Characterization of selected Bacillus isolates exhibiting broad spectrum antifungal activity.

Abstract

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. Ofparticular 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. A study was undertaken to further characterize selected Bacillus isolates that exhibit broad spectrum antifungal activity. Dual culture bioassays were used to screen seven selected Bacillus isolates for activity against four plant pathogenic fungi in vitro. All isolates were able to inhibit the pathogens to varying degrees. Two isolates, R29 and B81, were selected for further testing and characterization. Further bioassays were performed on five complex nutrient media which were adjusted to pH S.S and 7, and both incubated at 2SoC and 30°C" respectively. It was found that pH and media composition showed significant influences on the antifungal activities of the isolates tested, but that a SoC temperature difference in incubation temperature did not. Tryptone soy agar was found to give rise to the largest inhibition zones. Both isolates were tentatively identified using standard biochemical and morphological tests. Based on its phenotypic characteristics, R29 was identified as a strain of B. subtilis. B81 proved to be more difficult to assign to a specific group or species of Bacillus, though B. subtilis and B. licheniformis were considered to be the nearest candidates. Genomic DNA was extracted from both isolates and a portion of each of their 16s rDNA genes were amplified and sequenced for homology testing against the GeneBank database. Homology testing confirmed that both isolates were members of the genus Bacillus and most probably strains of B. subtilis. The DNA fragment used for sequencing proved to be too small to give conclusive identification of the isolates. Isolate R29 was selected for further characterization of its antifungal compound/so Growth curve studies using a defined synthetic medium showed that antifungal activity arose during the stationary phase and appeared to be closely linked to sporulation. The antifungal component of cell free culture supematant was extracted using various methods including thin layer chromatography, acid precipitation, hydrophobic interaction chromatography and methanol extractions. High performance liquid chromatography (HPLC) analysis of extracts from acid precipitation and hydrophobic interaction chromatography revealed two active peaks indicating that at least two antifungal compounds were produced. Methanol extracted samples produced the cleanest sample extract but only revealed one active peak from the HPLC fraction . Nuclear magnetic resonance analysis of purified samples indicated that the antifungal compound/s have aromatic complex and peptide structures. The extracted antifungal compounds were Protease K resistant and found to be thermostable at temperatures ranging 80-121oC, and, were active at pH ranges of 3-13. The antifungal compounds were found to exhibit similar properties to known antifungallipopeptides i.e. iturin A and fengycin A and B. Further characterization and identification of the active compounds is recommended usmg methods such as liquid chromatography mass spectrometer and matrix-assisted laser desorption ionisation time-of- flight. The results presented in this dissertation provide a basis from which antifungal compounds produced by strains ofBacillus can be further characterized.