SUMMARY In this study, growth and efficacy of two biological control agents, Pseudomonas fluorescens P-35 and Bacil- lus subtilis B-3, were evaluated in combinations of two carbon (sucrose and molasses) and two nitrogen (urea and yeast extract) sources to optimize control of Botrytis cinerea on apple. Nutrient broth was used as control. Re- sults indicated that the medium containing molasses and yeast extract (MY) in a 1:1 w/w ratio supported rapid growth and high cell yields in both strains. The biocon- trol efficacy of P-35 grown in MY was higher than when grown in the other media, and the percentage of decayed fruits was 38.6% as compared to the control (100%), af- ter ten days incubation. P-35 also showed good biocon- trol efficacy in MY after 20 days. Nutrient broth resulted in poor biocontrol efficacy for B-3 after ten days, as com- pared with the other media. For both strains molasses were a good carbon source in combination with yeast ex- tract, but antagonist growth was decreased when mo- lasses were complemented with urea. The results ob- tained could provide a reliable basis for mass multiplica- tion of biocontrol agents in fermentation processes. Key words: Biocontrol, bacterial biocontrol agents, Pseudomonas, Bacillus, postharvest decay, Botrytis cinerea. INTRODUCTION The application of fungicides against postharvest dis- ease of fruits to reduce decay has been increasingly com- promised by the development of pathogen resistance to many fungicides, the poverty of replacement fungicides, negative public attitudes regarding the safety of pesti- cides, and consequent restrictions on fungicide use (Janisiewicz and Korsten, 2002). Over the past 15 years, biological control has often been found to be effective Corresponding author: S Peighami-Ashnaei Fax: +98.261.2238529 E-mail: saharpeighamy@yahoo.com to combat major postharvest decays of fruits (Korsten et al., 1994; Fravel et al., 1998). Fungal pathogens are the main cause of postharvest losses in apple, grey mould, caused by Botrytis cinerea Pers. ex. Fr., being one of the most destructive (Romano et al., 1983). Most biocontrol agents (BAs) have varied perform- ance in different environmental conditions. Some of this variability has been attributed to differences in physical and chemical properties found in natural environments where biocontrol agents are applied (Thomashow and Weller, 1996; Duffy et al., 1997). The growth medium used to produce these agents, has a profound effect on them and their products. Mass production of BAs has become a focus of research and industrial development in the search for alternatives to chemical postharvest treatments (Lumsden and Lewis, 1989; Connick et al., 1990). The accurate incorporation of nutrients has im- proved the biomass production of BAs, but unexpect- edly did not enhance (Slininger et al., 1996) or even de- creased the biocontrol efficacy (Moënne-Loccoz et al., 1999). On a large scale, the medium should allow a maximum concentration of biomass and active products to be produced at a low price (Lewis, 1991). The aim of this paper was to find the carbon and ni- trogen sources that provide maximum biomass produc- tion of Pseudomonas fluorescens and Bacillus subtilis and minimizing the cost of media. MATERIALS AND METHODS Antagonists and pathogen preparation. From seven strains of P. fluorescens and seven of B. subtilis the two most inhibitory strains (P-35 and B-3) were selected for further investigation. P. fluorescens P-35 and B. subtilis B-3 were isolated from the roots of wheat and cucum- ber plants, respectively (Table 1) (Sarathchandra et al., 1993). The isolated bacteria were identified with bio- chemical and physiological tests (Schaad, 2001) and were transferred weekly on starch agar plates containing 5 g peptone, 5 g yeast extract and 3 g soluble starch (per liter of double distilled water). After growing for 24 h at 27±1°C, the plates were stored at 4˚C in the dark or frozen at -80°C at the Biological Control Laboratory of Journal of Plant Pathology (2009), 91 (1), 65-70 Edizioni ETS Pisa, 2009 65 INTERACTION OF DIFFERENT MEDIA ON PRODUCTION AND BIOCONTROL EFFICACY OF PSEUDOMONAS FLUORESCENS P-35 AND BACILLUS SUBTILIS B-3 AGAINST GREY MOULD OF APPLE S. Peighami-Ashnaei 1 , A. Sharifi-Tehrani 2 , M. Ahmadzadeh 3 and K. Behboudi 4 Department of Plant Protection, College of Horticultural Sciences and Plant Protection, University of Tehran, Karaj, Iran