Hugo Kuniyuki

Cytopathology of callus cells infected with grapevine leafroll-associated virus 3

ABSTRACT The cytopathology of grapevine Vitis spp.) callus tissue(infected with Grapevine leafroll-associated virus 3 (GLRaV-3),genus Vitivirus was studied in order to investigate the usefulnessof callus cultures to study grapevine leafroll-associated viruses.Ultrathin sections were made from in vitro callus obtained fromstems and shoots of GLRaV-3 infected grapevine plants. Calluswas composed of two types of tissue. Translucent, soft calluswas formed and composed of large loosely arranged cells,containing big vacuoles and a thin layer of cytoplasm. Other partsof the callus were brown-coloured and composed of smallcompactly arranged cells, which showed flexuous and rod-shapedclosterovirus-like particles, with 10-12 nm in diameter, at highermagnifications. Groups of vesiclesformed by a single membrane were also observed, with sizes ranging from 50-200 nm,containing fine fibrillar material, also typical of closterovirusinfections. Virus concentration was monitored byImmunosorbent electron microscopy (ISEM) tests, whichshowed that

Transmissão experimental do Grapevine virus B pela cochonilha Pseudococcus longispinus Targioni-Tozzetti (Hemiptera: Pseudococcidae)

In the State of Sao Paulo, Brazil, there are two isolates of Grapevine virus B (GVB) associated with grapevine corky bark disease (GCB). Although serologically similar, they induce distinct reaction on some grape varieties. They are called GVB-C for common isolate and GVB-I for isolate obtained from the variety Italia. The objective of this work was to verify the transmission of both GVB isolates from infected to healthy plants by the mealybug Pseudococcus longispinus. The transmission of the virus was determined by visual analysis of symptoms, ELISA and RT-PCR. In all transmission experiments, grape indicator plants that had been exposed to presumably viruliferous mealybugs reacted in 8-12 months with a typical symptoms of GCB. Healthy LN-33 plants, maintained around one GCB-C affected LN-33 plant, highly infested by the mealybug, became infected with incidence of 54.2% after four years. Experimental inoculation of healthy LN-33 plants with viruliferous mealybugs resulted in infection rates of 46.2% for GVB-C and 40.0% for GVB-I, after three years. Although P. longispinus occurs eventually in Sao Paulo State vineyards, preventive control measures for this insect must be taken on areas where healthy clones of scion and rootstock varieties are maintained.

Molecular Detection of Rupestris stem pitting-associated virus in Grapevines in Brazil

Deteccao por metodos moleculares do Rupestris stem pittingassociated virus em videiras no Brasil RT-PCR foi utilizada para amplificar parte do gene da replicase do Rupestris stem pitting-associated virus (RSPaV) a partir de videiras (Vitis spp.) no Brasil. O segmento amplificado foi clonado e sequenciado e, por comparacao da sequencia de nucleotideos e dos aminoacidos deduzidos, verificou-se que correspondiam, respectivamente, em 88% e 94% com as de isolados do RSPaV de outros paises.

Caracterização do gene da proteína capsidial do Grapevine virus A em videiras afetadas pela acanaladura do lenho de Kober no Estado de São Paulo

Universidade Estadual Paulista Instituto de Biociencias, Letras e Ciencias Exatas Departamento de Zoologia e Botânica

Detecção do Grapevine leafroll-associated virus 5 no Estado de São Paulo

ABSTRACT Grapevine leafroll is a complex disease that can be caused byany one of the nine serologically distinct closteroviruses, referredto as Grapevine leafroll-associated viruses 1 to 9 (GLRaV-1 toGLRaV-9). So far, it has been known the occurrence of GLRaV-1,GLRaV-2, GLRaV-3 and GLRaV-6 in Brazil. This work reports the Kuniyuki, H.; Rezende, J.A.M.; Gaspar, J.O.; Yuki, V.A. Detection of Grapevine leafroll-associated virus 5 in the State of Sao Paulo, Brazil. Summa Phytopathologica, v.34, n.4, p.366-367, 2008 Keywords: Vitis , Closteroviridae, GLRaV-5O enrolamento da folha da videira (“Grapevine leafroll disease” –GLR) e considerado uma das mais importantes viroses da videira ( Vitis spp.) nos principais paises produtores de uva, devido a elevadaincidencia e devido as significativas reducoes na producao e qualidadedos frutos. Os sintomas caracteristicos dessa doenca, mais evidentesem cultivares sensiveis de V. vinifera , incluem enrolamento dos bordosfoliares para baixo e coloracao avermelhada ou amarelada das areasinternervais, com as nervuras principais e secundarias e areas adjacentesdo parenquima permanecendo verdes (5).O GLR e atribuido a nove virus sorologicamente distintos, de1.400 a 2.200 nm de comprimento, denominados de

DAS-ELISA detection of Apple chlorotic leaf spot virus in dehydrated apple petals stored under refrigeration for 16 years

DAS-ELISA detection of Apple chlorotic leaf spot virus in dehydrated apple petals stored under refrigeration for 16 years Flower petals of eleven apple (Malus domestica) clones doubly infected with Apple chlorotic leaf spot virus (ACLSV) and Apple stem grooving virus (ASGV) were harvested in 1985, dehydrated in calcium chloride and stored in the refrigerator at 4-5 C. Petals from fourteen virus free clones were also stored at the same time. DAS-ELISA with antisera against both viruses detected ACLSV in petals from eight clones after sixteen years of storage. ASGV was not detected in any clone. All virus free clones tested negative for both viruses. Os virus da mancha clorotica da folha da macieira (Apple chlorotic leaf spot virus – ACLSV, Trichovirus) e do acanalamento do lenho da macieira (Apple stem grooving virus – ASGV, Capillovirus), assinalados inicialmente no Brasil com base em caracteristicas biologicas e da particula viral (Rev. Soc. Brasil. Fitopatol. 5:125. 1972), tiveram a identidade confirmada por meio de ISEM (Summa Phytopatol. 11:62. 1985) e RT-PCR (Fitopatol Bras. 24:444. 1999). Esses virus foram associados a necrose interna do porta-enxerto Maruba-kaido em mudas de macieira (Summa Phytopathol. 14:33. 1988; Fitopatol. Bras. 26:655. 2001), mas normalmente ocorrem sem causar sintomas diagnosticos. Petalas de flores de macieiras estabelecidas a ceu aberto no Centro Experimental Central do IAC, Campinas, SP foram coletadas em 18/9/1985 e colocadas em frascos de vidro de 10 ml, contendo 3,5 g de cloreto de calcio e sobre ele um chumaco de algodao, para evitar o contato direto do sal com as petalas. A desidratacao e a manutencao das petalas ocorreram em geladeira (4-5 oC). Onze amostras foram obtidas de plantas infetadas conjuntamente com o ACLSV e o ASGV, referentes a clones originarios de Paranapanema e Angatuba, SP. Catorze amostras representaram clones resultantes de termoterapia determinados livres dos dois virus por meio de testes biologicos e ISEM (Summa Phytopathol. 12:19. 1985). Em fevereiro de 2002, as amostras foram submetidas a deteccao por DAS-ELISA (kit Bioreba) para o ACLSV (duas replicacoes) e o ASGV (tres replicacoes), na ESALQ-USP, Piracicaba, SP. Valores medios de absorbância (A405 nm) dos extratos de petalas de pelo menos duas vezes o valor medio dos controles negativos foram considerados como positivos. Os resultados na Tabela 1 indicam que o ACLSV foi detectado em oito das onze amostras provenientes de plantas infetadas. Todas as amostras de clones de termoterapia apresentaram resultados negativos, confirmando os testes biologicos e de ISEM realizados anteriormente. Os resultados para o ASGV foram totalmente negativos, mesmo para petalas provenientes de plantas infetadas, indicando que o virus nao mais estava presente numa forma detectavel pelo metodo empregado. O ACLSV apresenta ponto final de inativacao termica e longevidade in vitro inferiores ao ASGV, o inverso do que ocorreu para o virus em petalas desidratadas mantidas em geladeira. Nao foram encontrados relatos sobre o comportamento desses virus apos anos de armazenagem. 03142 TABELA 1 Valores medios de absorbância (A405 nm) de DAS-ELISA na deteccao de Apple stem grooving virus (ASGV) e Apple chlorotic leaf spot virus (ACLSV) em petalas desidratadas de macieira armazenadas durante 16 anos em geladeira *Amostras consideradas positivas estao grifadas em negrito A405 nm* apos a incubacao indicada