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Study on Relative Bacterial Wilt Tolerance of Bt Eggplant Varieties and Their Non-bt Parents (2019--2020)

A. Saha
Biotechnology Division, BARI, Joydebpur, Gazipur

K. Nahar
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:

Bacterial wilt is one of the most important diseases of eggplant caused by Ralstonia solanacearum. Eggplant samples were collected from wilt-infected brinjal fields from 9 districts of Bangladesh. Bacteria were isolated from the wilted plant stem in autoclaved distilled water, cultured on TZC media and preserved in 40 percent glycerol stock solution in -80^oC. PCR of all the collected samples was carried out using the universal bacterial 16S rDNA primer set and the PCR products were sequenced. Based on sequence analysis and BLAST search four bacteria were identified as R. solanacearum. Phylogenetic analysis showed that four sequences were closely related with R. solanacearum and most of the sequences were closely related to the Enterobacter. These results suggested that the wilting symptom of eggplant might also be caused by pathogens other than R. solanacearum strain. Further study is needed to confirm this result. Next year artificial inoculation in Bteggplant will be carried out using identified samples

Keywords: Phylogenetic analysis, Gene sequences, Bacterial wilt, Eggplant.

Location:

Start Date: 00-00-2019

End Date: 00-00-2020

Program Area: Variety and Species

Commodity: Brinjal, Bacteria

Objectives:

The experiments were undertaken to collect and identify the bacterial wilt causing pathogen in eggplant.

Methodology:

The infected brinjal plant samples were collected from the Bt brinjal field of 9 different districts of Bangladesh. Infected eggplant stems were dipped in 70% ethanol for 30 sec and surface sterilized with 5% sodium hypochlorite (3 min), rinsed in sterilized distilled water three times. Each piece was placed and dipped in the tubes containing sterilized distilled water. The selected media called 2,3,5-triphenyltetrazolium chloride (TTC) medium was used to streak the bacterial suspension by using a sterilized wire loop. Large, irregular, round, fluidal and white single colonies with the pink center were collected from each plate and stored in 40% glycerol at -80⁰C for further use. The 16S rDNA genes of the 29 isolates were amplified by PCR in 50 μl reaction volumes containing 10µl 5X reaction buffer,1 µl dNTPs, 0.25 µl Taq DNA polymerase, 1 μl of 10 p moles of each primer (8F, 1492R), 36.75µl distilled water and as template bacterial colony has grown in TZC media. The reaction was performed in an automated thermocycler with an initial denaturation at 95°C for 2 min, followed by 30 cycles of denaturation at 95°C for 30 sec, annealing at 52°C for 30 sec, and extension at 72°C for 1.5 min, with a final extension at 72°C for 10 min. After the PCR amplicons of 16S rDNA gene were confirmed by electrophoresis. The PCR products were sent for sequencing.

Phylogenetic analysis of 16S rDNA gene sequences

Identities of the sequenced isolates were confirmed by using BLAST. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-Nei model (Tamura and Nei, 1993). The bootstrap consensus tree inferred from 100 replicates (Felsenstein, 1985) is taken to represent the evolutionary history of the taxa analyzed (Felsenstein, 1985). Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (100 replicates) are shown next to the branches (Felsenstein, 1985). Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach and then selecting the topology with a superior log likelihood value. The analysis involved 29 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 45 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 (Kumar et al., 2016).

Source of Information: Annual Research Report 2019-2020, Biotechnology Division, BARI, Joydebpur, Gazipur

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

PCR amplification using the universal bacterial 16S rDNA primer set 8F/1492R a single 1484 bp amplicon was obtained and confirmed by gel electrophoresis. The PCR products were sent for sequencing. By 16S rDNA sequence analysis and BLAST search, four bacteria were identified as R. solanacearum. These four bacteria shared identified of 99.85% with R. solanacearum sequence in GeneBank. Surprisingly maximum no. of sequences were not identical to any R. solanacearum strain. They shared identities of about >99% with Enterobacter species. A phylogenetic tree was constructed based on 16S rDNA sequence. Four sequences were closely related to R.solanacearum. Maximum sequences were closely related to the Enterobacters. These results suggested that the wilting symptom of eggplant also caused by pathogens other than R. solanacearum strain. Further study is needed to confirm this result.

Knowledge Management: Report/Proceedings