PRODUCTION REGULATION OF SECONDARY METABOLITES BY FUSARIC ACID AND THEIR ROLES IN SUPPRESSION OF FUSARIUM SPP.BY THE RHIZOBACTERIUM PSEUDOMONAS FLUORESCENS PF-5

Maria Carolina Quecine Quecine (ESALQ-USP); Teresa Kidarsa Kidarsa (USDA-ARS); Marcella D. Henkels Henkels (USDA-ARS); Brenda T. Shaffer Shaffer (USDA-ARS); Aline Aparecida Pizzirani-kleiner Pizzirani-kleiner (ESALQ-USP); Joyce Loper Loper (USDA-ARS)

Pseudomonas fluorescens strain Pf-5 is a rhizosphere bacterium that acts as a biocontrol agent of soilborne plant diseases, and produces at least 10 different secondary metabolites including several with antifungal properties. Previously, other workers showed that the mycotoxin fusaric acid (FA), which is produced by many Fusarium spp., inhibited the production of 2,4-diacetylphoroglucinol (2,4-DAPG) by Pseudomonas spp. The goal of this study was to determine the role of Pf-5 metabolites in the suppression of Fusarium spp. and to investigate FA effects on secondary metabolite production.  We derived site-directed mutants of Pf-5 with single and multiple mutations in the biosynthetic gene clusters for the antifungal metabolites 2,4-DAPG, pyrrolnitrin, pyoluteorin, hydrogen cyanide, rhizoxin, pyoverdine and enantio-pyochelin.  These mutants were tested for suppression of the pathogens Fusarium verticillioides from corn and sugarcane and Fusarium oxysporum f. sp. pisi from pea. Fusarium spp. were similar, but not identical, in their sensitivity to Pf-5 metabolites. Rhizoxin and 2,4-DAPG were found to be primarily responsible for fungal antagonism by Pf-5 on several culture media.  The influence of FA on secondary metabolism by Pf-5 was investigated using RT-qPCR and HPLC. Amendment of the culture medium with FA decreased 2,4-DAPG production and transcript levels of 2,4-DAPG biosynthetic genes. FA also influenced transcript abundance of pyoluteorin, rhizoxin, and pyoverdine biosynthesis genes. Similar results were confirmed by HPLC analysis. These results indicate that the mycotoxin influenced antibiotic production by Pf-5 at the transcriptional level. Our results demonstrated the importance of two compounds, rhizoxin and 2,4-DAPG, in suppression of Fusarium spp. by Pf-5, and confirmed that an inter-species signaling system mediated by FA influenced secondary metabolites production by the bacterial biological control organism.