Sanat Myti
Shahjalal University of Science and Technology, Dept. Genetic Engineering & Biotechnology, Sylhet-3114, Bangladesh.
Shabbir Ahmed Khandaker
Shahjalal University of Science and Technology, Dept. Genetic Engineering & Biotechnology, Sylhet-3114, Bangladesh
Shamim Akhter
Fruit Research Station , Bangladesh Agricultural Research Institute(BARI), Binodpur, Rajshahi-6206, Bangladesh
Alim Uddin
Fruit Research Station , Bangladesh Agricultural Research Institute(BARI), Binodpur, Rajshahi-6206, Bangladesh.
Mohammad Kamruzzaman
Shahjalal University of Science and Technology, Dept. Genetic Engineering & Biotechnology, Sylhet-3114, Bangladesh
Md. Omar Faruq
Shahjalal University of Science and Technology, Dept. Genetic Engineering & Biotechnology, Sylhet-3114, Bangladesh
Gokul Chandra Biswas
Shahjalal University of Science and Technology, Dept. Genetic Engineering & Biotechnology, Sylhet-3114, Bangladesh
Incidence, DAC-ELISA, CMV.
Plant Pathology Laboratory of Fruit Research Station, Bangladesh Agricultural Research Institute (BARI), Binodpur, Rajshahi
Pest Management
This research works has been conducted at Plant Pathology Laboratory of Fruit Research Station, Bangladesh Agricultural Research Institute (BARI), Binodpur, Rajshahi.To assess the incidence of CMV in chilli at two major chilli growing region namely Rangpurand Sylhet of Bangladesh.The presence of CMV has been confirmed by Direct Antibody Coated Enzyme-Linked Immune-Sorbent Assay (DAC-ELISA).
A field survey has been conducted in Rangpur and Sylhet districts during the month of March-June in 2013. Five locations in Sylhet namely Sadar, South Surma, Gowinghat, Kanaighat and Bishwanath and four locations in Rampur namely Sadder, Mithapukur, Pirgong and Kaunia has been selected for survey. Early, mid and late stage of plant growth of chilli has been surveyed in each location. During the survey different types of symptomatic chilli plants has been observed closely, the data on percent disease incidence and other parameter has been taken for further analysis. The symptomatic disease samples have been brought to Plant Pathology Lab at FRS for further study. The field survey was conducted by random selection of rows of plants grown in the field for estimating disease incidence. The growth and yield parameters were checked by selecting ten healthy plants and ten diseased plants of each random selected rows of that cultivated land. At first, the symptoms that appeared in chilli plants in field condition by viruses were closely and carefully observed and were identified by visual observation as described by Green and Kalloo (1994). The following formula was used to calculate the disease incidence:
Disease incidence (%) = (X1/X2) ×100 Where, X1=No. of infected plants, X2= No. of healthy plants. Data were recorded from each of the plot of an area where ten healthy and ten infected plants were sampling randomly for growth and yield discernments like plant height ( inch), fruit number, fruit weight (gm), fruit length (cm). The following formula was used to calculate the percent reduction of the growth and yield contributing characters.
R= (Y-Y1)/Y×100 Where, R= percent reduction of growth/ yield contributing character, Y=Growth/Yield contributing characters of healthy plants, Y1=Growth/yield contributing characters of infected plants.
The entire field collected samples were tested by DAC-ELISA according to Hobbs et al., (1987) for the evidence of CMV. The antisera of CMV, PVY, ChVMV were used to detect viruses from the field surveyed samples. Samples to be tested were extracted by grinding 1g of tissue with 10ml of coating buffer in a mortar and pestle. Then extracted samples were settled to centrifuge (5 min at 4000 rpm) and used the supernatant in the test and discarded the pellet. 200μl of each sample was added to the wells of the microtitre plate. Wrapping the plate by tightly in aluminum foil and then incubated the plate at 4°C overnight. After overnight incubation, the plate was washed three times with phosphate buffered saline +Tween 20 (0.05%) – PBST at three minutes interval for each wash. Firstly, the wells of the plate were filled with PBST and inverted to remove the buffer repeat twice, pat the plate dry on paper towels. 200μl of blocking buffer was added to each test well. The plate was wrapped again and incubated at 37°C for 1 hour. The plate was washed again by wash buffer for three times at three minutes interval for each wash. The virus specific antibody (Primary) was diluted to 1:2000 in 100 ml Conjugate buffer and added 200μl to each test well. The plate was wrapped again and incubated at 37°C for 1 hour. The plate was washed three times by washing buffer. The antibody-enzyme conjugate was diluted in 15 ml conjugate buffer and added 200μl to each test well. Plate was wrapped again by aluminum foil and incubated at 37°C for 1 hour. The plate was washed four times as washing buffer. An extra wash stage was done at this stage to ensure that all the unbound antibody-enzyme conjugate is removed from the wells. The substrate buffer was prepared just before use was done by adding pNPP tablet to substrate buffer (one 0.5mg tablet in 5ml of buffer). 200μl of prepared substrate buffer was added to each test well. The plate was wrapped again and incubated at 37°C for 1 hour. Preparing Stop solution was done by adding 30 gm NaOH into 250 ml dH2O. 50 μl stop solution was added to each well. The presence of virus had been confirmed by color developed in the microtitre plate.
International Journal of Biosciences ( IJB ) ISSN: 2220-6655 (Print) 2222- 5234 (Online) Vol. 5, No. 7, p. 40-49, 2014
Journal