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 A non-radioactive dot-blot nucleic acid hybridization method was evaluated for detecting citrus leaf blotch virus (CLBV, Flexiviridae: Citrivirus) in total RNA extracts from kumquat trees. DNA fragments partially encompassing the open reading frames encoding the polymerase (RdRp) and the movement (MP coat (CP) proteins of CLBV were used to generate digoxigenin (DIG-11-dUTP) probes by PCR. Probes were individually generated using cloned DNA fragments of the Cuban CLBV isolate as templates. In the assays, several working conditions, including the use of different membranes, hybridization and washing buffers were tested. The method detected up to 40 pg of a plasmid containing the complete CLBV genome, as well as viral genomic sequences in RNA extracts of naturally infected trees of 'Nagami' kumquat (Fortunella margarita) and 'Round' kumquat (F. japonica). Results support considering the method developed an appropriated tool to be used after its validation by the programs of sanitation, quarantine, and certification of citrus propagation materials in Cuba.

RESUMEN Se evaluó un método de hibridación de ácidos nucleicos no radioactivo en formato dot-blot para la detección precisa de citrus leaf blotch virus (CLBV: Citrivirus) en extractos de ARN totales de árboles de cítricos. Los fragmentos de ADN que conforman parcialmente los marcos de lectura abiertos que codifican la polimerasa (RdRp), la proteína de movimiento (MP) y la proteína de la cubierta (CP) de CLBV se utilizaron para generar sondas marcadas con digoxigenina (DIG-11-dUTP) mediante PCR. Para generar las sondas se utilizaron fragmentos de ADN clonados a partir del aislado de CLBV de Cuba. Se evaluaron varias condiciones de trabajo que incluyeron el uso de diferentes membranas, soluciones de hibridación y tampones de lavado. Con el método evaluado fue posible detectar hasta 40 pg de un plasmidio que contiene el genoma completo de CLBV, así como material genómico del virus en extractos de ARN de plantas de kumquat 'Nagami' (Fortunella margarita) y kumquat 'Round' (F. japonica) infectadas de forma natural con el virus. Los resultados evidenciaron que el método desarrollado es una herramienta útil y altamente recomendable, una vez sea validado, para su uso en los programas de saneamiento, cuarentena y certificación del material de propagación de cítricos en Cuba.

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.