Gluconic acid: An antifungal agent produced by Pseudomonas species in biological control of take-all Rajvinder Kaur a , John Macleod b , William Foley c , Murali Nayudu c, * a Department of Plant and Microbial Biology, University of California, 111 Koshland Hall, Berkeley, CA 94720-3102, USA b Research School of Chemistry, The Australian National University, Canberra, ACT 0200, Australia c School of Botany and Zoology, Faculty of Science, The Australian National University, Daley Road, ANU Campus Acton, Canberra, ACT 0200, Australia Received 12 October 2005; received in revised form 7 December 2005 Available online 30 January 2006 Abstract Pseudomonas strain AN5 (Ps. str. AN5), a non-fluorescent Australian bacterial isolate, is an effective biological control (biocontrol) agent of the take-all disease of wheat caused by the fungus Gaeumannomyces graminis var. tritici (Ggt). Ps. str. AN5 controls Ggt by producing an antifungal compound which was purified by thin layer and column chromatography, and identified by NMR and mass spectroscopic analysis to be D-gluconic acid. Commercially bought pure gluconic acid strongly inhibited Ggt. Two different transposon mutants of Ps. str. AN5 which had lost take-all biocontrol did not produce D-gluconic acid. Gluconic acid production was restored, along with take-all biocontrol, when one of these transposon mutants was complemented with the corresponding open reading frame from wild-type genomic DNA. Gluconic acid was detected in the rhizosphere of wheat roots treated with the wild-type Ps. str. AN5, but not in untreated wheat or wheat treated with a transposon mutant strain which had lost biocontrol. The antifungal compounds phen- azine-1-carboxylic acid and 2,4-diacetylphloroglucinol, produced by other Pseudomonads and previously shown to be effective in sup- pressing the take-all disease, were not detected in Ps. str. AN5 extracts. These results suggest that D-gluconic acid is the most significant antifungal agent produced by Ps. str. AN5 in biocontrol of take-all on wheat roots. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Pseudomonas; Gaeumannomyces graminis; Triticum; Wheat; Take-all; Antifungal; Biological control; Gluconic acid; Root disease; Australia 1. Introduction Pseudomonas bacteria produce different metabolites that can suppress fungal plant pathogens. The production of antibiotics (compounds which at very low concentrations, lg levels, inhibit microorganisms) such as phenazine and phloroglucinol by symbiotic fluorescent Pseudomonas bac- teria has been shown to provide a natural defense to the plant against fungal diseases such as take-all (Weller, 1988; Keel et al., 1992; Cook et al., 1995). Pseudomonas flu- orescens 2–79 and Pseudomonas aureofaciens 30–84 pro- duce the novel antifungal secondary metabolite phenazine-1-carboxylic acid (PCA). PCA produced by Pseudomonas on wheat roots has been shown to be a cru- cial factor in take-all disease suppression (Turner and Mes- senger, 1986; Thomashow and Weller, 1990; Thomashow et al., 1990; Pierson and Thomashow, 1992). The antibiotic 2,4-diacetylphloroglucinol (DPG), which has also been shown to suppress the take-all pathogen on plant roots, is produced by several Pseudomonads including Pseudomo- nas fluorescens strain CHA0 (Keel et al., 1992, 1996). Other antibiotics produced by Pseudomonads include pyoluteo- rin (Maurhofer et al., 1994), Pyrrolnitrin (Ligon et al., 2000) and oomycin A (James and Gutterson, 1986) which can suppress a range of different plant pathogenic fungi. Novel bacterial antifungal metabolites produced by Pseu- domonas species have also been identified: 3-(1-hexenyl)- 5-methyl-2-(5H)-furanone (Paulitz et al., 2000); N-mer- capto-4-formylcarbostyril (Fakhouri et al., 2001); aerugine 0031-9422/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2005.12.011 * Corresponding author. Tel.: +61 2 61253643; fax: +61 2 61259738. E-mail address: Murali.Nayudu@anu.edu.au (M. Nayudu). www.elsevier.com/locate/phytochem Phytochemistry 67 (2006) 595–604 PHYTOCHEMISTRY