Isolation and Identification of Antifungal Compounds from Bacillus subtilis C9 Inhibiting the Growth of Plant Pathogenic Fungi

Abstract

Antagonistic microorganisms against Rhizoctonia solani were isolated and their antifungal activities were investigated. Two hundred sixteen bacterial isolates were isolated from various soil samples and 19 isolates were found to antagonize the selected plant pathogenic fungi with varying degrees. Among them, isolate C9 was selected as an antagonistic microorganism with potential for use in further studies. Treatment with the selected isolate C9 resulted in significantly reduced incidence of stem-segment colonization by R. solani AG2-2(IV) in Zoysia grass and enhanced growth of grass. Through its biochemical, physiological, and 16S rDNA characteristics, the selected bacterium was identified as Bacillus subtilis subsp. subtilis. Mannitol (1%) and soytone (1%) were found to be the best carbon and nitrogen sources, respectively, for use in antibiotic production. An antibiotic compound, designated as DG4, was separated and purified from ethyl acetate extract of the culture broth of isolate C9. On the basis of spectral data, including proton nuclear magneric resonance (1H NMR), carbon nuclear magneric resonance (13C NMR), and mass analyses, its chemical structure was established as a stereoisomer of acetylbutanediol. Application of the ethyl acetate extract of isolate C9 to several plant pathogens resulted in dose-dependent inhibition. Treatment with the purified compound (an isomer of acetylbuanediol) resulted in significantly inhibited growth of tested pathogens. The cell free culture supernatant of isolate C9 showed a chitinase effect on chitin medium. Results from the present study demonstrated the significant potential of the purified compound from isolate C9 for use as a biocontrol agent as well as a plant growth promoter with the ability to trigger induced systemic resistance of plants.

Isolation, screening and growth optimization of antagonistic Bacillus subtilis MS21 from Thengapattanam estuary against plant fungal pathogens

“…Usama (2003) tested several carbon sources reported that the maximum growth of B. subtilis and β-glucanase production was obtained with lactose as sole carbon source. Similarly, in the present, work 1% beef extract was found to be an ideal nitrogen source while Islam et al, 2012 found 1% soytone as the ideal nitrogen source for the growth of antifungal compound producing Bacillus subtilis C9 against the plant pathogenic fungi Rhizoctonia solani (Figs.10 and 11). Joshi et al, 2008 found ammonium nitrate as a nitrogen source in minimal salt media enhanced the production of lichenysin (1 g/L) in B. licheniformis.…”

“…alone (kumari et al, 2017). Many B. subtilis strains have been reported for their effective biocontrol activity by producing antifungal compounds (Silo-Suh et al, 1994;Souto et al, 2004;Cazorla et al, 2007;Islam et al, 2012 andMardanova et al, 2017).…”

Purification and characterization of an antifungal protein from Bacillus subtilis XL62 isolated in Vietnam

“…The results show that the crude supernatant of B. subtilis XL62 has a significant antifungal activity on both fungal pathogens. Our results are in agreement with those of Islam et al who reported the antifungal activity of an ethyl acetate extract (2 mg/well) of B. subtilis C9, which inhibited the growth of R. solani AG2-2(IV), R. solani AG1-1(A), R. solani AG-4, and F. oxysporum mycelia by 88%, 32%, 72%, and 72%, respectively 1 . In addition, Ali et al showed that a culture filtrate from Bacillus sp.…”

“…Tan et al purified antifungal protein with molecular weight of 38 kDa from B. subtilis B25 26 . The difference in antifungal peptides produced by different strains of B. subtilis may be due to the fact that biosynthesis of antibiotics from microorganisms is often regulated by nutritional and environmental factors 1 . Using MALDI-TOF mass spectrometry to characterize the purified protein, we found that it is a chitin-binding protein.…”

“…Bacillus spp. offer several advantages over other organisms since they can form endospores and can tolerate extreme pH, temperature, and osmotic conditions 1 . Furthermore, they produce a diverse array of antifungal compounds, which range from small peptides, such as iturin 2,3 , surfactin 4,5 , fengymycin, bacilysin 6 , bacillomycin 7 , mycosubtilin 8 , and mycobacillin 9,10 , to volatile metabolites 11,12 .…”