5.2.19
INCREASED SUPPRESSION OF PYTHIUM SPLENDENS IN BEAN (PHASEOLUS VULGARIS) BY A SIDEROPHORE NEGATIVE MUTANT OF PSEUDOMONAS AERUGINOSA 7NSK2

JT TAMBONG, G VANNESTE, J POPPE and M HÖFTE

Laboratory of Phytopathology, University of Gent, Coupure Links 653, B-9000 Gent, Belgium

Background and objectives
Pythium spp. are the causal agents of pre- and post-emergence damping-off and root rot of many crops. Losses tend to be all-or-nothing. Seeds rot before or shortly after germination (pre-emergence) and newly emerged seedlings collapse (post-emergence damping-off). In beans, Pythium splendens can reduce seed emergence by more than 70% depending on soil moisture under greenhouse conditions. Pseudomonas aeruginosa 7NSK2 has been reported to suppress P. splendens-induced damping-off with the involvement of pyochelin and pyoverdine [1]. In addition, it was shown that P. aeruginosa PNA1 controls P. splendens by producing phenazines or anthranilate. In P. aeruginosa, the secondary metabolites pyochelin (together with its precursor, salicylic acid), anthranilate, and phenazine-antibiotics such as phenazine-1-carboxylate and pyocyanin are all synthesized starting from shikimic acid and chorismate. Surprisingly, a mutant of P. aeruginosa 7NSK2 unable to produce pyoverdine (pvd), pyochelin (pch) and salicylic acid (SA) (KMPCH567) showed a significantly better antagonism against P.salicylicsplendens than its parent, KMPCH (pvd-, pch-, SA+). To explain this antagonistic phenomenon, it was hypothesized that inactivation of the SA/pch branch in the chorismate pathway leads to an increased production of anthranilate and/or phenazine antibiotics.

Results and conclusions
KMPCH567 (57.3%) and 7NSK2 (56.9%) significantly increased percentage of surviving bean seedlings compared to Pythium control (22.5%) and KMPCH (39.0%) treatments. Thin-layer chromatography revealed that anthranilate was not excreted by either KMPCH567 or KMPCH, indicating that the observed antagonism could not be explained by anthranilate production. However, application of 50 µg synthetic anthranilate per g commercial substrate significantly increased the number of surviving seedlings (76.0%) compared to Pythium control (22.5%). In both KMPCH and KMPCH567, pyocyanin production was strongly increased compared to the wild type, 7NSK2 and a pyochelin-pyoverdin-producing mutant. This indicates that a pyochelin mutation leads to a shift in metabolite production, but it did not explain the difference in antagonism observed between KMPCH and KMPCH567. We are currently investigating whether some intermediate metabolites along the pyocyanin pathway such as phenazine-1-carboxylate [2] are involved in the antagonism of KMPCH567 against P. splendens.

References
1. Buyens S, Heungens K, Poppe J, Höfte M, 1996. Applied Environmental Microbiology 62, 865-871.
2. Essar DW, Eberly L, Hadero A, Crawford IR, 1990. Journal of Bacteriology 172, 884-900.