Antifungal lipopeptides from Bacillus strains isolated from rhizosphere of Citrus trees

The microbial ecology within the rhizosphere of Tylenchulus semipenetrans-infected Citrus L. trees was examined. Sixty bacterial strains were obtained from the roots of infected trees and from the eggs of T. semipenetrans. Among these strains some were obtained from the agar plates of two nematophagous fungi, Dactylellina gephyropaga (Drechsler) Ying Yang & Xing Z. Liu and Arthrobotrys conoides Drechsler. Bacterial strains were identified using 16S rDNA, gyrA and rpoB genes sequence analysis. Bacterial strains identified as Bacillus spp. were examined for their ability to synthesize surfactin, iturin, fengycin, kurstakin and bacillomycin using PCR amplification and sequencing of the encoding genes. Additionally, Bacillus strains were screened for their antifungal activity against F. solani, D. gephyropaga and A. conoides using the dual culture technique. Lipopeptide from whole cells and from supernatants of Bacillus spp. were screened using MALDI-TOF-MS analysis. The majority of the identified bacterial strains belong to the genus Bacillus with the predominance of B. cereus, B. thuringiensis, B. pumilus and B. subtilis. A total of fifteen Bacillus strains demonstrated an antifungal activity against F. solani, D. gephyropaga and A. conoides with the strongest effect found in B. amyloliquefaciens. The analysis of lipopeptides showed a high diversity of molecules, including majorly iturin C, bacillomycin D, fengycin A/B and Kurstakin found especially in B. subtilis strains. Moreover, MALDI-TOF-MS analysis showed that the responsible antibiotics for the antifungal Bacillus strains were associated with the presence of Surfactins/Pumilacidin and Fengycin A/B. Our results demonstrated the wide diversity of lipopeptides among Bacillus strains associated with citrus rhizosphere and demonstrated their antifungal ability. Our results extend the importance of Bacillus strains as potential candidates for antimicrobial activities due to their ability to synthesize and secrete cyclic lipopeptides.