Isolation and Characterization of Bacillus spp. with Antagonistic Activities for Biological Control

We isolated 47 different bacterial isolates from 13 different soil samples, five of them showed antifungal activities belonged to Gram positive bacteria Bacillus subtilis and were designated as B1,B2,B3,B4 and B5 and they were screened to choose the better one whose activity appear to be high against different kinds of plant and human pathogenic Isolation and Characterization of Bacillus spp. with Antagonistic Activities ... 70 fungi which it was the isolate B4. It's activity is due to the production of antifungal compound(s). Also the optimum conditions for the production of the antifungal compound were determined by growing the antifungal isolate at 28°C for 5 days at pH 7. The crude extract containing the active compound was extracted and tested against different types of pathogenic fungi and showed different values of activities by measuring the inhibition zone and it was found that the fungus Alternaria alternata was the most sensitive. The crude extract is soluble in water and organic solvents and the activity of the extract was not affected by heat treatment. Thus the isolate B4 and its extract could be used as a potent biological control agent against these pathogenic fungi rather than chemicals which have harmful side effects. INTRODUCTION The genus Bacillus is among the most widely distributed groups of microorganisms in nature. Though the main reservoir of Bacillus species is considered to be in soil. (1,2). Many Bacillus spp. are capable of producing antibiotics of these bacitracin, polymyxin, tyrocidin, gramicidine (1,3). Most of the antibiotics produced are classified as peptide antibiotics and exhibit a range of spectra. They are active against Gram-positive bacteria whereas some inhibit Gram-negative bacteria and others exhibit antifungal properties (4). The potential application of bacilli is their capability of producing antifungal compounds which could be used as biological control (5). Biological control based on microorganisms to suppress plant disease, offers a powerful alternative to synthetic chemicals. The abuse of chemical pesticides or fungicides to cure or prevent plant diseases has caused soil pollution and detrimental effects in humans. It is desirable to replace chemical pesticides with materials that possess the following three criteria: high specificity against the targeted plant pathogens; easy degradability after effective usage; and low mass production cost (6,7). The genus Bacillus is comprised of Gram-positive, rod-shaped, spore-forming bacteria which are well known for their ability to produce a diverse array of antimicrobial compounds. Of particular interest is the ability of certain strains to produce antifungal compounds. Such organisms have the potential for application in agriculture where they can be used as biocontrol agents against selected plant pathogenic fungi. The use of a gram-positive Bacillus species as a biocontrol agent is relatively rare, and has received less intensive study than the use of gram-negative bacteria (5). Omar Mu'ayad Al-Obaidy 71 Fungi are primary causes of grain loss, and some of them produce compounds that are toxic when consumed (8). Fungal diseases of plants are usually controlled by some combination of cultural practices, use of fungicides, and host plant resistance. Fungicides are the primary means of fungal disease control, but their use is currently controversial because investigation have indicated potentially undesirable environmental side effects (9). According to these, this study was undertaken to isolate and characterize Bacillus spp. that exhibit broad spectrum of antifungal activity that could be used as potent biological control agent. MATERIALS AND METHODS Pathogenic fungal strains Different pathogenic fungi were used in this study as test fungi, and as follows: aPenicillium nalgiovense, Fusarium graminearum, Alternaria alternata, Rhizoctonia solani were taken from Biology Department/College of Science/ Mosul University. bMacrophomina phaseolina, was taken from Plant Protection Department/College of Agriculture/ Mosul University. cHuman pathogenic Aspergillus niger, Trichophyton mentagrophytes, Trichophyton rubrum, Candida albicans and toxin-producing Aspergillus flavus from Biology Department/College of Science/ Mosul University. Collection of the samples Different soil samples (about 13 soil samples) were collected for the isolation of the Gram positive antifungal bacteria and these samples were taken from the rhizosphere soils of different garden plants in Mosul city such as the rhizosphere of flowers and orange trees. Isolation and identification of bacteria Serial dilutions were done for each soil sample (in which 1 gram of the sample is diluted in 9 ml of sterile distilled water, then 1ml of this dilution is added to another 9 ml of distilled water and the same thing is repeated until we reach the dilution of 10), then 0.1 ml of the dilutions 10 and 10 of each sample was cultured on 9cm Petri dish of nutrient agar and incubated at 30oC for 24 hour. After that each different single colonies were tested against pathogenic fungi, and the antifungal bacterial isolates were taken and recultured for identification and further studies. The suspected isolates showed preliminary antifungal activities were subjected to several microscopical tests (shape of cells, shape of spores, swelling of cells), morphological tests (color, size of colonies, Isolation and Characterization of Bacillus spp. with Antagonistic Activities ... 72 edge of colonies, consistency of colonies etc...), biochemical tests (citrate utilization, oxidaze, indol, catalase, gelatin hydrolysis) in addition to the Gram stain, pigment production, swarming on plate, motility test, blood hemolysis, and all of these tests were done according to (10,11,12,13). Antifungal bioassay test In order to examine the antagonistic properties of bacterial isolates against the pathogenic fungi, and according to (14), a dual culture technique was carried out which is the simplest method to detect antifungal activity. In this technique an agar block (5 mm diameter) of 5day-old culture of fungal pathogen was placed in the centre of plate containing potato dextrose agar (PDA) or Sabouraud dextrose agar in the case of human pathogenic fungi (15). A loopful of 24-h-old culture of the tested bacteria was inoculated at 2 cm juxtaposed to the pathogen. A cork borer plug of fungal pathogen was inoculated centrally on PDA (or Sabouraud dextrose agar) plates, and some plates were left without inoculation of the bacteria juxtaposed to the fungal pathogen served as control. The plates were incubated at 28± 1°C for 5 days and inhibition of fungal growth was measured (16). Determination of the optimum conditions for the production of antifungal compound(s) Different conditions of incubation periods (4,5,6,7,8 days), temperatures (28,29-37°C), and pH values (6,7,8) were used to determine the optimum conditions for the production of the antifungal compounds from the bacterial isolates. Extraction and partial characterization of the active antifungal compounds Extraction of the active antifungal compounds and testing their activities against the used fungi were carried out according to (16) and also in order to characterize the nature of these compounds, the following method was described: Nutrient broth (N.B.) medium was inoculated with a loopful of fresh (24-hour old) culture of the bacterial isolate and was incubated at 28±1°C for 5 days. It was then centrifuged at 7000 rpm. for 15 min, and the supernatant was finally passed through a millipore filter paper (0.2 μm porosity) to get cell-free culture filtrate that contain the antifungal compound (16). Several tests were done for partially characterizing the active crude extract such as heat treatment (100°C for 3 hours), nature of the compound(s) (protein or not),and solubility of the compound (s) in water and organic solvents (17,18). Omar Mu'ayad Al-Obaidy 73 RESULTS AND DISCUSSION Isolation of antifungal bacteria From the thirteen soil samples, 47 different bacterial isolates of suspected Gram positive bacteria were obtained. Each one of these isolates was preliminary tested against the fungus Alternaria alternata and Macrophomina phaseolina (later tested against the other fungi). Six different bacterial isolates showed an antifungal activities against the tested fungi. Identification of bacterial isolates The six bacterial isolates that showed antifungal activities were subjected to several morphological, physiological, and biochemical tests to identify these bacteria. The results revealed that five of the antifungal isolates belonged to the Gram positive bacteria and one belonged to the Gram negative bacteria. The Gram negative isolates were neglected, while the other five antifungal isolates appeared to belong to the genus Bacillus spp. There are many researches concerning the isolation of different antifungal Bacillus isolates such as B.subtilis and B.cereus etc. (19, 20), the results showed that all antifungal isolates were able to grow in medium containing NaCl at concentration up to 5 and 7 %, also the Gram stain and other morphological, physiological, and biochemical tests indicated that all the five antifungal isolates were belonged to the genus Bacillus subtilis and they were designated as B1, B2, B3, B4, and B5, and these results were exactly in agreement with (10,11,12,13). Antifungal Bioassay Test The Bacillus isolates showed preliminary antifungal activity against the fungus Alternaria alternata (figure 1) were screened to choose the best antifungal bacterial isolate and to complete the other further tests on it. It was found that the isolate B. subtilis (B4) is the most efficient because it showed the highest inhibition zone against the tested fungus and was 8 mm. while the bacterial isolates B1, B2, B3, and B5 showed inhibition zones of 5, 5, 4,and 3 mm respectively when tested against the same fungus. So the isolate B4 was