Sampling area and collection of samples
Twenty (20) root samples from cowpea [Vigna unguiculata (L.) Walp] plants were collected from various agricultural fields of Hathazari Upazila, Chittagong, Bangladesh. Collected root samples were sealed in sterile zipper bags and labeled. All samples were brought to the Molecular Biology Laboratory of the Department of Genetic Engineering and Biotechnology, University of Chittagong, Bangladesh, by maintaining cold chain and stored them in the refrigerator (4°C) for further analysis.
Isolation of Endophytic Bacteria from cowpea roots
The total procedure were done according to Anderson et al. (2008), where samples [healthy fresh roots of Vigna unguiculata (L.) Walp] were collected and cleaned under running tap water to remove debris and then air dried. About 25.0 gm of 2-3 cm length of roots were cut and surface sterilization was carried out by rinsing them in Tween-20 for 10 minutes, followed by further washing with dH2O for at least 7 times. After that, root samples were dipped into 70% alcohol for 30 seconds, and then the samples were washed with dH2O. Twenty (20.0) ml of 0.2% Hg2Cl2 solution was added to the samples and the beaker was put on a shaker at 240 rpm for 5 minutes at 27°C. Then the samples were washed again with dH2O for at least 7 times. The final root rinsed water, was used as control and spread onto nutrient agar plate (Addisu and Kiros, 2016), which contained (g/L) - peptone 5.00, beef extract 2.00, yeast extract 3.00, NaCl 5.00 and agar 18.00, where pH was adjusted to 7.0. For the isolation of endophytic bacteria, root pieces were further triturated in sterile Phosphate buffer saline (PBS) (Anderson et al., 2008) containing (g/L) - NaCl 8.00, KCl 0.20, Na2HPO4 1.44 and KH2PO4 0.24, where pH was adjusted to 7.4 and maintained at 28°C under 150 rpm agitation. All plates including control were incubated at 37°C for 5 days and the number of CFU was determined to estimate bacterial population density according to Addisu and Kiros (2016).
Following purification, morphologically distinct colonies were identified by observing colony characteristics such as gram nature, color, shape using a binocular biological microscope (XSZ-107BN), where colonies of similar morphological features were grouped into the same species (Castillo et al., 2003; Beiranvand et al., 2017). Then isolates were selected, cultured, purified and stored in the laboratory at -80°C in glycerol stock (50%) solution for further studies.
Phenotypic and biochemical characterization of endophytic bacterial isolates
Standard morphological and biochemical tests were performed for the identification of endophytic bacteria. They were characterized by gram staining and biochemical tests as described in the Cowan and Steel’s Manual for the identification of Medical Bacteria (Barrow and Feltham, 1993). For the activities of oxidase, catalase, coagulase, citrate and carbohydrate (Maltose, Sucrose, Mannitol, D-Xylose and L-Rhamnose) fermentation, isolates were biochemically analyzed (Barrow and Feltham, 1993). Then according to Bergey’s Manual of systemic Bacteriology the isolates were provisionally identified up to species level (Claus and Berkeley, 1986).
Determination of antibiotic sensitivity
Susceptibility of three (3) finally identified isolates to different antibacterial agents was measured in vitro by employing the modified Kirby-Bauer (Bauer et al., 1966) method. This method allows for the rapid determination of the efficiency of a drug by measuring the diameter of the zone of inhibition that results from diffusion of the agent into the medium surrounding the disc (Wayne, 2009). Commercially available eight (8) antibiotic discs (Himedia, India) were used for the tests.
Statistical analysis
Triplicate experiments were done in all the cases during isolation, biochemical analysis and antibiotic sensitivity tests of the selected isolates. The results were measured as the mean value ± standard deviation (SD) in triplicate. Data were captured into Microsoft Excel Software, version 2010 to calculate means and standard deviations.