizopetalus, Salvia leucantha, Malvaviscus arboreus and Annona muricata were analyzed. ClCSV infection resulted in similar tissue alterations caused by other BTV as Citrus Leprosies Virus Cytoplasmic (CiLV-C) and Nuclear (CiLV-N), Coffee Ringspot Virus (CoRSV), Solanum violaefolium Ringspot Virus (SvRSV) and Orchid Fleck Virus (OFV). Such symptoms consisted of hypertrophy and hyperplasia frequently followed by necrosis in palisade and spongy parenchyma tissues.

Plantas de campânula (Campanula medium) exibindo mosaico e necrose foliar e anéis em flores foram coletadas em uma estufa comercial de flores na região de Atibaia, SP. Suspeitando de possível etiologia viral, amostras de tecido lesionado foram analisadas por ensaios de transmissão mecânica, microscopia eletrônica e sorologia. Todos os resultados apontaram para a presença do Tomato spotted wilt virus (TSWV) como o responsável pelos sintomas. Esse é o primeiro relato deste patógeno em campânula no Brasil.

Plants of bellflower (Campanula medium) exhibiting symptoms of mosaic, leaf necrosis and flower ring spot were found in a commercial crop in a greenhouse in Atibaia, São Paulo State. Electron microscopy, serology and biological assays indicated the presence of Tomato spotted wilt virus (TSWV). This is the first report of this pathogen in bellflower plants in Brazil.

Os primeiros casos de plantas ornamentais encontradas naturalmente infetadas por vírus transmitidos por Brevipalpus (Acari: Tenuipalpidae) (VTB) foram registrados nos anos 1990 na região de Piracicaba, Estado de São Paulo, e ocorrências subseqüentes foram observadas em várias outras regiões do país e de outros países das Américas. Atualmente acham-se relatadas 37 espécies de ornamentais (para efeito de simplificação, orquídeas foram consideradas como única espécie) pertencentes a 18 famílias botânicas. Pelo fato de causarem apenas infecções localizadas, geralmente nas folhas, VTB em ornamentais não causam preocupações aos produtores, mas potencialmente podem causar perdas econômicas se ocorrerem explosões populacionais do ácaro vetor. Plantas ornamentais podem servir de reservatório de VTB de importância econômica como os vírus da leprose dos citros-tipo citoplasmático (CiLV-C), da mancha verde do maracujá (PFGSV) e da mancha anular do cafeeiro (CoRSV).

The first cases of ornamental plants found infected by Brevipalpus transmitted viruses (BTV) were described in the 1990's from the region of Piracicaba, State of São Paulo, Brazil; subsequent cases were from other regions in the country and other American countries. Currently, 37 ornamental plant species (for the sake of simplicity, orchids being considered as a single species), belonging to 18 families of dicotyledons, have been reported hosting BTV. Because of the non systemic type of infection of these viruses, the localized diseases they cause are unimportant usually, but they have the potential to cause economic losses if severe outbreaks of Brevipalpus mite populations occur. Some ornamentals may serve as reservoirs to BTV known to cause serious damage to food crops as Citrus leprosis virus- cytoplasmic type (CiLV-C), passion fruit green spot virus (PFGSV) and Coffee ringspot virus (CoRSV).

Um estudo da ontogênese das caneluras induzidas em ramos de laranjeiras doces suscetíveis por isolados severos do vírus da tristeza dos citros (Citrus tristeza vírus - CTV) foi feito usando-se como modelo pedúnculos florais e de frutos. O menor calibre destes órgãos permite um melhor acompanhamento do processo. As observações foram feitas em laranjeira cv. Pêra infetada pelo isolado severo Capão Bonito do CTV. Cinco fases do processo de formação de caneluras puderam ser deduzidas pelas análises anatômicas. As primeiras alterações são representadas pelo aparecimento de células adensadas, hipertrofia e hiperplasia no parênquima e câmbio do floema e uma desorganização generalizada desta área. Segue-se uma atividade intensa do câmbio do floema adjacente e sua expansão em direção ao xilema. Esta invasão do xilema resulta na ruptura do anel do xilema pela massa celular do floema constituída de células recém formadas de parede celular delgada. Esta invasão do floema em direção ao xilema inicia um processo de degeneração dos vasos e parênquima do xilema. Finalmente há um colapso completo da região do xilema invadida, que é substituída pela massa do floema, resultando na canelura, notada ao se remover a casca.

An ontogenetic study of the stem pitting was carried out, using as a model system the peduncle of flowers and fruits of sweet orange cv. Pêra infected by a very severe isolate, Capão Bonito, of Citrus tristeza virus (CTV). Anatomical and ultrastructural analysis revealed five phases of this process. It starts with the localized appearance of chromatic cells in the phloem parenchyma, hypertrophy of companion and cambial cells and overall disorganization of the phloem and deposition of a dense material. The next step is characterized by the invasion of the xylem by an abnormal mass of phloem parenchymal cells formed by the disorganized proliferation. These changes lead to the third phase, where xylem ring is interrupted by the invading abnormal phloem proliferation, which produces undifferentiated cells with thin cell wall. The irregular activity of the vascular cambium continues followed by the complete degeneration of the xylem complemented by the appearance of dense phenolic deposits. Finally, there is the complete collapse of the xylem in the invaded region, and the corresponding space is occupied by the phloem mass which correspond to the stem pitting.