Abdul Mannan AKANDA*
Faculty of Agriculture , Kyushu University, Fukuoka 812, Japan
Kazunori TSUNO*
Faculty of Agriculture , Kyushu University, Fukuoka 812, Japan
Satoshi WAKITO*
Faculty of Agriculture , Kyushu University, Fukuoka 812, Japan
Cucurbit-virus, Dried sample, DAS-ELISA, DIBA, Bangladesh.
Different locations of Bangladesh in 1986-87
Pest Management
Samples. In total 21 samples comprised of nine different species of cucurbitaceous crops were collected from different locations of Bangladesh in 1986-87. The leaves of the plant showing virus disease-like symptoms in the field were collected in small polyethylene bags and immediately processed for drying by lyophilization or with calcium chloride. These dried samples were carried to Japan and stored at 4•Ž until testing in 1989. Antisera. Polyclonal antisera of CMV-serotype Y (CMV-Y), PRSV-watermelon strain (PRSV-W), PRSV-papaya strain (PRSV-P), WMV 2, ZYMV, CGMMV-yodo strain (CGMMV-Y), CGMMV-watermelon strain (CGMMV-W) and SqMV were used in the experiments. PRSV-W, WMV 2 and ZYMV antisera were provided by Dr. N. Sako, Saga University, Japan. PRSV-P antiserum was generously given by Dr. D. Gonsalves of Cornell University, USA. Antisera of CMV-Y and SqMV were obtained from by Dr. T. Maeda, Okayama University, Japan and CGMMV (Y and W) antisera were also provided by Dr. M. Kameya, National Agriculture Research Center, Tsukuba, Japan. Inoculation test. All the 21 cucurbitaceous samples were inoculated separately to the respective host plants and also to some common local lesion hosts several times, following standard inoculation procedures described by Hill15). The inoculated plants were maintained in an air-conditioned greenhouse, temperature of which was controlled at 20-25•Ž. The buffer used for inoculation was 0.02M phosphate containing 0.5% sodium sulfite, pH7.2. Double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Purified anti-CMV-Y, -PRSV-W, -WMV 2 and -ZYMV ƒÁ-globulin and ƒÁ-globulin conjugated to alkaline phosphatase used in the experiment were provided by Dr. N. Sako of Saga University, Japan. The protocol of ELISA was as outlined by Clark and Adams6) with slight modifications. Wells of microtiter plate (Nunc, Denmark) were coated with ƒÁ-globulin (2ƒÊg/ml, 200ƒÊl/well) in 0.05M carbonate buffer (pH9.6) containing 0.02% sodium azide and incubated at 37•Ž for 3hr. The small amount (ca. 0.05g) of dried samples was macerated with glass rod on Parafilm in a few drops of PBS (0.02M phosphate buffer, 0.15M NaCl, 0.02% sodium azide) containing 0.05% Tween 20 and 2% polyvinylpyrrolidone (PVP Av. Mol. Wt. 40,000). An amount of 30ƒÊl aliquot was poured in each well containing 120ƒÊl sample buffer (PBS-Tween-PVP) and incubated for 4•Ž for 24hr. The enzyme conjugated ƒÁ-globulins were applied at 1:1,000 dilution in PBS-Tween-PVP with 2% bovine serum albumin (BSA). After adding conjugated ƒÁglobulin (100ƒÊl/well), the plate was incubated for 37•Ž for 3hr. In each well an amount of 100ƒÊl of substrate (p-nitrophenylphosphate, 1mg/ml) in 10% diethanolamine containing 0.02 % sodium azide(pH9.6)was applied and incubated for 1hr at 30?. The plant leaves infected with each virus were used as positive control. The reaction was stopped by adding 50ƒÊl of 3N NaOH per well. Unless otherwise stated, the microtiter plates were washed at least four times with PBS-Tween after each incubations. Absorbance values were measured by using an ELISA analyzer (Immuno Reader NJ-2000) at 405nm. The experiments were designed so that there was two observations for each sample per plate. Dot-immunobinding assay (DIBA). In DIBA, polyclonal antisera of CMV-Y, PRSV-W, PRSV-P, WMV 2, ZYMV, CGMMV-Y, CGMMV-W and SqMV were used. DIBA was essentially performed as described by Hibi and Saito14) with some modifications. Nitrocellulose membrane (NCM, Bio-Rad) was dipped in distilled water for 10min and dried on filter papers for 5min. They were then dotted with 2ƒÊl sample extracts prepared by homogenizing ca. 0.05g dried samples in 0.5ml TBS (0.02M Tris-HCl, 0.5M NaCl, pH7.5) and dried for 15min. The NCMs were incubated in blocking solution consisted of 2% BSA, 2% Triton X-100 in TBS for 1hr, then briefly washed with TBST (Tween 20 in TBS). Antiserum (1:4,000) diluted with 1% healthy leaf extract in TBST containing 0.2% BSA and 2% PVP (polyvinylpyrrolidone) was spotted on the NCM at the rate of 20ƒÊl/grid and incubated for 50min. The NCM was rinsed in TBST for three times with gentle shaking for 30min. Membranes were incubated for 1hr in conjugate solution prepared by diluting with antibody buffer (1:8,000) and washed in TBST as described previously. Finally, the NCM was reacted with color development solution for 30-60min as recommended by Banttari and Goodwin2). The reaction was stopped by washing the NCM in distilled water and then air dried for visual observation and storage. Unless otherwise stated, the NCMs were incubated at room temperature and all the operations were performed in plastic box. The experiments were repeated at least twice to confirm the results.
Ann. Phytopath. Soc. Japan 57: 499-505 (1991
Journal