Sample collection Infected leaves were collected from University of Rajshahi and identified by Bangladesh Fruit Research Institute, Binodpur, Rajshahi, Bangladesh.
Isolation and purification Wilt disease affected plant parts were sterilized by using 10% sodium hypochlorite dilute solution and rinsed thoroughly. Thereafter affected area were cut down by the help of sterilized scalpel and grind in mortar pestle and keep them into Luria-Berteani (LB)broth medium for overnight incubation of bacterial growth(Seal et al. 1993). This incubation was done for 12-16 hours at 37°C. The bacterial isolates were purified by streaking method on LB broth media.
Characterization of the isolated bacteria: Different morphological and biochemical tests were done to characterize the isolated bacteria
Gram staining: Isolated bacteria was picked by the help of loop and spread on a glass slide and then fixed by heat on a very low flame. The crystal violet solution (0.5%) was added onto the top of the glass slide and smear over the slide for 30 seconds and then rinsed with water. It was inundated with gram’s iodine solution for one minute, rinsed with water and then added decolorized with 95% ethanol until colorless runoff. After washing the slide, again added counter stained with safranin for at least 10 seconds, and washed with water. Finally, allow the slide for air dried at room temperature and observed under 100X microscope using immersion oil (Tan et al. 2015).
Motility test: Mild agar media was prepared in a test tube for motility test. Picked up one pure colonies on the nutrient media and integrated center of the mild agar medium to a depth of 1 inch by the help of sterilized needle (Schaad et al. 2001).
Catalase test: For this test, 3% hydrogen peroxide (H2O2) solution was used to observe the production of oxygen bubbles. One of the purified colonies as placed on a glass slide and drop of H2O2 solution on the top of the cell (Tan et al. 2015).
Potassium hydroxide (KOH): Isolated bacteria were aseptically took from the Petri plates with a tooth pick or an inoculating loop and then placed on a clean glass slide in a drop of 3% KOH solution. After that the mixture was mixed up for 10 seconds and observed the thread like slime (Adler et al. 1967).
Simmons citrate test: Citrate media were mixed up in distilled water and sterilized at 121°C for 20 minutes. Thereafter the media were poured into test tube for created slanted position and one of isolated colony was streaked into the surface of the media. This was kept at 37ºC for 16 hours (Suslow et al. 1982). Kovacs oxidase test: The Kovacs oxidase reagent (1% tetramethyl-p-phenyl diamine dihydrochloride) was prepared and kept in dark bottle. Then one drop of reagent was placed to a piece of filter paper, which placed on a glass Petri dish. Few numbers of isolated colonies were grazed on the paper saturated with 1% oxidase reagent solution and noted for creating the purple color in 20-60 seconds (Sun et al. 2011). Urease test: In this test, the urease media were prepared and then poured into test tubes. Then small amount of inoculum were placed on a tube and kept into incubator for 16 hours at 37°C (Suslow et al. 1982). Sulfur indole motility (SIM): For SIM test, media were prepared with distilled water and autoclaved at 121ºC for 20 minutes. Then media were placed on a test tube. After that, a few amount of inoculum were inoculated into the media test tube and incubated overnight at 37ºC for 16 hours. Finally, one drop of Kovac’s reagent was added and observed (Lelliott et al. 1987).
Antibiotic sensitivity test: Antibiotic sensitivity test was done by Kirby-Bauer disc diffusion method (Levin et al. 1970). In this test, isolated bacteria were grown in nutrient broth medium, then take 1ml of overnight inoculum and transferred on the nutrient agar plate allowed for drying. Fifteen antibiotics with different disc concentrations such as Amoxicillin, Erythromycin, Gentamycin, Chloramphenicol, Clarithromycin, Ampicillin, Tetracycline, Carbenicillin, Neomycin, Streptomycin, Azithromycin, Kanamycin, Doxycycline, Cefotaxime, and Penicillin were placed in the center of the petri plates and incubated overnight at 37ºC for 16 hours (Hudzicki et al. 2009).
Antibacterial activity: Four medicinal plants such asleaves of Ocimum sanctum (Tulsi), Bulb of Allium cepa (Onion), Root of Asparagus racemosus (Shatamooli), Burk of Terminalia arjuna (Arjun) were used for this investigation. These plant parts were carefully washed and dried in shade for several days, then it was greened into a fine powder (Jorgensen et al. 2015). The prepared powder was soaked in ethanol solvents (ratio 1:10, w/v) and shaking at 150 rpm for 24 hour. Filtrated extracts were dried at 40°C. The dried extracts were re-suspended in Phosphate Buffered Saline (PBS) to bring to 500 mg mL-1 concentration. The test was screened by disc diffusion method. For preparing aliquot 5 mm in diameter disc were used and 10, 20, 30 μl ethanol extract was soaked into each disc on the bacterial plate and placed on central of the plates for 30 minutes for proper diffusion and incubated at 37ºC for 16 hours (Biemer et al. 1973).