Suppression of root-knot disease by Pseudomonas fluorescens CHA0 in tomato: importance of bacterial secondary metabolite, 2,4-diacetylpholoroglucinol

The antimicrobial metabolites 2,4-diacetylphloroglucinol (2,4-DAPG) and pyoluteorin contribute to the ability of Pseudomonas fluorescens strain CHA0 to control plant diseases caused by soil-borne pathogens. P. fluorescens strain CHA0 and its derivatives CHA89 (antibiotics-deficient) and CHA0/pME3424 (antibiotics overproducing) were investigated as potential biocontrol agents against Meloidogyne javanica the root-knot nematode. Exposure of root-knot nematode to culture filtrates of P. fluorescens under in vitro conditions significantly reduced egg hatch and caused substantial mortality of M. javanica juveniles. Nutrient broth yeast extract (NBY) medium amended with 2% (w/v) glucose or 1 mM EDTA markedly repressed hatch inhibition activity of the strain CHA0 but not that of CHA0/pME3424 or CHA89. On the other hand, NBY medium amended with glucose significantly enhanced nematicidal activity of the strain CHA0/pME3424. Neither glucose nor EDTA had an influence on the nematicidal activity of the strains CHA0 and CHA89. Under in vitro conditions, antibiotic overproducing strain CHA0/pME3424 and CHA0 expressed phl‘-’lacZ reporter gene but strain CHA89 did not. Expression of the reporter gene reflects actual production of DAPG. In general, CHA0/pME3424 expressed reporter gene to a greater extent compared to its wild type counterpart CHA0. Regardless of the bacterial strains, reporter gene expression was markedly enhanced when NBY medium was amended with glucose but EDTA had no such effect. A positive correlation between the degree of juvenile mortality and extent of phl‘-’lacZ reporter gene expression was also observed in vitro. Strain CHA0 produced zones of 4-6 mm on MM medium containing gelatin while strain CHA0/pME3424 and CHA89 did not. When MM medium containing gelatin was amended with 2% glucose of 1 mM EDTA size of haloes produced by the strain CHA0 reduced to 2 mm. Under glasshouse conditions aqueous cell suspension of the strains CHA0 or CHA0/pME3424 at various inoculum levels (10⁷, 10⁸ or 10⁹ cfu ml⁻¹) significantly reduced root-knot development. CHA89 caused significant reduction in galling when applied at 10⁹ cfu ml⁻¹. To better understand the mechanism of nematode suppression, split root bioassay was performed. Split-root experiments, that guarantee a spatial separation of inducing agent and a challenging pathogen, showed that soil treatment of one half of the root system with cell suspension of CHA0 or CHA0/pME3424 resulted in a significant systemic induced resistance leading to reduction of M. javanica infection of tomato roots in the non-baterized nematode treated half. The results clearly suggest that the antibiotic 2,4-DAPG from P. fluorescens CHA0 act as the inducing agents of systemic resistance in tomato roots. Populations of CHA0 and its derivatives declined progressively by 10-fold between first and fourth harvests (0-21 days after inoculation). However, bacterial populations increased at final harvest (28 days after application).