Elsevier

Phytochemistry

Volume 49, Issue 6, 20 November 1998, Pages 1579-1583
Phytochemistry

Chlorosis-inducing products from Pseudomonas syringae pathovars: new N-coronafacoyl compounds

https://doi.org/10.1016/S0031-9422(98)00279-9Get rights and content

Abstract

Liquid cultures of Pseudomonas syringae pv. tomato strains that also produced the phytotoxin coronatine, were found to have a new chlorosis-inducing activity, not previously described. Bioassay-guided fractionation and HPLC analysis revealed two new peaks that were chlorosis-inducing on leaves of bean plants. Mass spectrometry and NMR analyses of the compounds led to the derivation of their structures as coronafacoyl-l-serine and coronafacoyl-l-threonine, respectively. The amino acid C-2 configurations were determined by GC analysis following hydrolysis with 6 M HCl. Both compounds have more polar and more acidic properties than coronatine, norcoronatine, and other known coronafacoyl conjugates with non-polar amino acids. Investigation of Pseudomonas syringae pv. glycinea strains that are known to be prolific producers of the coronatine family of compounds revealed the production of coronafacoylserine and coronafacoylthreonine also.

Introduction

The phytopathogenic bacterium Pseudomonas syringae pv. tomato is well known to produce the phytotoxin coronatine1, 2. We have previously reported[2]a divergence in the nature of chlorosis-inducing compounds produced by various strains of P. syringae pv. tomato, where some strains produced coronatine, and others did not, but instead an alternative and uncharacterised chlorosis-inducing component, which was refered to as tomatotoxin. In subsequent work we have found strains of P. syringae pv. tomato that produce both coronatine and tomatotoxin [unpublished data]. The `coronatine' complement from such strains generally contains the array of known coronatine related compounds such as those produced by P. syringae pv. glycinea [3, 4, and unpublished data], that is, in addition to coronatine (usually the major component), norcoronatine, coronafacic acid, coronafacoyl-l-valine, coronafacoyl-l-alloisoleucine and coronafacoyl-l-isoleucine. During a recent investigation of the chemistry of chlorosis-inducing products of P. syringae pv. tomato, we undertook a study of the products from a `dual producing' strain of P. syringae pv. tomato. We observed, during fractionations, additional chlorosis-inducing activity that was clearly arising from components different to both tomatotoxin and to any known coronatine-related compounds. The present paper describes the isolation, purification and characterisation of the compounds causing this chlorosis activity, and reports on their occurrence from several strains of P. syringae pv. glycinea.

Section snippets

Sequential extraction

The results reported relate entirely to P. syringae pv. tomato #3362 which we have previously found to produce coronatine[2]and the other known N-coronafacoyl compounds [unpublished data], that is, norcoronatine, coronafacoyl-l-valine, coronafacoyl-l-isoleucine and coronafacoyl-l-alloisoleucine. The ethyl acetate extracts of the concentrated culture supernatant, taken at various (decreasing) pH levels, differed markedly in their composition as judged by GC analysis and bioassay results. At the

Bacterial strains and culture

Strains of pathovars of P. syringae were taken from the International Collection of Microorganisms from Plants, recovered from the lyophilized state by plating onto King's medium B, and maintained both on slants of phosphate-salts agar and by storage at −80° in 15% glycerol. Liquid shake cultures were 600 ml vols in 2 l conical flasks, for 5 days at 18°, inoculated with 2 ml of a 24 hr yeast inoculum culture, in turn inoculated with cells from a 24 hr culture on a King's B slant[10]. P. syringae pv.

Acknowledgements

We express our gratitude to Dr Harry Young for compiling the mass spectral data, and to Mr Michael Walker, University of Auckland, for the NMR spectra.

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