Pseudomonas fluorescens strain CHAO suppresses Thielaviopsis basicola-induced black root rot of tobacco and Gaeumannomyces graminis var. tritici-induced take-all of wheat. Strain CHAO produces 2,4-diacetylphloroglucinol, a metabolite with antifungal, antibacterial, and phytotoxic activity. The role of this compound in disease suppression was tested under gnotobiotic conditions. A P. fluorescens mutant, obtained by Tn5 insertion, did not produce 2,4-diacetylphloroglucinol, showed diminished inhibition of T. basicola and of G. g. var. tritici in vitro, and had a reduced suppressive effect on tobacco black root rot and on take-all of wheat, compared with wild-type CHAO. Complementation of the mutant with an 11-kb DNA fragment from a genomic library of wild-type CHAO largely restored production of the metabolite, inhibition of the fungal pathogens in vitro and disease suppression. The Tn5 insertion was physically mapped using a 5.8-kb complementing fragment as a probe. 2,4-Diacetylphloroglucinol was shown to be produced in the rhizosphere of wheat by strain CHAO and by the complemented mutant, but not by the mutant defective in 2,4-diacetylphloroglucinol synthesis. These results support the importance of 2,4-diacetylphloroglucinol production by strain CHAO in the suppression of soilborne plant pathogens in the rhizosphere.

The minimal replicon of the Pseudomonas plasmid pVS1 was genetically defined and combined with the Escherichia coli p15A replicon, to provide a series of new, oligocopy cloning vectors (5.3 to 8.3 kb). Recombinant plasmids derived from these vectors were stable in growing and nongrowing cells of root-colonizing P. fluorescens strains incubated under different environmental conditions for more than 1 month.

Certain strains of fluorescent pseudomonads can effectively colonize plant roots and protect plants from diseases caused by a variety of root pathogens. Such beneficial or plant health-promoting strains are emerging as promising biocontrol agents. They are suited as soil inoculants either individually or in combination and may be compatible with current chemical pesticides (1, 2, 3, 4, 5, 6, 7, 8). In our biocontrol studies, we have focused on Pseudomonasfluorescens strain CHAO, an isolate from a suppressive soil in the western part of Switzerland (9). This strain was originally shown to colonize tobacco roots and to suppress black root rot, which is caused by the fungus Thielaviopsis basicola (9, 10). Subsequent work has established that disease suppression by strain CHAO displays little specificity with respect to the host plant and the pathogen. Protected plants include wheat, cucumber, sugar beet, cotton, flax, corn, and cress. Pathogenic action of at least the following fungal pathogens can be reduced by strain CHAO: Pythium ultimum, Gaeumannomyces graminis var. tritici (Ggt), Fusarium oxysporum f.s p. cucurbitaceae, Phomopsis sclerotioides, and Rhizoctonia solani (11, 12, 13, 14, 15; our unpublished data). Since the interactions between I! fluorescens, other organisms and the soil environment are extremely complex, it became important to develop reproducible methods that allow us to monitor the plant-beneficial effects of strain CHAO reliably and to analyze the traits that make it an effective biocontrol agent. In section 6.2 we will review some of our approaches to investigate the mechanisms by which strain CHAO achieves biological control.We are using strain CHAO as a model organism to study not only the mechanisms of disease suppression but also the ecological impact of introduced plant-beneficial bacteria (see section 6.3). In a parallel approach, we are investigating the potential applications of biological control agents to improve the yields of protected crops. We are testing a variety of strains, singly and in combination, for the development of greenhouse applications. A brief account of this work is presented in section 6.4. 68Disease Suppression by I! Fluorescens CHAO Mechanistic Studies on Biocontrol m i t s of Pseudomonas Fluorescens CHAO Chemical Identification of Extracellular MetabolitesMetabolites produced and excreted by I? fluorescens are assumed to be important biotic factors in the biological control of root diseases (2, 5, 16, 17, 18, 19, 20, 21). Until now, about a dozen low molecular weight compounds have been identified in culture supernatants of Rfluorescens CHAO ( Table 1). These products can be broadly classified into two groups: siderophores and secondary metabolites. The siderophores (e. g. , iron chelators) pyoverdine (pseudobactin), salicylate and pyochelin are all produced by I? fluorexens CHAO (22, 23 ; our unpublished results) and by other fluorescent pseudomonads when these bacteria are grown under ironlimiting conditions (24; reviewed by Loper & Buyer [20] and O'Sullivan...

Pseudomonas fluorescens CHA0 produces a variety of secondary metabolites, in particular the antibiotics pyoluteorin and 2,4-diacetylphloroglucinol, and protects various plants from diseases caused by soilborne pathogenic fungi. The rpoD gene encoding the housekeeping sigma factor 70 of P. fluorescens was sequenced. The deduced RpoD protein showed 83% identity with RpoD of Pseudomonas aeruginosa and 67% identity with RpoD of Escherichia coli. Attempts to inactivate the single chromosomal rpoD gene of strain CHA0 were unsuccessful, indicating an essential role of this gene. When rpoD was carried by an IncP vector in strain CHA0, the production of both antibiotics was increased severalfold and, in parallel, protection of cucumber against disease caused by Pythium ultimum was improved, in comparison with strain CHA0.

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