Jahan, S. M. H.
Professor
Department of Entomology, Patuakhali Science and Technology University, Dumki,Patuakhali-8602, Bangladesh.
Lee, K-Y
Department of Agricultural Biology, Kyungpook National University, Daegu 702-701, Korea,
Holader, M. I. A.
Senior Scientific Officer
On-Farm Research Division, Bangladesh Agricultural Research Institute, Patuakhali-8600, Bangladesh.
Bashar H. M. K
Scientific Officer
On-Farm Research Division, Bangladesh Agricultural Research Institute, Patuakhali-8600, Bangladesh.
Hasan G. N.
Scientific Officer
On-Farm Research Division, Bangladesh Agricultural Research Institute, Patuakhali-8600, Bangladesh.
Molecular divergence, Intra-specific variation, Aleurodicus dispersus, Endosymbiont, Bemisia tabaci, Cardinium
Pest Management
Adult individuals of B. tabaci and other whiteflies were collected from various host plants, such as tomato, pepper, ridge gourd, bean, okra, eggplant, and guava grown in Bangladesh, Myanmar, Nepal, China, Philippines and Korea. In Bangladesh, we collected whiteflies from bean, okra, and eggplant as of the southern, northern and eastern parts of the country during 2011-2012, immediately preserved samples in 99% ethanol, and stored them at -200C for further molecular analysis. Adults of B. tabaci B and Q biotypes were collected from cucumber, sweet melon, and tomato plants which grown throughout Republic of Korea in 2010 in order to compare the morphologies and genetic sequences of foreign whiteflies. This research work has been performed from January 2011 to June 2012 at the insect molecular physiology lab in the Republic of Korea. Different genotypic group of B. tabaci was determined by amplification the gene of mtCOI region causing specific fragments from extracted genomic DNA samples . The presence of Cardinium in whiteflies was determined using specific primer sets for Cardinium by amplification of 16S rDNA gene fragments as described by Chiel et al., 2007. The occurrence of Tomato yellow leaf curl virus (TYLCV) on farmhouses of various horticultural crops was surveyed in different geographical regions. Acquisition of this virus by B. tabaci was determined using a TYLCV-specific primer set that can amplify conserved intergenic sequences. Specific primer sets for biotypes, endosymbionts, and TYLCV are listed. Polymerase chain reaction (PCR) reactions for Cardinium were performed in a 20 l mixture containing 5× SuperTaq PCR buffer (10 mM Tris-HCL, 40 mM KCl, 1.5 mM MgCl2, pH 9.0), 2.5 mM dNTPs, 0.5 μM of each primer, 1 unit of SuperTaq DNA polymerase (SuperBio Co, Korea), and 1 g DNA of whitefly as a template. The mixtures were amplified using a PTC-200 thermal cycler with 5 min of initial denaturation at 95°C, 35 cycles of annealing (1 min at 94°C, 1 min at 58°C, 1 min at 72°C), and 10 min of extension at 72°C. The PCR products were visualized on a 1.0% agarose gel containing ethidium bromide. Expected PCR products were excised from the gel and purified using the Wizard PCR preps DNA purification system and sequenced either directly or by cloning into pGEM-T easy plasmid vector.
DNA sequence analysis: Sequences of the PCR products were determined using a Big Dye Terminator Cycle Sequencing Kit and analyzed using a 3730XL DNA Sequencer.Databases were searched using the BLAST algorithm in NCBI, and sequences were aligned using the Multiple Sequence Comparison by Log-Expectation (MUSCLE) program .The 16S ribosomal DNA sequences of Cardinium were analyzed using Bayesian MrBayes 3.0 software. Four Metropolises-coupled Markov Chain Monte Carlo (MCMC) chains were run until standard divergence of the split frequencies become lower than 0.01. All sequences were analyzed over 10 million generations, and four sequences were sampled every 100 generations. The first 25% of burn-in (SUMP and SUMT) cycles were discarded prior to the construction of consensus tree, which were visualized by MEGA 4.0 .
Bangladesh J. Agril. Res. 40(1): 121-135, March 2015 (ISSN 0258-7122)
Journal