5.2.9
PRODUCTION OF ANTIFUNGAL SUBSTANCES BY PSEUDOMONAS FLUORESCENS W8a ISOLATED FROM WHEAT ROOTS OF CONTINUOUSLY CULTURED FIELD PLANTS

Y HOMMA1, J TAZAWA-ISOGAMI1, T NAKAYAMA2, K KATO3 and M MATSUZAKI1

1Hokkaido National Agricultural Experiment Station, Shinsei, Memuro, Kasai, Hokkaido 082-0071, Japan; 2National Agricultural Ressearch Center, Kannondai, Tsukuba 305-8666, Japan; 3National Institute of Agro-Biological Resources, Kannondai, Tsukuba 305-8602, Japan

Background and objectives
Take-all of wheat, caused by Gaeumannomyces graminis var. tritici (Ggt), is widely known to decline for the first several years when wheat has been continuously cultured for a long time [1]. It is of interest to know the mechanisms of natural biological control in soil which is suppressive to Ggt in order to be able to develop natural biological control methods based on antagonisitc organisms. Recently, the role of antibiotic production in biological control has been discussed based on genetic and molecular aspects. Examples of such antibiotics produced by Pseudomonas sp. are phenazine-1-carboxylic acid and its derivatives [2], and 2,4-diacetylphloroglucinole [3] which are active in the suppression of Ggt. However, the role of the antibiotic pyrrolnitrin, produced by Pseudomonas sp., has not been reported in the biological control of wheat take-all.

P. fluorescens strain W8a was obtained from roots of wheat which had been cultured continuously for a long time and was suppressive to wheat take-all in a field test. This paper deals with the biological characteristics of the soil from which the strain was isolated, and the production of the antifungal substances by the strain, in order to establish the role of pyrrolnitrin production in the suppression of wheat take-all by seed coating with the strain.

Results and conclusions
P. fluorescens strain W8a was isolated from wheat roots of field plants continuously cultured for 23 years in Memuro, Hokkaido, Japan. The field soil was suppressive to wheat take-all caused by Ggt. The suppressiveness was reduced by heat treatment by aerated steam at temperatures greater than 55C. Strain W8a was effective in suppressing take-all in field tests and was antagonistic to Ggt in culture. Antifungal substances were extracted from PPGS culture filtrate of the strain, fractionated in several steps of silica gel column chromatography, and purified by HPLC. One of the substances was identified to pyrrolnitrin, based on the characteristics of Rf value of TLC, the spectrum of NMR and EI-MS comparing with the authentic sample. The strain also produced a fluorescent substance in KB culture and HCN detected by the method of Casteric and Casteric [4]. The suppressive capacity of mutants induced by insertion of the transposon mini-Tn5 were deficient in production of pyrrolnitrin; the production of fluorescent substances was less than that of the parent strain. These results suggest that pyrrolnitrin is involved in biological control of wheat take-all disease. These antifungal substances were tested to clarify the mechanism of the suppression of wheat take-all.

References
1. Cook RJ, Baker KF, 1983. APS, St Paul, Minesota, pp. 256-258.
2. Thomashow LS, Weller DM, 1988. Journal of Bacteriology 170, 3499-3508.
3. Keel C, Schnider U, Maurhofer M et al., 1992. Molecular Plant-Microbe Interactions 5, 4-13.
4. Casteric KF, Casteric PA, 1983. Applied and Environmental Microbiology 45, 701-702.