Biological control of Ralstonia solanaciarum and Erumia carotovora by use of locally isolated bacteria

A total of 990 isolates, 430 of which are Bacilli and other bacteria and 560 actinomycetes, revived from the stock culture of the Antibiotics Laboratory, BIOTECH, UPLB and some isolated from different soil samples, were used in this study. Of these, a total of 143 and 135 Bacillus isolates inhibited R. solanacearum T731 and T523, respectively. Thirty six of these isolates showed high activity (24 mm zone of inhibition) against the test organisms by agar plug assay. Shake flask experiment were conducted to validate the activity of the Bacillus isolates in liquid medium. The problem, however, was that, growth of Bacillus around the cup cylinders after incubation interfered with the measurements of the zones of inhibition. Thus, the results obtained from the agar plug assay served as basis in selecting the best isolates to be used in the greenhouse experiment. Among the 560 actinomycetes assayed, a total of 192 isolates showed activity against R.solanacearum T731 and T523 by agar plug method. Among these, a total of 12 and 8 isolates showed high activity( 24 mm zone of inhibition) against the test organisms T731 and T523, respectively, by cup cylinder assay. All the Bacillus and actinomycete isolates had higher activity than the standard antibiotic Kanamycin monosulfate and gentamycin was conducted to optimize the medium for production and the concentration of inoculum. Rich nutrient broth (RNB) proved to be the best medium being able to support growth and bioactive production of BC152 and BB142 giving 28.0 mm and 24.40 mm, respectively. Zone of inhibition against Ralstonia solanacearum was highest at 10% concentration of BC152 and BB142 with inhibition of 27.6 mm and 24.06 mm, respectively. Production of the biocontrol agents was done in 2.6 L bioreactors. Bacillus BB142 had the highest zone inhibition of 25.9 mm at the 4th day of fermentation with aeration of 1vvm and agitation rate at 250 rpm. BC152 on the other hand showed higher activity at the 4th day of fermentation with 29.9 mm inhibition of the growth of R. solanacearum by cup cylinder assay. Production conditions for BC152 was set at 1vvm aeration and 300rpm agitation rate. Actinomycetes AB 240 and AE21 were selected for greenhouse experiment to evaluate their effectiveness as biocontrol agents against R. solanacearum. These isolates, however, were not effective as biocontrol agents because they were unable to inhibit the infection of R. solanacearum in natural environment with unsterile soil as potting medium. The natural microflora of the soil and added R. solanacearum caused wilting of the transplanted tomato plants. Hence, this part of the study was not pursued at this time. On the other hand, Bacillus isolates BC152 and BB142 are effective biocontrol agents in the greenhouse experiment conducted. These two isolates prevented the wilting of tomato plants inoculated with bacterial wilt-causing R. solanacearum. Morphological and biochemical characterization of the best Bacillus isolates BC152 and BB142 identified them as Bacillus subtilis.