Submitted to: Applied Microbiology and Biotechnology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 12/16/2004
Publication Date: 2/18/2005
Citation: Slininger, P.J., Shea Andersh, M.A. 2005. Proline-based modulation of 2,4-diacetylphloroglucinol and viable cell yields in cultures of Pseudomonas fluorescens wild-type and over-producing strains. Journal of Industrial Microbiology and Biotechnology 68:630-638. Available: http://www.springerlink.com/index/0.1007/s00253-005-1907-4. Interpretive Summary: The compound 2,4-diacetylphloroglucinol (DAPG) is an important antifungal antibiotic produced on the roots of wheat by Pseudomonads, which are beneficial bacteria responsible for the natural biological control phenomenon known as "take-all decline." Take-all, one of the most devastating root diseases of wheat world-wide, is incited by the fungus Gaeumannomyces graminis var. tritici. A biological control approach to this pest problem appears promising because no economical chemical or cultural means exist for reliable control. While DAPG production drives the biocontrol process of root-colonizing bacteria, too much accumulation of DAPG in liquid production cultures followed by too much DAPG delivered in wheat seed coatings may be detrimental to both biocontrol product yield and quality. In studies to develop production culture and seed formulation technologies for P. fluorescens strains as biological control agents, the impact of culture metabolites DAPG and 2,4,6-trihydroxyacetophenone (THA) on cell growth rate, viability, and seed germination rate were studied. Inhibitory concentration ranges of DAPG and THA were observed and defined; and the inclusion of proline in the cultivation medium to supply part of the carbon and nitrogen needed for cell growth was discovered to minimize THA and DAPG accumulations. These findings are fundamental to developing biocontrol agent manufacturing strategies that are needed to supply commercial seed inoculants of DAPG-producing bacteria for take-all control.
Technical Abstract: The antifungal compound 2,4-diacetylphloroglucinol (DAPG) is produced in the rhizosphere of wheat by Pseudomonad populations responsible for the natural biological control phenomenon known as 'take-all decline.' Studies were conducted to elucidate the impact of DAPG and its co-product 2,4,6-trihydroxyacetophenone (THA) on the production of Pseudomonas fluorescens for biological control. Increasing DAPG from 0.1 to 0.5 g/L and THA from 0.05 to 0.5 g/L significantly inhibited the growth and lowered the yield of viable bacteria in liquid cultures. On further examination of these metabolites applied in seed coatings, levels of DAPG and THA exceeding 0.05 mg/g seed significantly reduced wheat germination percentages. The three-way interaction of DAPG, THA, and culture medium ingredients was significant, and greatest seed germination loss (40-50%) was observed when 0.5 mg DAPG and 0.25 mg THA were combined in a coating of 0.5 mL culture medium/g seed. Based on results of Biolog Microplate GN, flask and fermentor screens of carbon sources, proline was found to optimize the viable cell yields of the P. fluorescens strains tested. The combination of proline with glucose and urea as C and N sources in growth media could be optimized to minimize DAPG production and maximize the vitality of P. fluorescens cultures, Q8R1-96 and the DAPG-over-producer Q69c-80:miniTn5:phl20. In production cultures, proline supply rate offers a potentially useful means to optimize biological control agent yield and quality.