Detection de extracellular metabolities with antifungal activity produced of Bacillus sp. by bioautography method

DELFINI C D,; VILLEGAS LB; TERESCHUK M L; BAIGORÍ M

ROSARIO

Congreso; IX CONGRESO ARGENTINO DE MICROBIOLOGIA GENERAL; 2013

SAMIGE

Bacterium characterized as belonging to Bacillus genus with chitinase activity was previously isolated from bat guano and it was called Bacillus sp. PA 14. Sequence analysis of the 16S ribosomal RNA (rRNA) gene of this strain showed high identity (99%) with Bacillus atrophaeus. Cell-free supernatants of Bacillus sp. PA 14 cultures exhibited antifungical activity against several phytopathogenic fungi, principally against member of Colletotrichum genus, the causative agent of berries anthracnose diseases. Following this line of research, the present work illustrates the application of bioautography techniques, in order the directly view how many fractions with antifungical activity are present in the supernatants of Bacillus sp. PA14 culture. Bioautography on thin-layer chromatographic (TLC) plates is a means of detecting the biological activity of a sample which migrated on the plate with a solvent. TLC was carried out with silica gel plates 60 F 254 and cell-free supernatants of Bacillus sp. PA14 obtained with SPI-chitin (SPI-Q) and Standar Nutrient (SN) media were spotted over a TLC plates. To test the stability of antifungal activity, it was included supernatants that remained at room temperature during 30 days. Buthanol:acetic acid: water was used as solvent. After migration, TLC was observed under different wavelengths: 259 and 360 nm. Duplicate TLC plate was placed on sabouraud agar inoculated with Colletotrichum acutatum and was incubated at 30 °C for 48h. The results showed two and three spots when TLC plate was exposed at 259 nm and 360 nm respectively in supernatants obtained with Bacillus sp. PA14 grown in SN, and only one in those obtained with SPI-Q. After the fungi growth inhibition two spots were visible after incubation in supernatants SN medium and only one in SPI-Q. After 30 days, the supernatants showed a slight decrease in antifungal activity Bioautographic methods combine chromatographic separation and in situ activity determination. It is a fast and economic technique that in two steps verified the presence of more than one metabolite responsible of antifungal activity in Bacillus sp. PA 14 supernatants. The presence of extracellular metabolites allows recovery and handling them easier and cheaper than if they were intracellular metabolites. These results suggest the feasibility of using Bacillus sp. PA14 supernatants, on field tests for Colletotrichum growth inhibition. The next goal will be to purify and characterize these metabolites.