Plate-trapped Antigen Enzyme-linked Immunosorbent Assay Research Articles

Quantification ofBotrytisin Grape Juice Determined by a Monoclonal Antibody-based Immunoassay

Monoclonal antibodies to <i>Botrytis</i>//, raised in a previous study, were used to develop a plate—trapped antigen enzyme linked immunosorbent assay ( PTA-ELISA) to detect and quantify <i>Botrytis</i> antigens in juice from infected grapes. Using a dilution series of extracts from freezedried preparations of <i>B. cinerea</i> grown in liquid culture on grape juice and juice from artificially infected and healthy Cabernet Sauvignon and Semillon grapes, we have shown that BC-12.CA4, a genus-specific antibody, can be used in micro-titer PTA-ELISA tests to measure levels of infection from 0 to 5%. Forty-one samples of juice from 15 different cultivars from seven different wineries in the Central Valley region in California were tested along with juice from healthy-looking grapes of the same cultivars. No cross-reaction was found with any of the cultivars tested for juice from healthy-looking berries taken from loads with low levels of infection. Using BC-12.CA4 immunoassay levels of rot were generally higher than visual assessments of bunch rot particularly with machine-harvested grapes at the end of the season. However, in early season samples immunoassay estimates were, in some cases, lower than the visual assessments. It is strongly suspected that in the latter case fungi other than Botrytis were the main cause of bunch rot. Preliminary studies with another antibody, that recognizes both <i>B. cinerea</i> and <i>Aspergillus niger</i>, confirmed that the bunch rot in these cases was mostly due to fungi other than Botrytis.

Wilt Pathogens of Solanaceae in Tanzania: Clavibacter michiganensis subsp. michiganensis, Pseudomonas corrugata, and Ralstonia solanacearum.

Surveys of vegetables in the southern and northern growing regions of mainland Tanzania and Zanzibar during 1997 and 1998 indicated the presence of three wilt pathogens of tomato. Clavibacter michiganensis subsp. michiganensis (cause of bacterial canker) was isolated from wilting plants in the southern highlands and Lushoto District (Tanga Region, north) on selective King's medium B with polymyxin B (1). The identity of the isolates was confirmed by cultural, morphological, and biochemical characteristics and a plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA) kit (Pathoset 113-08, Adgen, Auchincruive, U.K.). Pathogenicity was confirmed by host inoculation. In addition, the bacterium was detected directly by the same methods in several sources of seed, including commercial farmers' saved seed and seed extracted from infected plants. Of 61 tomato seed lots tested, 18 samples were positive. Ralstonia solanacearum (cause of bacterial wilt) was isolated from tomato and potato by semiselective media and detected directly in stem and tuber tissues, respectively, by polymerase chain reaction and ELISA (4) in all vegetable-growing areas surveyed. In 1998, R. solanacearum was detected for the first time in Zanzibar on tomato and eggplant. Pathogenicity was confirmed by inoculation on and reisolation from tomato seedlings of cv. Money Maker. Only biovar 3 (2) occurred in tomato. Biovar 3 also was found in midaltitude potato. Biovar 2 has been found only in potato plants grown above 1,500 m. Pseudomonas corrugata (cause of pith necrosis) was isolated from tomato on semiselective media at only one location. From wilted tomato plants in the southern highlands, 38% of samples tested positive for C. michiganensis subsp. michiganensis and ≈ 10% for R. solanacearum. Of samples collected from the northern highlands, 43% tested positive for R. solanacearum. Wilt incidence of ≈35% was observed in tomato fields where the bacterial wilt pathogen was isolated in the northern highlands compared with gt;90% incidence and almost total crop loss in tomato fields of the southern highlands infected with bacterial canker. Although all three pathogens caused systemic wilt of plants, bacterial canker occasionally caused downward turning of lower leaves, unilateral wilting, and marginal necrosis of leaflets as well as fruit spotting. In general, wilts caused by C. michiganensis subsp. michiganensis, R. solanacearum, and P. corrugata were not readily differentiated in Tanzania prior to this research. Plants with pith browning had often been assumed to be infected by P. corrugata, until R. solanacearum was isolated (3) frequently from such plants.

Cayaponia tibiricae: New Host of Zucchini Yellow Mosaic Virus in Brazil.

Cayaponia tibiricae Cogn. (CT) is a wild Cucurbitaceae species found in secondary forests in the State of São Paulo, Brazil. The species has indefinite growth and bears oblong dark green fruits, 15 to 20 mm long (1,2). CT plants showing yellow mosaic symptoms were found in Atibaia County. Extracts from symptomatic plants were rub inoculated to zucchini squash (Cucurbita pepo L.) and Chenopodium amaranticolor Coste & Reyn. Zucchini squash plants developed severe yellow mosaic with intense leaf malformation, while C. amaranticolor showed necrotic local lesions. Extracts from naturally infected CT, zucchini squash, and C. amaranticolor were tested by plate trapped antigen-enzyme-linked immunosorbent assay (PTA-ELISA) with antisera against papaya ringspot virus type W (PRSV-W), zucchini yellow mosaic virus (ZYMV), zucchini lethal chlorosis virus (ZLCV), watermelon mosaic virus 2 (WMV-2), and cucumber mosaic virus (CMV). All samples were positive in PTA-ELISA only with ZYMV antiserum. Also, in Western blot (immunoblot) assay, ZYMV antiserum labeled a protein of approximately 36 kDa. Electron microscopic examination of ultrathin sections from infected CT tissue revealed the presence of pinwheel inclusions typical of potyvirus (type 1) infection in the cytoplasm of the cell. CT seedlings were susceptible to mechanical inoculation with the ZYMV isolated from this species. This is the first report of CT as a natural host for ZYMV in Brazil.

Screening Carica papaya × C. cauliflora hybrids for resistance to papaya ringspot virus‐type P

Carica papaya, C. cauliflora and interspecific hybrids of these species were screened for resistance to two Australian isolates (338, 445) of papaya ringspot virus‐type P (PRSV‐P). Plants were manually inoculated with PRSV‐P in the glasshouse and the reaction assessed 30 days later by back‐inoculation to susceptible Cucurbita pepo and by a plate‐trapped antigen‐enzyme linked immunosorbent assay (PTA‐ELISA). Both parents and interspecific hybrids were also planted adjacent to infected C. papaya and 30 days later tested for PRSV‐P by PTA‐ELISA. All interspecific hybrid and C. cauliflora plants manually inoculated in the glasshouse or planted in the field failed to become infected, whereas C. papaya plants, in both situations, were infected by PRSV‐P. In addition, the surviving interspecific hybrid and C. cauliflora plants tested negative, while all C. papaya plants were positive for PRSV‐P in both the back‐inoculation and PTA‐ELISA tests. Thus, the interspecific hybrid and C. cauliflora plants were resistant to the Australian PRSV‐P isolates.