IDENTIFICATION OF GENES RESPONSIBLE OF THE POTENT ANTIFUNGAL ACTIVITY OF Burkholderia ambifaria T16

FELDMAN, N. ; ÁLVAREZ, F.; VINACOUR, M.; SIMONETTI, E.; RUIZ, JIMENA A

Congreso; XIII CONGRESO ARGENTINO DE MICROBIOLOGÍA GENERAL SAMIGE; 2018

Burkholderia ambifaria T16 is a bacterial strain isolated from the rhizosphere of barley which showed the ability to inhibit mycelial growth and conidial germination of several phytopathogenic fungi, including different Fusarium spp. With the aim to identify the genes responsible of this potent antifungal activity, an insertional mini-Tn5 library was constructed in B. ambifaria T16 by triparental mating. A total of 8,250 mutants were screened for the ability to inhibit growth of F. oxysporum by using an overlay assay with homogenized mycelium. The insertion sites of the mini-Tn5 were mapped in 20 mutants, which showed reduced or null antifungal activity, by an arbitrary primed polymerase chain reaction (AP-PCR) method. In half of these mutants, the mini-Tn5 was inserted in a modular non ribosomal peptide synthetase (NRPS) gene cluster, encoding proteins involved in the biosynthesis of cyclic antifungal lipopeptides (CLPs) known as occidiofungins/burkholdines (bks). In these mutants, the mini-Tn5 insertion abolished completely the ability of Burkholderia ambifaria T16 to inhibit mycelial growth of F. oxysporum. When these mutants were tested against other important pathogenic fungi, such as F. graminearum, Macrophomina phaseolina and Candida albicans, the antifungal activity was significantly reduced compared to the wild type strain, but not completely abolished. These results suggest that besides burkholdines, other compounds produced by B. ambifaria T16 are capable to inhibit growth of F. graminearum, M. phaseolina and C. albicans. Nevertheless, for all fungi analyzed, burkholdines were responsible for most of the antifungal activity. The identification and characterization of NRPS gene clusters responsible of strong antifungal activity against phytopathogenic fungi would provide key information for engineered biosynthesis of innovative antifungal compounds.