Inclusion bodies induced by Bean rugose mosaic comovirus seen under light microscopy

Two types of inclusion bodies were consistently observed under light microscopy in bean (Phaseolus vulgaris) leaf tissue infected with Bean rugose mosaic virus (BRMV), a species of the genus Comovirus, family Comoviridae. One type consisted of vacuolated inclusions found mainly in the cytoplasm of epidermal cells. The other type consisted of abundant crystalloid inclusions of different sizes and shapes found consistently in glandular hairs, guard cells, phloem tissue, xylem elements and occasionally in epidermal and mesophyll tissues. The two types of inclusion bodies stained with Azure A and Luxol Brilliant Green Bl-Calcomine Orange 2RS (O-G), and were similar to those seen to be caused by other species of comoviruses.Se observaron dos tipos de inclusiones virales, mediante microscopia de luz, en hojas de plantas de frijol (Phaseolus vulgaris) previamente infectadas con el virus del mosaico rugoso del frijol (“Bean rugose mosaic virus”, BRMV), especie del género Comovirus, familia Comoviridae. Se hallaron inclusiones vesiculadas, principalmente en el citoplasma de células de la epidermis, y abundantes inclusiones cristalinas de diferentes formas y tamaños siempre en células guarda, tricomas glandulares, floema, elementos del xilema y ocasionalmente en células epidérmicas y del mesófilo. Ambos tipos de inclusiones tiñeron con Azure A y con la tinción, verde naranja (Luxol Brilliant Green BL-Calcomine Orange 2 RS) conocida como OG, y son similares a las inclusiones inducidas por otras especies del género Comovirus.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Biología Celular y Molecular (CIBCM

Detection of CEVd in an orange grove using the Arizona 861-S1 selection of the ‘Etrog’ citron

La prueba de infectividad empleada tradicionalmente para el viroide de la exocortis consiste en injertar yemas de los árboles por evaluar en clones sensibles, como el cidro Arizona 861-S1, el cual ha sido previamente injertado en un patrón vigoroso (limón rugoso). En este trabajo informamos una variación de este tipo de prueba. aprovechando las condiciones favorables de temperatura y humedad que se dan en los países tropicales, se pueden obtener fácilmente y en menor tiempoi síntomas de viroides en el campo, injertando yemas del cidro Etrog directamente en los árboles que se desea analizar. Esta prueba será de utilidad para productores y viveristas, ya que contarán con un medio sencillo y barato para discriminar los árboles en el campo, mientras que con el método tradicional es necesario contar con una gran cantidad de árboles del cidro injertados sobre limón rugoso, y se requiere, además, de áreas o instalaciones adecuadas para el mantenimiento de estos. Sin embargo, la prueba de detección que se describe no cuenta con la sensibilildad y especificidad de los análisis moleculares. Estos son indispensables para todos aquellos árboles en los que las yemas del cidro injertadas no muestren síntomas, ya que árboles con baja concentración de viroides podrían escapar a la prueba de infectividad.Indexing for CEVd in symptomless hosts is usually done by graft inoculation on sensitive clones such as Etrog citron Arizona 861-S1, budded on a vigorous rootstock (rough lemon). We report here a modification of this test, consisting of grafting trees directly in the filed instead of using plants in greehouse conditions. The favorable temperature and humidity conditions in the Tropics allow symptom development in the fiels when Etrog citron is employed as an indicator. The Etrog citron buds are grafted directly to the trees to be tested. This test is simple, inexpensive and allows earlier detection of trees infected with viroids than more traditional indexing methods. etrog trees can supply budwood for testing a large number of trees in the field, whereas the traditional method requires many young Etrog trees grafted on rough lemon and adequate greehouse concentrations may escape detection therefore, laboratory analyses are required for trees in which the Etrog indicator does not slowly symptoms.Universidad de Costa Rica/[801-94-905]/UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Biología Celular y Molecular (CIBCM)UCR::Vicerrectoría de Docencia::Ciencias Agroalimentarias::Facultad de Ciencias Agroalimentarias::Escuela de AgronomíaUCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiologí

First Report of an Aster Yellows Subgroup 16SrI-B Phytoplasma Infecting Chayote in Costa Rica

An outbreak of a witches' broom disease affected approximately 20% of plants in several chayote (Sechium edule (Jacq.) Schwartz) fields in the commercial production area of the Ujarrás Valley, Cartago Province, Costa Rica during 2000 and 2001. Affected chayote plants exhibited symptoms, including basal proliferation with severe foliage reduction, aborted flowers, and deformed fruits, suggestive of phytoplasmal infection. Two other symptomatic cucurbit species growing near the chayote fields were also identified. These species were tacaco plants (S. tacaco (Pitt.) C. Jeffrey), an edible cucurbit for domestic marketing in Costa Rica, showing severe size reduction of leaves and fruits, and Rytidostylis carthaginensis (Jacq.) Kuntze, a weed in chayote and tacaco fields, exhibiting abnormal tendril proliferation. Plants were analyzed for phytoplasma infection by a nested polymerase chain reaction (PCR) assay, using the universal rRNA primer pair P1/P7 followed by R16F2n/R16R2 (2). Phytoplasmas were detected in all symptomatic samples (18 chayote, 6 tacaco, and 3 weed) tested but were undetectable in all asymptomatic samples (10 chayote, 6 tacaco, and 2 weed). Restriction fragment length polymorphism (RFLP) analysis of PCR products (16S rDNA sequences) by separate digestion with eight restriction enzymes (RsaI, HhaI, KpnI, BfaI, HaeIII, HpaII, AluI, MseI) revealed that a phytoplasma belonging to subgroup 16SrI-B in the aster yellows phytoplasma group (16SrI) was associated with chayote witches' broom (CWB). The same or very similar phytoplasmas were found in both symptomatic tacaco and R. carthaginensis plants. Phylogenetic analysis of 16SrDNA sequences also confirmed the CWB phytoplasma to be most similar to members of subgroup 16SrI-B. Similar diseases in chayote and other cucurbits have been reported in Brazil (3), Taiwan (1), and Mexico (4). The CWB phytoplasma differs from the phytoplasma (16SrIII-J subgroup) associated with chayote in Brazil. The identities of phytoplasmas associated with cucurbits in Taiwan and Mexico are unknown. The occurrence of an aster yellows group phytoplasma in chayote may pose a potential threat to continued production and exportation of this cash crop. To our knowledge, this is the first report of 16SrI-B subgroup phytoplasmas in naturally infected cucurbits in Costa Rica.Universidad de Costa Rica/[801-A5-582]/UCR/Costa RicaInternational Atomic Energy Agency/[]/IAEA/AustriaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Biología Celular y Molecular (CIBCM

First Report of Orchid fleck virus in Costa Rica

copyright 2002 American Phytopathology Society JournalsOrchid fleck virus (OFV), a tentative member of the family Rhabdoviridae, infects orchids in several countries. The virus is vectored worldwide by the mite Brevipalpus californicus (Banks) (Acari: Tenuipalpidae). Eleven plants of Oncidium spp. and one plant each of the genera Cymbidium and Maxillaria exhibiting numerous yellow flecks and necrotic ringspot lesions on leaves were collected in two private orchid collections in Costa Rica. Presence of OFV was assessed by plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA) using an antiserum developed against an OFV isolate in Japan (2), analyses of ultrathin sections of the host cell with transmission electron microscopy (TEM), and reverse transcription polymerase chain reaction (RT-PCR) amplification using specific primers for the viral nucleocapsid gene (1). Eight of eleven Oncidium samples, and both Cymbidium and Maxillaria samples tested positive for OFV with PTA-ELISA having A values ranging from 3.9 to 14.6 times higher than negative controls. Thin sections from individual samples of Cymbidium, Oncidium, and Maxillaria revealed electronlucent intranuclear viroplasm and short, rodlike particles (40 to 50 × 100 nm) in the nucleus or cytoplasm typical of OFV-infected cells. RTPCR amplifications from one sample of each genera resulted in PCR product bands of approximately 800 bp. The Cymbidium RT-PCR product was cloned into a pGEM-T-Easy expression vector and sequenced using an ABI 3700 sequencer. The 619-bp nucleocapsid gene consensus sequence had 98% homology with the OFV isolate 0023 identified in Germany (GenBank Accession No. AF343870) (1). However, it had only approximately 85% nucleocapsid gene homology with other OFV isolates available through GenBank, including those from countries geographically closer to Costa Rica, such as Brazil (1).To our knowledge, this is the first report of OFV infecting orchids in Costa Rica.Universidad de Costa Rica/[801-A1-801]/UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Biología Celular y Molecular (CIBCM

First Report of Citrus variegation virus in Palestine Sweet Lime, as Coffee Shade in Costa Rica

Symptoms resembling those of Citrus variegation virus (CVV) were observed in old Palestine sweet lime trees (Citrus limettioides Tan.) used as coffee shade in the Central Valley in Costa Rica. The symptoms include leaf flecking, mosaic, malformation, and dwarfing. This disease was transmitted by grafting to “Valencia” sweet orange (C. sinensis L.), Etrog Citron (C. medica L.), and sweet lime under greenhouse conditions. Symptoms were severe in Palestine sweet lime all year long, but sweet orange and Etrog citron were asymptomatic under high temperature. Reverse transcription polymerase chain reaction (RT-PCR) was used to confirm the presence of CVV in young leaves from sweet lime trees collected from field, and other Citrus spp. grafted and maintained in the greenhouse at University of Costa Rica. Fresh tissue from trees infected with CVV obtained from the USDA (Riverside, California), was used as positive control. Furthermore, the sequence obtained (605 bp) was analyzed by Blastn algorithm which showed 97% homology with CVV RNA 3 (GenBank Accession No. AF434912). Additionally, small scale virus purification was carried out and isometric particles (26 to 32 nm) were observed under the electron microscope. To our knowledge, this is the first report of the presence of CVV in Costa Rica infecting Palestine sweet limes. Also, we remark on the potential of Palestine sweet lime as a host plant indicator in biological indexing of CVVMinisterio de Ciencia y Tecnología, Fondo Reinserción/[222-2004]/MICIT- CONICIT/Costa RicaUniversidad de Costa Rica/[801-A5-506]/UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Biología Celular y Molecular (CIBCM)UCR::Vicerrectoría de Docencia::Ciencias Agroalimentarias::Facultad de Ciencias Agroalimentarias::Escuela de AgronomíaUCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de Biologí

Occurrence of Squash yellow mild mottle virus and Pepper golden mosaic virus in Potential New Hosts in Costa Rica

Leaf samples of Solanum lycopersicum, Capsicum annuum, Cucurbita moschata, Cucurbita pepo, Sechium edule and Erythrina spp. were collected. All samples were positive for begomoviruses using polymerase chain reaction and degenerate primers. A sequence of ~1,100 bp was obtained from the genomic component DNA-A of 14 samples. In addition, one sequence of ~580 bp corresponding to the coat protein (AV1) was obtained from a chayote (S. edule) leaf sample. The presence of Squash yellow mild mottle virus (SYMMoV) and Pepper golden mosaic virus (PepGMV) were confirmed. The host range reported for SYMMoV includes species of the Cucurbitaceae, Caricaceae and Fabaceae families. This report extends the host range of SYMMoV to include the Solanaceae family, and extends the host range of PepGMV to include C. moschata, C. pepo and the Fabaceae Erythrina spp. This is the first report of a begomovirus (PepGMV) infecting chayote in the Western Hemisphere.Consejo Nacional para Investigaciones Científicas y Tecnológicas/[]/CONICIT/Costa RicaMinisterio de Ciencia, Tecnología y Telecomunicaciones/[]/MICITT/Costa RicaFondo Especial de Educación Superior/[]/FEES/Costa RicaUniversidad de Costa Rica/[]/UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Biología Celular y Molecular (CIBCM)UCR::Vicerrectoría de Docencia::Ciencias Agroalimentarias::Facultad de Ciencias Agroalimentarias::Escuela de AgronomíaUCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de Biologí

Inclusion bodies induced by bean rugose mosaic virus seen under light microscopy

Two types of inclusion bodies were consistently observed under light microscopy in bean (Phaseolus vulgaris) leaf tissue infected with bean rugose mosaic virus (BRMV), a species of the genus Comovirus, family Comoviridae. One type consisted of vacuolated inclusions found mainly in the cytoplasm of epidermal cells. The other type consisted of abundant crystalloid inclusions of different sizes and shapes found consistently in glandular hairs, guard cells, phloem tissue, xylem elements and occasionally in epidermal and mesophyll tissues. The two types of inclusion bodies stained with Azure A and Luxol Brilliant Green Bl-Calcomine Orange 2RS (O-G), and were similar to those seen to be caused by other species of comoviruses.<br>Se observaron dos tipos de inclusiones virales, mediante microscopia de luz, en hojas de plantas de frijol (Phaseolus vulgaris) previamente infectadas con el virus del mosaico rugoso del frijol ("bean rugose mosaic comovirus", BRMV), especie del género Comovirus, familia Comoviridae. Se hallaron inclusiones vesiculadas, principalmente en el citoplasma de células de la epidermis, y abundantes inclusiones cristalinas de diferentes formas y tamaños siempre en células guarda, tricomas glandulares, floema, elementos del xilema y ocasionalmente en células epidérmicas y del mesófilo. Ambos tipos de inclusiones tiñeron con Azure A y con la tinción, verde naranja (Luxol Brilliant Green BL-Calcomine Orange 2 RS) conocida como OG, y son similares a las inclusiones inducidas por otras especies del género Comovirus

Ultrastructural alterations in mouse capillary blood vessels after experimental injection of venom from the snake Bothrops asper (Terciopelo)

Histological and ultrastructural alterations in capillary blood vessels were studied at various time intervals after im injection of 50 micrograms of Bothrops asper snake venom in mouse gastrocnemius muscle. Hemorrhage was observed as early as 5 min after envenomation, as abundant erythrocytes appeared in the interstitial space. Ultrastructural observations revealed two different patterns of pathological changes: in the majority of damaged capillaries, endothelial cells had blebs and cytoplasmic projections pinching off to the lumen. This phenomenon was observed together with a decrease in the number of pinocytotic vesicles, with endothelial cells becoming very thin. As an apparent consequence of this process, some endothelial cells had evident gaps in their continuity. In addition, basal laminae surrounding these capillaries were altered and discontinuous. Other endothelial cells underwent a morphologically different process of degeneration, characterized by swelling of the endoplasmic reticulum and of the cytosol. These cells had a diffuse appearance and their basal laminae were discontinuous or absent. No major changes in the intercellular junctions were noticed in damaged endothelial cells. Samples obtained 30 and 60 min after venom injection were devoid of normal capillaries in many areas, and only diffuse remnants of their structure were found. Many altered capillaries had platelet aggregates and fibrin, the latter also being observed in the interstitial space. It is concluded that B. asper venom induces rapid and drastic pathological effects on capillaries leading to hemorrhage per rhexis i.e., erythrocytes probably escape through gaps in damaged endothelial cells and not through widened intercellular junctions.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiologí

Confirmación de la presencia de Xylella fastidiosa infectando vid (Vitis vinifera) en Costa Rica

Copyright 2008 by the Brazilian Phytopathological Society. www.sbfito.com.brIn 2003, symptoms of Pierce Disease (PD) were observed in two vineyards established in two different localities in San Jose province (Santa Ana and La Uruca) and in La Garita, Alajuela province. Twenty four symptomatic plants (15 from Santa Ana, five from La Uruca and four from La Garita) were analyzed by DAS-ELISA using antibodies against Xylella fastidiosa. Sixteen plants (nine from Santa Ana, five from La Uruca, and two from La Garita) were positives for the bacterium. Isolation attempts were made from petioles of these plants, and seven isolates were obtained in liquid PW. Aliquots of liquid media were inoculated on solid PW. Ten days after inoculation, circular and convex colonies, with entire margin, smooth and opalescent, of 0.5-1mm in diameter were observed. From each isolate three colonies were selected and considered as different clones. Every clone was analyzed by DAS-ELISA with positive results. Clones were Gram negative, catalase positive and oxidase negative. The bacteria were rod shaped, and showed a typically rippled cell wall. DNA of each clone was extracted and used as template in PCR with primers 272-1/272-2 and RST31/RST33; products of 500pb and 733pb were obtained, respectively. These results confirmed the presence of X. fastidiosa causing PD in grapevine plants in different vineyards in Costa Rica.Durante el año 2003, se observaron síntomas del mal de Pierce (PD) en dos plantaciones de vid en la provincia de San José (Santa Ana y La Uruca) y en otra en La Garita, provincia de Alajuela. De un total de 24 plantas sintomáticas analizadas mediante DAS-ELISA usando anticuerpos para Xylella fastidiosa, se detectaron 16 plantas positivas (nueve de Santa Ana, cinco de La Uruca y dos de La Garita). A partir de diferentes intentos de aislamiento de los peciolos en medio líquido PW, se obtuvieron siete aislamientos. Alícuotas de medio líquido se inocularon en medio PW sólido. Diez días después de la inoculación, se observaron colonias circulares, cónvexas, lisas y opalescentes, con márgenes enteros, con diámetro entre 0.5-1mm. De cada aislamiento se seleccionaron tres colonias, las cuales fueron consideradas como clones diferentes. Todos los clones presentaron valores positivos mediante DAS-ELISA. Los clones presentaron bacilos Gram negativo, catalasa positivo y oxidasa negativo. Los bacilos al observarse al microscopio electrónico de transmisión presentaron la pared rugosa previamente informada para X. fastidiosa. Al amplificar el ADN extraido de cada clon mediante la técnica de la PCR con los imprimadores 272-1/272-2 y RST31/RST33, se obtuvieron productos de 500pb y 733pb, respectivamente. Estos resultados confirman la presencia de X. fastidiosa causando mal de Pierce en vid en Costa Rica.Costa Rica-USA Foundation grant 16-01CTUniversidad de Costa Rica/[801-A2-528]/UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Biología Celular y Molecular (CIBCM

Aplicación del método de coagulación de plasma al estudio ultraestructural de especímenes biológicos

Copyright © 1991, Revista Biología TropicalBiological particulate specimens, including Saccharomyces cerevisiae yeast, bovine spermatozoa and human blood cells (normal erythrocytes and leukemic cells) were processed for scanning and transmission electron microscopy using the coagulated plasma technique. The specimens were suspended in frozen and thawed plasma; later, coagulation was induced by adding CaCI2. The clot was cut into small pieces and processed as tissue fragments. The technique is a useful tool when processing biological particulate specimens for electron microscopy.Universidad de Costa Rica/[803-88-418]/UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Biología Celular y Molecular (CIBCM)UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiologí