Isolation and purification of bacteria
In 2018 (September-December), eight black rotted orange fruits were collected from RDA market, Rajshahi, Bangladesh. Bacterial pure colonies were isolated and purified from infected fruits as described previously. Briefly, the infected skin of the fruits were excised with a sterile scalpel and disinfected superficially using 70% alcohol for one minute, sodium hypochlorite for one minute, and finally was rinsed three times in sterile distilled water. The samples were then placed in 100 ml of Luria-Bertani (LB) broth medium and incubated at 37°C overnight. Bacteria was streaked onto a fresh LB agar plate and incubated for 16 hours at 37°C. After that, eight different single colonies were picked up by loop and streaked on fresh medium plate for pure culture. Finally, two pure culture (BS1 and BS2) were selected and preserved in 80% glycerol-water solution at -80°C. The two pure cultures were characterized and identified using different approaches, as below.
Morphological characterization
The two isolated bacterial strains were observed through several morphological tests. Gram staining and motility of bacteria were conducted as previously described.
Molecular characterization
The two bacterial isolates (BS1 and BS2) were sub-cultured on LB liquid medium and incubated at 30°C for 16 hours with continuous shaking. Genomic DNA was extracted using an automated DNA extractor (Model: Maxwell 16; Promega, USA) and suspended into TE buffer. Afterwards, quantity and quality of the isolated DNA was checked using NanoDrop Spectrophotometer (Model: ND2000; Thermo Scientific, USA).
The isolated bacterial DNA was amplified using the universal bacterial 16S rRNA gene PCR primers (27F 5’-AGAGTTTGATCCTGGCTCAG3’ and 1492R 5’-CGGTTACCTTGTTACGACTT-3’), as previously described; genomic DNA was used as a template. The PCR reaction was performed using the method by Hassan et al. (2018), using hot start green master mix (dNTPs, Buffer, MgCl2, Taq Pol; Cat: M7432; Promega, USA).
A 20 µl reaction mix was used, containing master mixture 10 µl, T DNA (concentration 25-65 ng/µl) 1 µl, each primer (concentration 10-20 pMol) 1 µl and nuclease free water 7 µl. Thermo-cycling parameters were 95°C for 3 minutes, 32 cycles of 95°C for 30 seconds, 48°C for 30 seconds and 72°C for 90 seconds; a final extension step at 72°C was added for 5 minutes, using a PCR machine (Gene Atlas, Model: G2; Astec, Japan). PCR products were run on 1% agarose gel (Cat: V3125; Promega, USA) and visualized under alpha imager UV trans-illumination (Model: mini; Protein Simple, USA) with 0.5% ethidium bromide solution (Cat: H5041; Promega, USA) in 1xTAE buffer (Cat: V4251; Promega, USA) using a1kb DNA ladder (Cat: G5711; Promega, USA). PCR products were purified from the agarose gel, using SV gel and PCR clean up system (Cat: A9281; Promega, USA). Purified PCR products were sequenced commercially by Sanger sequencing (Apical Scientific, Malaysia). Sequences were used in a search using the NCBI BLAST tool. Obtained sequences were submitted to GenBank (see Data availability) and compared with the GenBank database.
In vitro antimicrobial screening of the plant extracts
To evaluate antibacterial activities of the isolates, Allium sativum, Hibiscus rosa-sinensis, Ocimum sanctum, Allium cepa, Zingiber montanum and Psidium guajava plants were collected from various places (Kajle and Binodpur villages) in Rajshahi district. Plants extraction was performed using the method described by Kader et al. (2018). Different parts of selected plants (bulb of A. sativum and A. cepa; flowers of H. rosa-sinensis; and leaves of O. sanctum, Z. montanum, and P. guajava) were cut into small pieces, air-dried, and ground by blender to form a fine powder. The dried powder of the selected plants (100gm of each plant) were rinsed in methanol (500ml) using a conical flask and kept in a shaking incubator for 15 days. The liquid contents were pressed through Markin cloth followed by filtration using Whatman no. 1 filter paper. Obtained filtered liquids were dehydrated in vacuo to leave a blackish and sticky mass. The extracts were preserved in a refrigerator at 4°C using glass vials.
For antibacterial screening, BS1 and BS2 bacterial cultures were inoculated on LB agar plates. Subsequently, discs of filter paper (6 mm in diameter) were saturated with the plant extracts at the concentration of 30 µg/disc and were impregnated on the surface of plates. The plates were incubated at 37°C for 16 hours. Inhibition zones were measured with the help of a millimeter scale.
Antagonistic control
For evaluation of antagonistic effects, five soil bacteria, Rhizobium phaseoli, Rhizobium leguminosarum, Escherichia coli, Bacillus subtilis and Brevibacillus borstelensis were used against isolated bacterial strains (BS1 and BS2). Pure cultures of the soil bacteria were kindly provided by Dr. Md. Salah Uddin, Associate Professor and Director, Microbiology Lab., Department of Genetic Engineering and Biotechnology, University of Rajshahi, Rajshahi, 6205, as the part of a collaboration.
To assess the antagonistic effects, agar well diffusion method was used, as previously described. The agar plate’s surface was inoculated by spreading a volume of the microbial inoculum over the entire agar surface. A 6 mm diameter hole was made aseptically with a cork borer. In total, 40 µl/hole bacterial suspension (108 CFU/ml) of the isolates were introduced into the well and were incubated at 37°C for 16 hours. The antibacterial agents diffuse in the LB agar medium that inhibit the growth of the microbial strain tested of soil bacteria against two plant pathogens. The test samples were determined by measuring the diameter of zones of inhibition in term of mm with a transparent scale.
Data analysis
In the study, all the experiments and test were replicated thrice. All data were expressed as the mean value and standard deviation (M ± SD) using Microsoft Excel software 2013.