Agricultural Research Management Information System

Home
Research Summary
Search
About Us
- ARMIS
- Brochure
Contact Us
Report
User Request
Data Input
Help
- Operation Manual
  - PDF
  - Video
- Program Area & Commodity
We have reached 37600 number of research entries at this moment.

- Logout

Research Detail

Home
Research
Detail

Secondary Endosymbionts Distribution in Bangladesh Whitefly, Bemisia Tabaci (gennadius) (homoptera: Aleyrodidae)

S.M.H. Jahan
Department of Entomology, Patuakhali Science and Technology University, Dumki, Patuakhali, Bangladesh

M.A. Rahman
Department of Entomology, Patuakhali Science and Technology University, Dumki, Patuakhali, Bangladesh

M. Asaduzzaman
Department of Entomology, Patuakhali Science and Technology University, Dumki, Patuakhali, Bangladesh

K.Y. Lee
Department of Agricultural Biology, Kyungpook National University, Daegu 702-701, Korea

Abstract/ Executive Summary:

The sweetpotato whitefly, Bemisia tabaci, harbors of all most six secondary endosymbionts such as Arsenophonus, Cardinium, Fritschea, Hamiltonella, Rickettsia and Wolbachia. These bacteria play important roles to insect physiology. Bemisia tabaci is a species complex composed of more than 24 biotypes, which may diverge from each other both hereditarily and morphologically. The presence of secondary endosymbiont into the whiteflies was varied from biotype to biotype and strain to strain. Secondary endosymbionts infection occurrence in B. tabaci from different host-plants at different places in Bangladesh was determined by Polymerase Chain Reaction (PCR), in order to test for correlation between bacterial composition to biotype, host-plants and TYLCV transmission. Arsenophonus, Cardinium, Hamiltonella and Wolbachia were detected in all of the populations of the whiteflies from different places in Bangladesh that were collected from different host-plants, at the same time Fritschea and Rickettsia did not found in any whitefly populations of Bangladesh. No significant differences in secondary endosymbionts were found among host-plants within the whitefly population in Bangladesh. Secondary endosymbionts recommends a potential contribution of these bacteria to host-plant traits such as TYLCV transmission, insecticide resistance and host ranges.

Keywords: Arsenophonus, Bemisia tabaci, Cardinium, Hamiltonella, Wolbachia

Location: Department of Entomology, Patuakhali Science and Technology University, Dumki, Patuakhali

Start Date: 00-00-2010

End Date: 00-00-2010

Program Area: Pest Management

Commodity: Diseases

Objectives:

1. The genetic differences among the B. tabaci populations from different host-plants in Bangladesh were investigated, for determination of secondary endosymbiont in B. tabaci.

Methodology:

Whiteflies collection: Samples of Adult B. tabaci were collected from different places on different host-plants (bean, ridge gourd, pepper, tomato and eggplant) from Bangladesh in 2010 and were immediately preserved in 99% ethanol (absolute alcohol) and stored at -20°C. DNA extraction: Total genomic DNA was extracted from individual B. tabaci according to protocol supplied by Invitrogen Purelink Genomic DNA mini kit. After removing the sample from ethanol had been washed with double-distilled water to remove alcohol. Individual whiteflies were homogenized in 180 µl genomic digestion buffer using a 1.5 ml microcentrifuge tube and micropestle (homogenizer). Then added 200 µl genomic lysis/ binding buffer (1% SDS, 10 mM Tris-HCl, pH 8.0, 25 mM EDTA, 25 mM NaCl, Proteinase K 200 mg/ml) and after that immediately added 200 µl absolute ethanol. Subsequently added wash buffer into the genomic column and finally added 20 µl genomic elusion buffer (Invitrogen Purelink, Carisbad, CA, USA). Samples were centrifuged (12000 rpm for 1 minute) and incubated at RT (20°C) for 1 min. After that the supernatants/pellets were directly used for PCR amplification for detecting secondary endosymbionts or were stored at -20°C for later use. Total genomic DNA was extracted from each individual for further analysis. Primer design and PCR amplification: The presence of secondary endosymbionts in the whitefly populations in Bangladesh was determined using the primers listed. PCR reactions were performed in a 20 µl 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 µg of DNA as a template. The mixtures were amplified in a PTC-200 thermal cycler (MJ Research, Watertown, MA, USA) with a 3 min initial denaturation at 95°C, 35 cycles (30 sec at 94°C, 30 sec at 52~60°C, 30 sec at 72°C), and finally by a 10 min extension at 72°C. Annealing temperatures of each gene were listed. Gel-electrophoresis: Amplified PCR products (5 µl) were electrophoresis using 1.0% agarose gels with 1X TAE at 100 V for 30 mins with 100bp ladder DNA marker and the gels were then stained by 10 µl Ethidium Bromide for 20 mins. When bands with the expected size were visible on the gels, then the rest of 15 µl of PCR products were used for sequencing. (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 (Promega, Madison, WI, USA) and sequenced either directly or by cloning into the pGEM-T easy plasmid vector (Promega, Madison, WI, USA).) Sequence analysis: The sequences of PCR products were determined using the Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, USA) and analyzed by 3730XL DNA Sequencer (Applied Biosystems, Foster City, USA). Databases were searched using the BLAST algorithm in NCBI and sequences were aligned using the MUSCLE program. Mitochondrial COI sequences of B. tabaci were analyzed using MrBayes 3.0 software. Bayesian software MrBayes 3.0 four metropolises coupled with Markov Chain Monte Carlo (MCMC) chains were run, stopping when the standard divergence of split frequencies was less than 0.01. All sequences were analyzed over 10 million generations and four were sampled every 100 generations and the first 25% burn-in (SUMP and SUMT) cycles were discarded prior to the construction of the consensus tree. Consensus trees were visualized with MEGA 4.0

Source of Information: J. Agrofor. Environ. 5 (1): 45-50, 2011; ISSN 1995-6983

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

To determine endosymbiont infection of B. tabaci, the presence of 6 endosymbiotic bacteria in Bangladesh populations of B. tabaci from different host-plants were examined by PCR analysis of 16S or 23S rDNA sequences. Arsenophonus, Cardinium, Hamiltonella and Wolbachia were detected in all the tested populations of Bangladesh indigenous biotypes. However, Rickettsia and Fritschea were not detected in any populations of Bangladesh.

Knowledge Management: Journal