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Study on Relative Bacterial Wilt Tolerance of Bt Eggplant Varieties and Their Non-bt Parent Lines (2017--2018)

A. Saha
Biotechnology Division, BARI, Joydebpur, Gazipur

K. Nahar
Biotechnology Division, BARI, Joydebpur, Gazipur

S. Ahmed
Biotechnology Division, BARI, Joydebpur, Gazipur

R. Islam
Biotechnology Division, BARI, Joydebpur, Gazipur

D. Khanam
Biotechnology Division, BARI, Joydebpur, Gazipur

M.A.Y. Akhond
Biotechnology Division, BARI, Joydebpur, Gazipur

Abstract/ Executive Summary:

Thirty-six wilt-affected brinjal plant samples were collected from 6 different locations of Bangladesh from July 2017 to May 2018 for studying the diversity of the Ralstonia solanacearum strains available in Bangladesh. From the collected samples bacteria were isolated by collecting the bacterial ooze in autoclaved distilled water. Then the bacteria were cultured in TZC medium and 30 virulent isolates were identified by morphological observation. Pure cultures of virulent isolates were done in TZC media in 28^oC and liquid culture was done in CPG media with continuous shaking in the incubator. Then the bacterial samples were preserved in 40 percent glycerol stock solution in -80^oC for further molecular study.

Keywords: Brinjal, Bacterial wilt, Molecular characterization

Location:

Start Date: 00-00-2017

End Date: 00-00-2018

Program Area: Variety and Species

Commodity: Eggplant

Objectives:

To know the diversity of that bacterial wilt found all over the country and characterize both in morphological and molecular level.

Methodology:

Collection of bacterial wilt-affected brinjal plants was done from July 2017 to May 2018. The plant samples plants showing typical symptoms of bacterial wilt were collected from 6 different locations of Bangladesh. The collected plant samples were packed in polythene bags and kept at 4⁰C for isolation of the causal organism. The small pieces of discolored vascular tissue measuring 5-6 cm in length were cut from the discolored stem. Surface sterilization was done by washing the stem with jet and trix solution and immersing in one percent sodium hypochloride for 30 seconds followed by repeated washing in sterile distilled water. Then a small part of discolored vascular tissues of plant samples was cut and they were kept in the glass beaker containing autoclaved sterile distill water taking care that the infected tissue was in contact with the water surface. After five minutes the water in the glass beaker became turbid due to the oozing of bacterial cells from the cut ends of the diseased tissue and thus confirming the bacterial nature of the wilted plants. With help of a sterile loop bacterial suspension was poured onto the surface of solidified Triphenyl Tetrazolium Chloride agar (TZC) medium (Kelman, 1954) in sterilized Petri plates. The inoculated plates were incubated at 28^oC for 48 hours. At the end of the incubation period, the plates were observed for the development of both the virulent and avirulent colonies of R.solanacearum. The virulent colonies are irregularly shaped, fluidal, dull-white colonies with a slight red center. Whereas, avirulent colonies were small, round, convex, butyrous with large red pigment and narrow bluish margins as described by Kelman (1954). For the pure culture of this bacteria typical virulent colonies of R. solanacearum on TZC medium were picked and streaked separately on TZC medium in sterilized Petri plates. The plates were incubated at 28^oC for 44 to 48 hours. The well-separated virulent colonies of R. solanacearum were picked up with a sterile inoculated loop and suspended in liquid CPG (Casaninoacid-Peptone-Glucose) broth in sterile bottles. The inoculated flasks were incubated for 24 hours at 28 ^oC in continuous shaking at 300 RPM. After the liquid culture, 50 ml of the bacteria and 50 ml of 40 percent glycerol solution were taken in a screwcap tube in sterile condition at the clean beach for each sample and stored in -80^o.

Source of Information: Annual Research Report 2017 2018, Biotechnology Division, BARI, Joydebpur, Gazipur

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Representative samples of R. solanacearum infected brinjal plants were collected from Bt brinjal field of 6 different districts of Bangladesh. The infected plant samples showing typical symptoms were collected. At the time of extraction of bacteria in autoclaved sterile distill water from the plant sample, the samples that did not show any bacterial ooze were discarded. Typical virulent colonies of R. solanacearum developed within 48 hours in TZC media that were well-separated, fluidal, dull-white colored with slight pink to the red-colored center were selected for further procedure. The samples which did not produce virulent colonies were discarded. The well-separated colonies were picked up and purified further by single colony isolation technique and after liquid culture in CPG media the bacterial samples were stored at -80^o C in 40 percent glycerol stock solution; this will serve as a stock culture for further culture. All the bacterial isolates collected from Rangpur ,Gazipur and Chittagong were successfully isolated and cultured, but 3 samples from Jamalpur, 1 sample from Moulavibazar, and 2 samples from Jessore did not show typical symptom of virulent R. solanacearumstrain during extraction in sterile water and in culture media, so they were not allowed for further procedure.

Knowledge Management: Report/Proceedings