Passionfruit woodiness virus (PWV) is the most important virus on passionfruit crops in several states of Brazil and in other parts of the world. Studies were made to evaluate the practical application of the indirect DAS-ELISA, using antibodies produced in two different species of animals, for diagnosis and quantitative studies with PWV. A PWV isolate collected in Vera Cruz country, State of São Paulo, was purified from systemically infected Canavalia ensiformis (Leguminosae) , with an average yield of 1.82 mg of virus per gram of tissue. Purified virus was injected in New Zealand rabbit and chicken (line IPEG), using 100 µg of virus per injection. A total of 6 injections for the rabbit and 3 for the chicken were applied intramuscularly, at one week interval. Rabbit IgG was purified by precipitation with saturated ammonium sulfate followed by ion exchange chromatography. Chicken IgY (lgG) was directly purified from the yolk of the egg. Indirect DAS-ELISA tests with different concentration of IgG and IgY, at different combinations, were carried out to establish the most sensitive and accurate design for the technique. Absorbance values higher than twice the average absorbance for the healthy control were considered positive. Under this circumstance, the best results were obtained using 10 µg/ml of IgG to coat the ELISA plate and 1 µg/ml of IgY to complete the double sandwich. Studies on the sensitivity of the indirect DAS-ELISA, using known concentrations of purified PWV (10, 20, 40, 80, 160, 320, 640, 1.280, 2.560 e 5.120 ng/ml) and the same criterium mentioned previously for separation of positive and negative serological reactions were also done. Under this conditions it was found that the technique detected up to 20 ng of virus/ml. Quantitative evaluations of PWV were made on plants of Passiflora edulis f. flavicarpa, P. giberti, P. cincinnata, Crotalaria juncea and Phaseolus vulgaris cv. Black Turtle 2. Plants were inoculated separately with three isolates of PWV (Vera Cruz, Brasília and Minas Gerais), under greenhouse conditions. Discs from three expanded upper leaves of each plant were harvested fifteen days after inoculation and ground in PBS-Tween, diluted 1:500. All samples were tested on indirect DAS-ELISA, using the same known concentrations of purified PWV as standard. Absorbance values obtained with the standard concentrations of PWV were used for linear regression analysis and further estimation of the virus present in the samples. Estimated concentrations of PWV (µg/g of tissue) varied from 61.0 to 1,232.0 for P. edulis f. flavicarpa, from 45.0 to 669.5 for P. giberti, from 25.0 to 1,820.0 for P. cincinnata, from 43.8 to 99.1 for G. juncea and from 48.4 a 84.6 for P. vulgaris cv. Black Turtle 2. Based on these data it is suggested the use of indirect DAS-ELISA for diagnosis of passionfruit woodiness and preliminary screening of passionfruit plants resistant or tolerant to the disease, based on the concentration of virus in their tissue. O vírus do endurecimento dos frutos ("Passionfruit woodiness virus" - PWV) é um dos principais vírus que causam problemas à cultura do maracujazeiro em diversas regiões do país e do mundo. Neste trabalho estudou-se a aplicação da técnica de DAS-ELISA indireto, com anticorpos produzidos em coelho e galinha, para fins de diagnóstico e estudos quantitativos do PWV. Um isolado do PWV obtido de plantas de maracujazeiro de Vera Cruz, SP, foi purificado a partir de plantas de feijão-de-porco (Canavalia ensiformis D.C.), com produção média de 1,82 mg de vírus por 100 g de tecido. Suspensões do PWV purificado foram injetadas em coelho da raça Nova Zelândia e em galinha da linhagem IPEG, na proporção de 100 µg de vírus por injeção. As injeções, num total de 6 para o coelho e de 3 para a galinha, foram intramusculares e a intervalos de duas e uma semana, respectivamente. A IgG do antissoro obtida do coelho foi purificada por meio da precipitação com sulfato de amônio saturado e cromatografia de troca iônica. A IgY do antissoro da galinha foi obtida diretamente a partir da gema do ovo. Os testes para a padronização do DAS-ELISA indireto foram feitos com diferentes concentrações de IgG e de IgY, em diferentes combinações. Considerou-se positiva toda a reação cujo valor da absorbância foi superior ao dobro da média da absorbância do extrato da planta sabia (controle). Nessa condição, os melhores resultados foram obtidos utilizando-se 10 µg/ml da IgG para a captura do antígeno (PWV) e 1 µg/ml da IgY para completar o sanduíche da reação sorológica. Avaliações da sensibilidade do DAS-ELISA indireto, foram feitas com concentrações conhecidas do vírus purificado (10, 20, 40, 80, 160, 320, 640, 1.280, 2.560 e 5.120 ng/ml) e utilizando-se o mesmo critério acima mencionado para separar reações positivas e negativas. Nessas condições a técnica foi altamente sensível para a detecção de até 20ng/ml. Para os estudos quantitativos do PWV foram utilizadas plantas de Passiflora edulis f. flavicarpa, P. giberti, P. cincinnata, Crotalaria juncea e Phaseolus vulgaris cv. Black Turtle 2. As plantas foram inoculadas mecanicamente com três isolados do PWV (Vera Cruz, Brasília e Minas Gerais), separadamente, e mantidas em casa de vegetação. Quinze dias depois foram retiradas amostras de três folhas desenvolvidas de cada planta e maceradas em PBS-Tween, na diluição de 1:500. As amostras foram testadas através da DAS-ELISA indireto, juntamente com as mesmas concentrações conhecidas do PWS purificado. Os valores médios de absorbância obtidos para cada concentração conhecida do vírus foram utilizados para obtenção de uma equação de regressão linear para posterior quantificação do vírus. As concentrações do PWV nas diferentes espécies, estimadas em µg/g de tecido, variaram de aproximadamente 61,0 a 1.232,5 para P. edulis f. flavicarpa, de 45,0 a 669,5 para P. giberti, de 25,0 a 1.820,0 para P. cincinnata, de 43,8 a 99,1 para C. juncea e de 48,4 a 84,6 para P. vulgaris cv. Black Turtle 2. Diante desses resultados, sugere-se a utilização da técnica de DAS-ELISA indireto nos trabalhos de diagnóstico do endurecimento dos frutos e nos estudos de seleção preliminar de plantas de maracujazeiro resistentes e/ou tolerantes à doença, através da quantificação do vírus nos tecidos das mesmas.

Passionfruit woodiness virus (PWV) is the most important virus on passionfruit crops in several states of Brazil and in other parts of the world. Studies were made to evaluate the practical application of the indirect DAS-ELISA, using antibodies produced in two different species of animals, for diagnosis and quantitative studies with PWV. A PWV isolate collected in Vera Cruz country, State of São Paulo, was purified from systemically infected Canavalia ensiformis (Leguminosae) , with an average yield of 1.82 mg of virus per gram of tissue. Purified virus was injected in New Zealand rabbit and chicken (line IPEG), using 100 µg of virus per injection. A total of 6 injections for the rabbit and 3 for the chicken were applied intramuscularly, at one week interval. Rabbit IgG was purified by precipitation with saturated ammonium sulfate followed by ion exchange chromatography. Chicken IgY (lgG) was directly purified from the yolk of the egg. Indirect DAS-ELISA tests with different concentration of IgG and IgY, at different combinations, were carried out to establish the most sensitive and accurate design for the technique. Absorbance values higher than twice the average absorbance for the healthy control were considered positive. Under this circumstance, the best results were obtained using 10 µg/ml of IgG to coat the ELISA plate and 1 µg/ml of IgY to complete the double sandwich. Studies on the sensitivity of the indirect DAS-ELISA, using known concentrations of purified PWV (10, 20, 40, 80, 160, 320, 640, 1.280, 2.560 e 5.120 ng/ml) and the same criterium mentioned previously for separation of positive and negative serological reactions were also done. Under this conditions it was found that the technique detected up to 20 ng of virus/ml. Quantitative evaluations of PWV were made on plants of Passiflora edulis f. flavicarpa, P. giberti, P. cincinnata, Crotalaria juncea and Phaseolus vulgaris cv. Black Turtle 2. Plants were inoculated separately with three isolates of PWV (Vera Cruz, Brasília and Minas Gerais), under greenhouse conditions. Discs from three expanded upper leaves of each plant were harvested fifteen days after inoculation and ground in PBS-Tween, diluted 1:500. All samples were tested on indirect DAS-ELISA, using the same known concentrations of purified PWV as standard. Absorbance values obtained with the standard concentrations of PWV were used for linear regression analysis and further estimation of the virus present in the samples. Estimated concentrations of PWV (µg/g of tissue) varied from 61.0 to 1,232.0 for P. edulis f. flavicarpa, from 45.0 to 669.5 for P. giberti, from 25.0 to 1,820.0 for P. cincinnata, from 43.8 to 99.1 for G. juncea and from 48.4 a 84.6 for P. vulgaris cv. Black Turtle 2. Based on these data it is suggested the use of indirect DAS-ELISA for diagnosis of passionfruit woodiness and preliminary screening of passionfruit plants resistant or tolerant to the disease, based on the concentration of virus in their tissue.