INTERNATIONAL JOURNAL OF AGRICULTURE & BIOLOGY ISSN Print: 1560–8530; ISSN Online: 1814–9596 08–075/SAE/2008/10–5–573–576 http://www.fspublishers.org Full Length Article To cite this paper: Ashnaei, S.P., A.S. Tehrani, M. Ahmadzadeh and K. Behboudi, 2008. Production of Pseudomonas fluorescens P-5 and P-6 for bean damping-off disease. Int. J. Agri. Biol., 10: 573–6 Production of Pseudomonas fluorescens P-5 and P-6 for Bean Damping-off Disease SAMIRA PEIGHAMI-ASHNAEI 1 , ABBAS SHARIFI-TEHRANI, MASOOD AHMADZADEH AND KEIVAN BEHBOUDI Department of Plant Protection, Faculty of Horticultural Science and Plant Protection, University of Tehran, Karaj, Iran 1 Corresponding author’s e-mail: saharpeighamy@yahoo.com ABSTRACT Growth and efficacy of two biological control agents, Pseudomonas fluorescens Flügge, P-5 and P-6, were evaluated in combinations of two carbon (sucrose & molasses) and two nitrogen (urea & yeast extract) sources to optimize control of Rhizoctonia solani Kühn, which is a causal agent of bean damping-off. Both strains were grown in five liquid media including: sucrose + yeast extract, molasses + yeast extract (2:1 w/w), molasses + yeast extract (1:1 w/w), molasses + urea and nutrient broth at an initial inoculation of 1×10 5 CFU mL -1 . Cells from overnight cultures were used to inoculate soil at 1×10 9 CFU cm -3 soil. At the same time, fungal inoculum was added to soil at the rate of 2 mg cm -3 soil. The medium containing molasses and yeast extract (1:1 w/w) supported rapid growth and high cell yields in both strains. In greenhouse conditions, the influence of the media on the biocontrol efficacy of P-5 and P-6 was the same and P. fluorescens P-6 in molasses + yeast extract (in two different ratios) reduced the severity of disease from 90.9% to less than 28%. On the other hand, there were significant differences on bean growth promotion in greenhouse conditions, after one month. Strain P-5 in molasses + yeast extract (1:1 w/w) was more effective on bean growth promotion as compared to the other media. Key Words: Production; Pseudomonas fluorescens; Rhizoctonia solani; Growth; Biocontrol efficacy INTRODUCTION Alternative strategies for disease management include the use of bacteria that show benefic effects on plants and these bacteria are known as plant growth-promoting rhizobacteria (PGPR). The positive effects of PGPR are normally divided into two categories: growth promotion and biological control (Kloepper, 1997). Also, certain root- colonizing bacteria can protect plants from soil-borne pathogens when used as inoculants (Keel et al., 1989; Slininger et al., 1996). However, the properties of the formulation used to deliver these bio-control agents can influence the success of the inoculation (Shah-Smith & Burns, 1997). Formulation of bio-control agents have been designed to promote their survival in soil (Trevors et al., 1992) and colonization of the rhizosphere, effective disease suppression (Slininger et al., 1996). Most of the bio-control strains such as PGPR bacteria have varied performance in different environmental conditions. Some of this variability has been attributed to differences in physical and chemical properties of the natural environments, where bio-control agents are applied (Thomashow & Weller, 1996; Duffy et al., 1997). The laboratory medium has a profound effect on bio-control agents and products, including ability to grow and effectiveness in disease control. The accurate incorporation of nutrients can improve the biomass production of bio- control agents, but unexpectedly did not enhance (Slininger et al., 1996) or even decreased the bio-control efficacy (Moënne-Loccoz et al., 1999). On a large scale, the medium should allow a maximum concentration of biomass and a high quality culture to be produced at a low price (Lewis, 1991). The aim of the present study was to find the carbon and nitrogen sources that should provide maximum biomass production of P. fluorescens P-5 and P-6 with minimum cost of media, whilst maintaining or promoting bio-control efficacy. MATERIALS AND METHODS Antagonists and pathogen preparation. From seven strains of P. fluorescens, only two strains P-5 and P-6 were selected for this investigation (Table I). These strains showed the inhibitory zone in a dual culture assay against R. solani on potato dextrose agar (PDA). Both of the strains were grown weekly on starch agar (Costa et al., 2001) plates containing (per liter): 5 g peptone, 5 g yeast extract and 3 g soluble starch. After growing for 24 h at 27˚C, the plates were stored at 4˚C. R. solani was grown at 24˚C on potato dextrose agar. Microorganisms belong to the laboratory of biological control of University of Tehran, Iran. Inoculation and growth condition. Both of the strains were cultured in 250 mL conical flasks, using 50 mL of