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Edizioni ETS Pisa, 2009
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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-Ashnaei1, A. Sharifi-Tehrani2, M. Ahmadzadeh3 and K. Behboudi4 Department of Plant Protection, College of Horticultural Sciences and Plant Protection, University of Tehran, Karaj, Iran
SUMMARY
In this study, growth and efficacy of two biological control agents, Pseudomonas fluorescens P-35 and Bacillus 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. Results 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 biocontrol 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%), after ten days incubation. P-35 also showed good biocontrol efficacy in MY after 20 days. Nutrient broth resulted in poor biocontrol efficacy for B-3 after ten days, as compared with the other media. For both strains molasses were a good carbon source in combination with yeast extract, but antagonist growth was decreased when molasses were complemented with urea. The results obtained could provide a reliable basis for mass multiplication of biocontrol agents in fermentation processes. Key words: Biocontrol, bacterial biocontrol agents, Pseudomonas, Bacillus, postharvest decay, Botrytis cinerea.
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 performance 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 improved the biomass production of BAs, but unexpectedly did not enhance (Slininger et al., 1996) or even decreased 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 nitrogen sources that provide maximum biomass production of Pseudomonas fluorescens and Bacillus subtilis and minimizing the cost of media.
INTRODUCTION MATERIALS AND METHODS
The application of fungicides against postharvest disease of fruits to reduce decay has been increasingly compromised by the development of pathogen resistance to many fungicides, the poverty of replacement fungicides, negative public attitudes regarding the safety of pesticides, 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:
[email protected]
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 cucumber plants, respectively (Table 1) (Sarathchandra et al., 1993). The isolated bacteria were identified with biochemical 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
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the University of Tehran, for reference purposes. B. cinerea was isolated from rotting apples (cv. Golden Delicious) and identified by Dr. K. Behboudi of the Department Mycology and Plant Pathology of the University of Teheran. It was grown at 24˚C on potato dextrose agar and stored at 4˚C. Inoculation and growth conditions. Strains P-35 and B-3 were produced in 250 ml conical flasks, using 50 ml of liquid autoclaved medium (in all liquid cultures, the pH was adjusted to 6.9). The flasks were inoculated with 1-day-old bacterial cultures at an initial concentration of 1×105 cfu ml-1 and incubated with shaking (using a rotary shaker at 140 rpm) at 27˚C. Samples were taken after 20 h incubation and populations of the two strains were estimated in duplicate on starch agar by dilution plating. The plates were incubated at 27±1˚C for 24 h and viable populations (in plates) recorded (cfu ml-1). Each flask assay was conducted in three replicates and two independent assays were carried out for each medium. Culture media. Five different media were used, containing: (i) sucrose (l0 gl-1) + yeast extract (5 g l-1); (ii) molasses (20 g l-1) + yeast extract (10 g l-1); (iii) molasses (20 g l-1) + urea (2 g l-1); (iv) molasses (12 g l-1) + yeast extract (12 g l-1); (v) nutrient broth (NB) (8 g l-1). Molasses were provided by a sugar factory in Ghazvin
(Iran) and contained significant amounts of sucrose, glucose and fructose, salts such as sodium, potassium, calcium, oxalate and chloride with a Purity (Q) of 60% and Brix (°Bx) of 80 %. Efficacy assay. Strains P-35 and B-3 were grown in 50 ml of five different media at 27±1°C, harvested after 20 h and centrifuged twice at 7,000 g for 10 min. Supernatants were discarded and the pellet resuspended in 50 ml sterile distilled water and homogenized. Bacterial concentration in the suspensions was adjusted to 2×108 cfu ml-1 using a spectrophotometer (absorbance at 600 nm). B. cinerea was grown for 10 days on PDA at 25±1˚C. ‘Golden Delicious’ apples were inoculated by dipping a stainless steel rod with a probe tip 1 mm wide and 2 mm long into a 25µl suspension of 106 spores ml-1 and wounding each fruit at two separate spots, according to Smilanick et al. (1999). A 25µl suspension of each strain, containing 2×108 cfu ml-1 grown in each of the above culture media was then applied to each wound. Treated fruits were incubated at 20±1˚C and 85±5% relative humidity. Data were recorded as the percentage of infected wounds after 10 and 20 days of incubation. There were six replicates per treatment and three apples constituted a single replicate. The experiment was carried out twice. Statistical analysis was performed on the pooled data using SAS software (Complete Random Design). Data for final populations (cfu ml-1) and the in-
Table 1. Identity of bacterial strains. Strain
Species
Host(a)
Origin
P-5
Pseudomonas fluorescens
Onion (Allium cepa)
Ghareh Tapeh, Iran
P-6
P. fluorescens
Pea (Pisum sativum)
Karaj, Iran
P-8
P. fluorescens
Pistachio (Pistacia vera)
Kerman, Iran
P-14
P. fluorescens
Wheat (Triticum aestivum)
Karaj, Iran
P-30
P. fluorescens
Wheat (T. aestivum)
Karaj, Iran
P-35
P. fluorescens
P-59
P. fluorescens
Onion (A. cepa)
Ghareh Tapeh, Iran
B-1
Bacillus subtilis
Rape (Brassica napus)
Karaj, Iran
B-3
B. subtilis
Cucumber (Cucumis sativus)
Karaj, Iran
B-12
B. subtilis
Onion (A. cepa)
Khosro Shahr, Iran
B-13
B. subtilis
Pea (P. sativum)
Karaj, Iran
B-15
B. subtilis
Soybean (Glycine max)
Gorgan, Iran
B-16
B. subtilis
Pea (P. sativum)
Karaj, Iran
B-19
B. subtilis
Pistachio (P. vera)
Sirjan, Iran
(a)
Wheat (T. aestivum)
Host from the rhizosphere of which bacterial strains was isolated.
Karaj, Iran
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cidence of grey mould was studied by analysis of variance applied to √x+1/2, followed by Duncan’s multiple range test for separation of means when the variance was statistically significant (P≤0.01).
RESULTS
Control of B. cinerea with 14 bacterial antagonists in dual-culture assay. Results of this experiment (which are the averages of three repetitions) are presented in Table 2. Of fourteen bacterial antagonists tested, P. fluorescens P-35 and B. subtilis B-3 were the most effective in controlling B. cinerea on PDA. Growth assay. The growth of P. fluorescens P-35 in MY medium was significantly higher (4.7×109 cfu ml-1) than in the other media. This medium also supported rapid growth and high cell yields (4.9×109 cfu ml-1) for B. subtilis B-3. Final growth obtained MY medium was also high for both strains (1.9×109 and 2.3×109 cfu ml-1 for P-35 and B-3, respectively). In NB, growth of P-35 (2.1×108 cfu ml-1) was limited as compared to the other media. The growth of B-3 was similar in molasses+urea and NB. These two media provided poor growth for B3 (2.9×108 and 2.1×108 cfu ml-1 in molasses+urea and NB, respectively) (Fig. 1). Biocontrol efficacy assay. After ten days incubation, strains P-35 and B-3 significantly inhibited pathogenicity of B. cinerea on apples. The biocontrol efficacy of P35 grown in MY medium was higher than that of this strain grown in the other media, and the percentage of decayed fruits was 38.6% after ten days. Also, B. subtilis B-3 in this medium reduced the severity of grey mould from 100% (inoculated control) to less than 26.9%. The influence of molasses + urea and NB media on the biocontrol efficacy of P-35 was lower than the other media and the severity of disease was more than 73% in both media. NB showed poor biocontrol efficacy in B-3 as compared to the other media. The overall results obtained indicated that B. subtilis B-3 was superior to P. fluorescens P-35 in inhibiting grey mould (Fig. 2). After twenty days incubation, P-35 reduced the severity of grey mould on apple from 100% (inoculated control) to less than 70%, in sucrose+yeast extract and MY media. Influence of molasses+urea on biocontrol efficacy of P-35 was essentially similar to the control. Strain B-3 showed considerable biocontrol efficacy in molasses+ urea and MY media and reduced the severity of grey mould from 100% (inoculated control) to less than 45 %, after 20 days. Influence of MY and NB media on the biocontrol efficacy of B-3 were poor, after 20 days. As the first ten days, B. subtilis B-3 was more effective in controlling grey mould on apple as compared with P. fluorescens P-35 (Fig. 3).
Fig. 1. Growth (CFU ml-1) of P. fluorescens P-35 and B. subtilis B-3 in five different media [sucrose+yeast extract (Suc. Yeast); molasses+yeast extract (2:1 w/w) (Mol. Yeast 2:1); molasses+urea (Mol. Urea); molasses+yeast extract (1:1 w/w) (Mol. Yeast 1:1); NB]. Biocontrol agent growth was tested in in 50 ml conical flasks at 27˚C, shaken at 140 rpm for 20 h. Columns with different letters are significantly different for each strain using Duncan’s multiple range test (P≤0.01).
Fig. 2. Postharvest decay of apples (cv. Golden Delicious) by grey mould after inoculation of biocontrol agents P. fluorescens P-35 and B. subtilis B-3 (2x108 cfu ml-1) when the control agents were grown in five different media viz: sucrose+yeast extract (Suc. Yeast); molasses+ yeast extract (2:1 w/w) (Mol. Yeast 2:1); molasses+urea (Mol. Urea); molasses+yeast extract (1:1 w/w) (Mol. Yeast 1:1) and NB. Decay was assessed after 10 days at 20˚C. For further details, see text. Columns with different letters are significantly different for each strain, using Duncan’s multiple range test (P≤0.01).
Fig. 3. As in Fig. 2 but assessed after 20 days at 20˚C.
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Table 2. Inhibition halos for P. fluorescens P-35 and B. subtilis B-3 against B. cinerea. Bacterial strains were tested on potato dextrose agar (PDA). Bacterial strain Inhibition halo (mm)(a) Duncan’s(b) P-5 10.1 b P-6 9.6 b 0 e P-8 P-14 7.3 c P-30 6.6 c P-35 15 a P-59 5 d B-1 9.3 b B-3 13 a B-12 6.6 c B-13 0 e B-15 10 b B-16 10.3 b B-19 5.1 d (a) Means for bacterial antagonists are of three replicates of two experiments. (b) Means followed by the same letter are significantly equal, according to Duncan’s multiple range test (P≤0.01).
DISCUSSION
Recognition of the environmental factors that regulate the growth and biocontrol efficacy of antagonist bacteria is an essential step towards advancing the level and reliability of their biocontrol potential (Duffy and Defago, 1999). Commercial production of disease-suppressive strains of bacteria such as P. fluorescens and B. subtilis as biocontol agents in postharvest diseases requires low cost and high biomass production while maintaining their biocontrol efficacy (Costa et al., 2001). Our results show that yeast extract as a nitrogen source supports rapid growth and higher cell yields in all of the strains as compared to urea. Yeast extract contains amino acids and peptides, water-soluble vitamins and carbohydrates (Peppler, 1982; Crueger and Crueger, 1993), which makes it an excellent substrate for many microorganisms (Smith et al., 1975). Costa et al. (2001) and Dharani-Aiyer (2004) showed that yeast extract was the best organic nitrogen source for antagonist bacteria. Nohata and Kurane (1997) considered yeast extract to be too expensive for an industrial process, so it should preferably be replaced by a cheaper industrial product having similar growth characteristics, that should be determined in an economic and technological study. Molasses showed good yield efficacy in both strains. The molasses used in our experiment contained a high sucrose concentration (50%) which may account for the high biomass obtained, because the combination of yeast extract and commercial sucrose also gave high final growth of bacteria. However, a combination of molasses and urea decreased bacterial growth. Luna et al. (2002) and Costa et al. (2001) showed that a molassesbased medium may be used for production of bacterial
BAs. Also, the low cost of molasses allows its concentration to be increased in media without reducing its suitability for commercial use. A high concentration of this substrate (40 g l-1), however, did not significantly improve bacterial growth, which could mean that it became toxic (Costa et al., 2001). The results of the growth assay, indicated that molasses is a suitable carbon source in combination with yeast extract, but not with urea. These observations are in accordance with the results of Costa et al. (2001) who studied Pantoea agglomerans as BA of Penicillium in oranges. Also, Luna et al. (2002) showed that maximum growth of B. subtilis R14 was obtained when C/N ratio was 1:1. Our results, proved that the combination of molasses + yeast extract medium in 1:1 ratio was enough to produce a considerable biomass of both strains. Apparently a C:N (1:1) ratio produces optimal growth for bacteria of two different genera (Pseudomonas and Bacillus) and this may hold true for other genera. The pH is another important parameter for bacterial growth. As a general principle (Mossel et al., 1991) bacterial growth decreases at more acidic pH values so that in our experiments the pH of the medium was adjusted to 6.9 to avoid negative effects on growth. The results, obtained in the biocontrol efficacy assay, showed that all of the media used performed well with both strains compared to NB. These observations are in accordance with the results of Costa et al. (2001) and Fuchs et al. (1999). Although by-products such as molasses are inexpensive, they have some disadvantages for they are not as standardized as purified products and also may contain impurities that need to be removed before fermentation (Stanbury et al., 1995). Moreover, their composition may vary according to the season and origin.
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This study has shown that antagonist bacteria such as P. fluorescens and B. subtilis can be produced in different media, using various nitrogen and carbon sources, while maintaining the efficacy as BAs. By-products such as molasses can serve as an economic culture medium, as suggested by the encouraging results obtained with the present results. Future research will concentrate on optimizing growth conditions and possible incorporation of other nutrients into formulations to obtain an even higher biomass.
ACKNOWLEDGEMENTS
The authors are grateful to the Center of Excellence of Biological Control of Plant Protection, University of Tehran, for providing facilities to carry out the trials.
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