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.

Pseudomonas fluorescens CHA0 colonizes plant roots, produces several secondary metabolites in stationary growth phase, and suppresses a number of plant diseases, including Thielaviopsis basicola-induced black root rot of tobacco. We discovered that mutations in a P. fluorescens gene named gacA (for global antibiotic and cyanide control) pleiotropically block the production of the secondary metabolites 2,4-diacetylphloroglucinol (Phl), HCN, and pyoluteorin. The gacA mutants of strain CHA0 have a drastically reduced ability to suppress black root rot under gnotobiotic conditions, supporting the previous observations that the antibiotic Phl and HCN individually contribute to the suppression of black root rot. The gacA gene is directly followed by a uvrC gene. Double gacA-uvrC mutations render P. fluorescens sensitive to UV irradiation. The gacA-uvrC cluster is homologous to the orf-2 (= uvrY)-uvrC operon of Escherichia coli. The gacA gene specifies a trans-active 24-kDa protein. Sequence data indicate that the GacA protein is a response regulator in the FixJ/DegU family of two-component regulatory systems. Expression of the gacA gene itself was increased in stationary phase. We propose that GacA, perhaps activated by conditions of restricted growth, functions as a global regulator of secondary metabolism in P. fluorescens.

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...

The secondary metabolite hydrogen cyanide (HCN) is produced byPseudomonas fluorescens from glycine, essentially under microaerophilic conditions. The genetic basis of HCN synthesis inP. fluorescens CHA0 was investigated. The contiguous structural genes hcnABC encoding HCN synthase were expressed from the T7 promoter in Escherichia coli, resulting in HCN production in this bacterium. Analysis of the nucleotide sequence of the hcnABC genes showed that each HCN synthase subunit was similar to known enzymes involved in hydrogen transfer, i.e., to formate dehydrogenase (for HcnA) or amino acid oxidases (for HcnB and HcnC). These similarities and the presence of flavin adenine dinucleotide- or NAD(P)-binding motifs in HcnB and HcnC suggest that HCN synthase may act as a dehydrogenase in the reaction leading from glycine to HCN and CO2. The hcnApromoter was mapped by primer extension; the −40 sequence (TTGGC … .ATCAA) resembled the consensus FNR (fumarate and nitrate reductase regulator) binding sequence (TTGAT … .ATCAA). The gene encoding the FNR-like protein ANR (anaerobic regulator) was cloned from P. fluorescens CHA0 and sequenced. ANR of strain CHA0 was most similar to ANR of P. aeruginosa and CydR of Azotobacter vinelandii. An anr mutant of P. fluorescens (CHA21) produced little HCN and was unable to express an hcnA-lacZ translational fusion, whereas in wild-type strain CHA0, microaerophilic conditions strongly favored the expression of the hcnA-lacZ fusion. Mutant CHA21 as well as an hcn deletion mutant were impaired in their capacity to suppress black root rot of tobacco, a disease caused by Thielaviopsis basicola, under gnotobiotic conditions. This effect was most pronounced in water-saturated artificial soil, where the anr mutant had lost about 30% of disease suppression ability, compared with wild-type strain CHA0. These results show that the anaerobic regulator ANR is required for cyanide synthesis in the strictly aerobic strain CHA0 and suggest that ANR-mediated cyanogenesis contributes to the suppression of black root rot.