Biological Control 37 (2006) 329337 www.elsevier.

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Evaluation of formulations of Bacillus licheniformis for the biological control of tomato gray mold caused by Botrytis cinerea
Jae Pil Lee a, Seon-Woo Lee a, Choul Sung Kim a, Ji Hee Son a, Ju Hee Song a, Kwang Youll Lee a, Hyun Ju Kim a, Soon Je Jung b, Byung Ju Moon a,
b a Division of Applied Biology, College of Natural Resources and Life Science, Dong-A University, Busan 604-714, Republic of Korea Division of Horticultural Plant Biotechnology, College of Natural Resources and Life Science, Dong-A University, Busan 604-714, Republic of Korea

Received 22 August 2005; accepted 10 January 2006 Available online 23 February 2006

Abstract Bacillus licheniformis N1, which has previously exhibited potential as a biological control agent, was investigated to develop a biofungicide to control the gray mold of tomato caused by Botrytis cinerea. Various formulations of B. licheniformis N1 were developed using fermentation cultures of the bacteria in Biji medium, and their ability to control gray mold on tomato plants was evaluated. The results of pot experiments led to the selection of the wettable powder formulation N1E, based on corn starch and olive oil, for evaluation of the disease control activity of this bacterium after both artiWcial infection of the pathogen and natural disease occurrence under production conditions. In plastic-house artiWcial infection experiments, a 100-fold diluted N1E treatment was found to be the optimum biofungicide spray formulation. This treatment resulted in the signiWcant reduction of symptom development when N1E was applied before Bo. cinerea infection, but not after the infection. Both artiWcial infection experiments in a plastic house and natural infection experiments under production conditions revealed that the N1E signiWcantly reduced disease severity on tomato plants and Xowers. The disease control value of N1E on tomato plants was 90.5% under production conditions, as compared to the 77% conferred by a chemical fungicide, the mixture of carbendazim and diethofencarb (1:1). The prevention of Xower infection by N1E resulted in increased numbers of tomato fruits on each plant. N1E treatment also had growth promotion activity, which showed the increased number of tomato fruits compared to fungicide treatment and non-treated control and the increased fruit size compared the non-treated control under production conditions. This study suggests that the corn starch-based formulation of B. licheniformis developed using liquid fermentation will be an eVective tool in the biological control of tomato gray mold. 2006 Elsevier Inc. All rights reserved.
Keywords: Bacillus licheniformis; Botrytis cinerea; Tomato gray mold; Formulation; Biological control

1. Introduction Gray mold disease caused by Botrytis cinerea Pers.: Fr. is one of the most serious plant diseases aVecting a large number of vegetable plants, including tomato. This fungal pathogen infects leaves, stems, Xowers, and fruits of plants, either by direct penetration or through wounds caused by cultivation practices (Hausbeck and Pennypacker, 1991; McKeen, 1974; ONeill et al., 1997; Peng

Corresponding author. Fax: +82 51 200 7505. E-mail address: bjmoon@dau.ac.kr (B.J. Moon).

et al., 1996). Infection is promoted by high humidity, free moisture on the plant surface, and low temperatures (Eden et al., 1996; Morgan, 1984, 1985; ONeill et al., 1997). Several fungicides eVectively control gray mold, but Bo. cinerea frequently acquires resistance against fungicides (Elad et al., 1992; Katan et al., 1989; Locke and Fletcher, 1988; Yourman and JeVers, 1999). Furthermore, for environmental reasons, there is increasing concern about pesticide use. Biological control has been shown to be eVective in many crops (Elad et al., 1996). Biological control using natural antagonistic microorganisms has been extensively studied, and some fungi, and bacteria have been

1049-9644/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2006.01.001

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demonstrated to be eVective against gray mold (Dik and Elad, 1999; Dik et al., 1999; Elad et al., 1994; Saligkarias et al., 2002; Sutton and Peng, 1993). Yeasts applied to tomato leaves have shown remarkable biocontrol activity (Elad et al., 1994). However, the commercial biological control agents that control gray mold on tomato plants are limited to a few fungal and bacterial strains (Utkhede and Mathur, 2002). Several strains of the genus Bacillus have received much attention as biological control agents because of their advantages over Gram-negative bacteria and fungal biological control agents. Bacillus species have been reported to be eVective in the biocontrol of multiple plant diseases owing to their production of several broad-spectrum antibiotics and their longer shelf lives as a result of their ability to form endospores (Emmert and Handelsman, 1999). Several strains of Bacillus species have been successfully developed as commercial biofungicides. Bacillus subtilis GB03, the cotton-adapted variant of B. subtilis A13, is registered as a seed treatment and as a liquid formulation of endospores under the trade name of Kodiak (Gustafson, Dallas, USA) for use in the control of damping-oV of cotton and other soil-borne diseases (Brannen and Kenney, 1997; MahaVee and Backman, 1993). Another strain, B. subtilis QST-713, which produces more than 30 lipopeptide antibiotics, is marketed under the trade name of Serenade, mainly for the control of foliar diseases (AgraQuest Davis, USA). The Serenade biofungicide was developed as a wettable powder (WP) formulation. Increasing numbers of Bacillus strains have been found to be eVective in the control of foliar diseases, including gray molds (Collins and Jacobsen, 2003; Gueldner et al., 1988; Hang et al., 2005; Paul et al., 1998; Tour et al., 2004). Although many microorganisms have exhibited promising biological control activity results on a small scale, it remains a major challenge to successfully formulate biological control organisms. Successful scale-up production of the organisms and eVective formulation is essential for the stable and economical development of biofungicides (Schisler et al., 2004). Sporulating Gram-positive bacteria such as Bacillus species may be amenable to formulation because they produce heat- and desiccation-tolerant spores (Emmert and Handelsman, 1999). We previously, described the isolation and identiWcation of the bacterium Bacillus licheniformis N1, which was shown to be a highly eVective agent against gray mold infection of perilla plants (Moon et al., 2002; Son et al., 2002). Our previous study showed that dried soybean curd residue, a waste product of tofu production, is a good component of liquid medium for the mass production of B. licheniformis N1 (Lee and Moon, 2001). In the present study, we investigated the eVects of corn starch-based formulations of B. licheniformis N1 on the control of gray mold on tomato plants. We report an eVective WP formulation that uses corn starch as the major carrier and shows good biological control activity in a plastic house and under production conditions.

2. Materials and methods 2.1. Microorganisms and cultivation Bacillus licheniformis N1 was previously isolated from the phyllosphere of perilla plants grown in Busan, Korea, and identiWed based on biochemical characteristics and 16S rDNA sequences (GenBank Accession No. AF492486; Son et al., 2002). B. licheniformis N1 was routinely grown on nutrient agar at 30 C and cultured in Biji medium (5% dried soybean curd residue in distilled water) for mass production. Soybean curd residue (Biji in Korean) was obtained from a Korean traditional tofu factory. Dried soybean curd residue was produced by completely drying the wet soybean curd residue in a drying oven, milling it in a Waring blender, and Wnally sieving the powder to remove particulates larger than 1.5 mm. Bo. cinerea LVF12, which causes tomato gray mold, was routinely grown on potato dextrose agar (PDA), and conidial suspensions were prepared as previously described (Son et al., 2001). BrieXy, Bo. cinerea LVF12 was grown on PDA for 15 days at 25 C in a 12-hr light period to trigger conidia formation. The conidia were suspended in a 30% tomato juice solution supplemented with 0.1 M KH2PO4 to a concentration of 1.6 106 conidia/ml. Commercial tomato juice was purchased from the Gaya tomato juice company in Korea. 2.2. Formulations of microbial fungicides To generate various formulations of B. licheniformis N1, 5 ml overnight culture of bacterial cells were Wrst inoculated into 400 ml of Biji broth in a 1 L Xask and shaken at 300 rpm at 35 C overnight. The grown 400 ml culture of bacterial cells was added directly to 4 L of Biji broth in a 7-L jar fermenter with 10 ml of 10-fold diluted antifoam emulsion (DB-110A, Dowcorning) and grown for 3 days at 300 rpm, pH 7.0 (automatic control with 1 M HCl and 1 M NaOH), and 35 C. The grown bacterial cultures (approximately 4 1011 cfu/ml) were thoroughly mixed with the materials described in Table 1 to generate diVerent formulations. For the WP formulation, the mixture was completely dried at 55 C in a drying oven for 48 h and subsequently ground in a Waring blender to form a powder. The grinding process was carried out intermittently to minimize heat generation. The powder was then sieved through a 200-mesh Wlter to produce the WP formulation, which was stored at 4 C until use. To generate the soluble concentrate (SL) formulations of B. licheniformis N1, a bacterial fermentation culture was centrifuged to separate the bacteria (approximately 1.6 1015 cells per 4 L culture) from the culture supernatant. The bacterial pellet was resuspended in an equal volume of saline (0.75% NaCl) and then mixed with the components indicated in Table 1 to generate the N1W2 formulation. The culture supernatant was simply mixed with the indicated components to produce the N1W1 formulation. The SL formulations were stored at 4 C until use. To

J.P. Lee et al. / Biological Control 37 (2006) 329337 Table 1 Compositions of various formulations of Bacillus licheniformis N1 Type Wettable powder Formulation N1A N1B N1C N1D N1E N1K Soluble concentrate N1W1 N1W2 N1W3 EmulsiWable concentrate N1O1 N1O2 Composition per liter of bacterial culture broth Corn starch, 600 g; olive oil, 50 ml Corn starch, 600 g Corn starch, 600 g; olive oil, 75 ml; sucrose, 50 g Corn starch, 400 g; olive oil, 25 ml; sucrose, 50 g Corn starch, 400 g; olive oil, 50 ml; sucrose, 50 g Corn starch, 400 g; modiWed corn starch, 100 g Supernatant: corn starch, 50 g; Tween 20, 8 ml; ethanol, 5 ml Cell pellet: corn starch, 50 g; Tween 20, 8 ml; ethanol, 5 ml Corn starch, 100 g; modiWed corn starch, 100 g SunXower oil, 1 L; sucrose, 75 g Corn oil, 1 L; sucrose, 75 g; Tween 80, 0.75 ml

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severity value was calculated using the following formula: Disease severity (%) D (( (the number of diseased leaves disease severity index))/(4 the number of leaves rated)) 100. The disease control value was calculated using the following formula: Disease control value (%) D ((AB)/A) 100, where A is the disease severity triggered by pathogen inoculation alone and B is the disease severity after various treatments, including chemical fungicides or formulated biofungicides. Under production conditions, the natural disease severity was rated without pathogen inoculation. 2.4. Pot experiments Pot experiments were performed to select the formulation of B. licheniformis N1 that most eYciently controlled tomato gray mold. Tomato plants were grown in 15-cmdiameter pots in a plastic house until the seven-leaf stage. The various biofungicide formulations were diluted 100fold with tap water before being sprayed on the tomato plants. The 100-fold dilutions contained 1 g of WP formulation, 1 ml of SL formulation, or 1 ml of EC formulation product suspended in 100 ml of tap water. The plants were inoculated with the fungal pathogen by spraying the conidial suspension on tomato leaves until runoV 1 day after chemical or microbial fungicide treatment, and the plants were maintained in a controlled growth room for 24 h (20 2 C, 90% RH). The inoculated plants were then transferred into a plastic house (25 5 C). The best formulations were evaluated for disease control activity against gray mold on tomato Xowers. The formulations N1E and N1W3 and a chemical fungicide were applied to tomato Xowers, and the fungal pathogens were inoculated 1 day after the treatment with the biofungicide or chemical fungicide by spraying conidial solutions on tomato Xowers. Each experiment included Wve plants per treatment with Wve replications. The disease severity was rated at 7 and 3 days after pathogen inoculation of tomato leaves and tomato Xowers, respectively. The number of fertilized fruits per plant was also determined in the Xower disease control experiment. 2.5. Evaluation of biofungicides in a plastic house Plastic-house experiments were conducted with the N1E formulation of B. licheniformis N1 to investigate the optimum dilution and frequency of treatment for the eVective control of tomato gray mold. Tomato plants were Wrst grown in a pot in a plastic house for 25 days and then transplanted into natural soil in a plastic house. The tomato plants were grown in the plastic house until the seven-leaf stage, after which they were treated with chemical fungicide or the N1E biofungicide dilutions as described above. N1E dilutions of 25-, 50-, 100-, 200-, 400-, and 800-fold were tested for disease control activity. To compare the disease control activities of N1E and the chemical fungicide, N1E (100-fold dilution) and the chemical

generate the emulsiWable concentrate (EC) formulations of B. licheniformis N1, bacterial cultures from the fermenter were thoroughly mixed with the components listed in Table 1. The EC formulations were stored at 4 C until use. A control formulation lacking bacterial cells was prepared with Biji broth mixed with corn starch for WP formulation. Most of formulations were used for biological control experiments within at least 2 months after formulation. Fungal pathogen inoculation and the control formulation lacking bacterial cells were used as controls for pot experiments and plastic-house experiments. 2.3. Plant material, screening process, and disease control values The tomato plants used in this study, of the cultivar Kwang-Myeong (Dongbu Hannong, Korea), were grown in a plastic house using routine cultivation practices. Pathogen inoculation was performed as previously described (Son et al., 2001). BrieXy, conidial suspensions of Bo. cinerea LVF12 in 30% tomato juice with 1 M KH2PO4 were sprayed on tomato plants maintained in pots or in a plastic house. The disease severity on tomato leaves and Xowers was rated on various days after pathogen inoculation. The fungicide used in this study for the control treatment (Dong-Bang Agro, Korea) contained equal amounts of diethofencarb (25%) and carbendazim (25%) and was used after 1000-fold dilution with tap water. Tomato plants of the same stages were treated with the formulated microbial fungicides after dilutions with various amounts of tap water. The disease severity index of gray mold on the tomato plants was deWned as the percentage of leaf area that was diseased, where 0 D no disease symptoms, 1 D 0.15%, 2 D 5.120%, 3 D 20.140%, and 4 D 40.1100%. The disease

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fungicide (1000-fold dilution of diethofencarb and carbendazim) were sprayed on tomato plants three times at 7-day intervals. The tomato plants were then sprayed with a Bo. cinerea conidial suspension at 1 day after the chemical or microbial fungicide treatment. The disease severity was rated until 3 weeks after pathogen inoculation. In the plastic house, each plot consisted of 10 tomato plants planted in an area of 2 m 50 cm. The three treatments were arranged in a completely randomized design, with Wve replicates per treatment. To determine whether the gray mold control eVect of the N1E treatment was curative or preventive, the N1E formulation was applied to tomato plants before or after fungal pathogen inoculation. Each experiment included Wve plants per treatment with Wve replications. The disease severity was usually rated at 7 days after pathogen inoculation. 2.6. Trials under production conditions A Weld experiment was conducted from August to October 2001 in a farmers plastic house under production conditions at Kang-Seo, Busan, Korea, utilizing naturally occurring gray mold on tomato plants. The tomato cultivar Kwang-Myeong was planted on July 10, 2001 and transplanted into a plastic house on August 5, 2001. The tomato plants were grown in the plastic house using routine cultivation practices. The three treatments were the N1E formulation of B. licheniformis N1, the diethofencarb and carbendazim fungicide, and a control. The N1E biofungicide sprayed on tomato plants was diluted 100-fold, and the chemical fungicide was diluted 1000-fold before treatment. The tomato plants sprayed with N1E or the chemical fungicide were at the growth stage, at which tomato plants begin to produce the Wrst tomato Xower cluster. From among several candidate plastic houses, we chose an experimental plastic house containing tomato plants showing an initial appearance of gray mold symptoms, on which a heavy disease occurrence was expected. The Wrst sprayings of the chemical fungicide and N1E were performed on August 14, 2001. The treatment was repeated three times at 7-day intervals, and the disease severity was rated at 7 days after the Wnal treatment. The disease severity was converted into disease control values as described in Section 2.3. The growth promotion activity of the N1E was also investigated under production conditions by determining the number of fruits per plant, the average fruit weight, and the average fruit size. The tomato fruits were harvested every 7 days after the Wnal spraying of each treatment, until the Wfth fruit cluster stage. In the experimental plastic house, each plot consisted of 10 tomato plants planted in an area of 2 m 50 cm. The three treatments were arranged in a completely randomized design with Wve replicates per treatment. Natural disease occurrence without any treatment was a control in this experiment. 2.7. Statistical analysis Data from the pot experiments, the plastic-house experiments, and the Weld trials under production conditions were

analyzed using analysis of variance (ANOVA) in a complete randomized design. Duncans multiple range test was used to compare the means of the treatments in each experiment. All statistical analyses were conducted using SAS/ STAT software (SAS Institute, 1989). 3. Results 3.1. Selection of the corn starch-based B. licheniformis N1 formulation N1E To select the most eVective microbial carrier, several minerals and organic materials were tested for plant surface attachment and biological control activity, including zeolite, ceramics, vermiculite, clay, corn starch, Xour, rice Xour, soybean powder, and powders of various plants. Our preliminary results revealed that corn starch was the most eYcient carrier material to deliver biocontrol bacteria onto the plant surface (data not shown). Therefore, our formulation of B. licheniformis N1 used corn starch as a major component. Our previous studies showed that Biji medium supported the growth of B. licheniformis N1 to 4 1011 cfu/ ml after 72 h of fermentation (data not shown). The prepared formulations, in WP, SL, and EC forms, were tested for tomato gray mold control activity in pots in a plastic house. At a 100-fold dilution, the N1E formulation showed the highest control activity, at 92.0%, which was higher, though not signiWcantly, than the activity of the chemical fungicide (Fig. 1). The N1W3 SL formulation showed a level of disease control activity similar to that of the chemical fungicide. Repeat experiments showed the same results. The control of gray mold on tomato Xowers by N1E was signiWcantly higher than that conferred by N1W3 or the chemical fungicide, resulting in higher number of fertilized tomato fruits per plant at 7 days after pathogen inoculation in the pot experiments (Table 2). Therefore, N1E was selected to evaluate the eVect of B. licheniformis N1 as a biofungicide in plastic-house experiments by artiWcial infection and under production conditions using natural disease occurrence. The number of bacteria in the 100-fold diluted N1E was 7 109 cfu/ml, which indicated 7% loss of viability from the rehydration process, since the number of bacteria in the original N1E was 7.5 1011 cfu/g of N1E (data not shown). 3.2. EVect of N1E dilution and preventive activity in a plastic house Various dilutions of N1E resulted in diVerent levels of disease control on tomato plants. Treatments with N1E diluted 25- to 200-fold with tap water all showed disease control values ranging from 90.7 to 72.9%. Treatments with 400- and 800-fold dilutions of N1E gave disease control activities of only 49.2 and 13.8% (Fig. 2). Although the disease control activity of the 200-fold dilution was 72.9%, it diVered signiWcantly from that of the 100-fold dilution. Treatment with N1E at a 100-fold dilution resulted in a disease control value

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Fig. 1. EVect of various formulations of Bacillus licheniformis N1 on the control of the gray mold Botrytis cinerea on tomato plants in pots in a plastic house. D + C represents a mix of the chemical fungicides diethofencarb and carbendazim. Pathogen and control indicate artiWcial inoculation of the fungal pathogen and no treatment, respectively. Error bars represent the standard deviation of Wve replications.

Table 2 Control of gray mold on tomato petals and comparison of fertilized tomato fruits at seven days after N1E, N1W3, and chemical fungicide treatments and pathogen inoculation Treatmenta N1E N1W3 D+C Pathogen alone (None) Control Disease control value (%) on petalsb 89 bd 39.4 d 47.3 c 0.5 e 100 a Number of fertilized fruitsc 3.2 a 1.8 b 1.9 b 0.5 c 1.7 b

the N1E treatment had a preventive disease control eVect but not a curative eVect. 3.3. Control of gray mold of tomato by N1E in a plastic house The disease control values on tomato plants treated with N1E or the chemical fungicide (diethofencarb+ carbendazim) were compared over 3 weeks, during which three consecutive treatments were applied. The optimum frequency to treat tomato plants with the biofungicide N1E was determined by testing both 3- and 7-day intervals. Three consecutive treatments at either 3- or 7-day intervals gave a higher disease control activity than two consecutive treatments or a single treatment (data not shown). There was no signiWcant diVerence in disease control activity in plants treated with N1E at 3- and 7-day intervals. Therefore, for subsequent plastic-house experiments, the 100-fold diluted N1E formulation was applied over three consecutive weeks at 7-day intervals starting at 1 day before inoculation with the fungal pathogen. The gray mold control activity triggered by N1E was stably maintained, with a disease control value of over 90% until 3 weeks after the Wnal N1E treatment, whereas the eVect of chemical treatment slowly decreased to 79% (Fig. 4). The negative control formulation prepared without bacterial cells did not suppress disease development or aVect plant growth. 3.4. Control of gray mold of tomato by N1E under production conditions The Weld experiments showed that the natural disease occurrence during the experimentation period was 92% of disease severity, indicating a heavy infection of gray mold disease on the tomato plants in the farmers plastic house, in which no control practices were used. The disease control value of the N1E treatment was 90.5%, much more eVective

a D + C indicates treatment with a mix of the chemical fungicides diethofencarb and carbendazim. Pathogen alone indicates inoculation with Botrytis cinerea LVF12 only. b The disease control value of each treatment was evaluated by rating the disease severity three days after Botrytis cinerea inoculation. c The number of fertilized tomato fruits from each treatment was counted at seven days after pathogen inoculation. d Means in the same column followed by the same letter are not signiWcantly diVerent (P < 0.05) based on Duncans multiple range test.

of over 80%, which was not signiWcantly diVerent from the eVects of treatment with the 25- or 50-fold dilutions. Therefore, the 100-fold dilution of N1E, prepared by suspending 1 g of N1E formulation in 100 ml of tap water, was chosen as the optimum concentration of N1E for a continuous Weld evaluation of the N1E formulation. Biofungicide treatment before, after, or simultaneously with inoculation of Bo. cinerea on tomato plants revealed the preventive eVect of N1E against gray mold on tomato plants. The N1E treatment after pathogen inoculation or simultaneous treatment with the pathogen did not result in acceptable disease control activity (Fig. 3), whereas the N1E treatment at least 1 or 2 days before pathogen inoculation led to 83.8 and 60.5% disease control value, respectively. Therefore, 1 day before fungal infection was determined to be the best spraying time for N1E treatment in plastic-house experiments. This result also indicated that

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Fig. 2. EVect of dilutions of the N1E formulation on the control of the gray mold Botrytis cinerea on tomato plants in pots in a plastic house. Error bars represent the standard deviation of Wve replications.

Fig. 3. Preventive and curative eVects on the control of gray mold on tomato plants in pots in a plastic house evaluated by varying the time of spraying of N1E formulations. () indicates N1E treatment before pathogen inoculation and (+) indicates treatment after pathogen inoculation. Pathogen and control indicate artiWcial inoculation of the fungal pathogen and no treatment, respectively. Error bars represent the standard deviation of Wve replications.

than that of the chemical fungicide, which gave a disease control value of 77% (Fig. 5). This result was similar to that obtained in the plastic house and pot experiments that utilized artiWcial infection of the fungal pathogen. From the time we observed the initial development of gray mold disease symptoms on tomato plants in our experimental plastic house, the natural occurrence of gray mold in the farmers plastic house was suYciently high to estimate the eVect of each treatment on disease control. 3.5. Promotion of tomato yield under production conditions by N1E The tomato fruit production and disease control on Xowers during natural disease occurrence were compared after chemical fungicide treatment, biofungicide treatment,

and no treatment (Table 3). The disease control value on tomato Xowers after N1E treatment was 82%, which was signiWcantly greater than that obtained using the chemical fungicide (48.3%). The number of tomato fruits per plant was signiWcantly higher after the N1E treatment than after the chemical fungicide treatment. The average weight of individual tomato fruits was similar after the biofungicide and chemical fungicide treatments, but both were much higher than the fruit weight in the control. The tomato fruit lengths and widths were similar after all of the treatments, except that the fruit width in the control was slightly lower, though not signiWcantly, than with the other treatments. Similarly, the tomato leaves treated with N1E became thicker and shinier, and the overall growth of these plants was greater than in the tomato plants treated with the chemical fungicide (data not shown).

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Fig. 4. Control of gray mold on tomato plants by the N1E formulation in a plastic house. D + C represents a mix of the chemical fungicides diethofencarb and carbendazim. Pathogen and control indicate artiWcial inoculation of the fungal pathogen and no treatment, respectively. Black, gray, and open bars indicate the disease control values at one, two, and three weeks after pathogen inoculation, respectively. Error bars represent the standard deviation of Wve replications.

Fig. 5. Control of gray mold on tomato plants by the N1E formulation under production conditions. D + C represents a mix of the chemical fungicides diethofencarb and carbendazim. Error bars represent the standard deviation of Wve replications.

4. Discussion Gram-positive bacteria, especially Bacillus species, have received much attention as eVective biological control agents and have some formulation advantages over Gram-negative bacteria (Emmert and Handelsman, 1999; Schisler et al., 2004). Here, we investigated the biological control activity of the corn starch-based WP formulation of B. licheniformis N1 against gray mold on tomato plants. We have previously found that Biji medium, which is composed of dried soybean curd residue, supports bacterial growth up to 4 1011 cfu/ml after 72 h of fermenta-

tion in liquid culture. Soybean curd residue is a waste product from the tofu industry in Korea and Japan. It is known to be a good fermentation medium, rich in protein, fat, carbohydrates, and Wber (Ohno et al., 1996). Since this industrial waste is frequently contaminated with microorganisms, it is not useful for long-term storage as a fermentation medium. However, soybean curd residue is often used as a solid fermentation medium for production of antibiotics by B. subtilis (Ohno et al., 1995, 1996). In this study, we successfully developed a liquid fermentation method using dried soybean curd residue. Since the dried soybean curd residue was free of microbial contamination at 4 C for at least 1 year, it was possible to use the material for liquid fermentation. Because Biji medium contains only 5% dried soybean curd residue, the cost for liquid fermentation of B. licheniformis N1 is much lower than if other media are used. Devising an eVective formulation product would enhance the biological activity of antagonistic bacteria and it is an essential for the stabilization of biofungicides (Schisler et al., 2004). To develop a successful formulation protocol for biological control agents, selection of an appropriate carrier material is important. The carrier should contribute to the attachment and longevity of the biological control activity in the Weld (Fravel et al., 1998). The eVective N1E formulation of B. licheniformis N1 contained corn starch and olive oil. Starch becomes gelatinized, forming a cross-linked structure in which the biocontrol agent can be trapped (Fravel et al., 1998). It may enhance the attachment of the bacteria to the plant leaf surface and protect the biocontrol organisms from UV illumination (Burges and Jones, 1998). Oil may serve as a food base with the sugar in N1E and regulate the water availability in the formulation. It has been suggested that the addition of oil aVects the viability of Bo. cinerea

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Table 3 Tomato growth promotion and yield increase after biofungicide treatment under production conditions at Kang-Seo, Busan Treatmenta Flower disease control value (%)b
d

Tomato fruit production under production conditionsc No. of fruits Weight (g) 132.9 a 121.9 a 68.3 b Length (cm) 5.4 a 5.1 a 4.4 a Width (cm) 6.4 a 6.5 a 5.0 b

N1E D+C Control

a b

82.0 a 48.3 b 1.0 c

16.2 a 9.2 b 2.8 c

N1E, wettable powder type; D + C, diethofencarb + carbendazim; Control, Disease developed from existing levels of Botrytis cinerea inoculum. The disease control value on tomato Xowers under production conditions was investigated at seven days after the Wnal spraying of each treatment. c The number of tomato fruits per plant was investigated until the Wfth fruit cluster stage. The fruit weight, length, and width were measured until the Wfth fruit cluster stage and averaged per fruit. d Means in the same column followed by the same letter are not signiWcantly diVerent (P < 0.05) based on Duncans multiple range test.

conidia (Fravel et al., 1998). However, the absence of a curative eVect by N1E suggests that B. licheniformis N1 cannot attack the fungal pathogen if it is inside the plant. Therefore, the absence of a curative eVect by N1E indicated that the oil in the N1E had no direct eVect on Bo. cinerea inside the plant or on the plant surface. Our formulation of B. licheniformis N1 fermented in Biji medium was cost-eVective, owing to the simple addition of corn starch, olive oil, and sucrose. At a 100-fold dilution, the N1E formulation was highly eVective in the control of gray mold of tomato plants, as compared to the mixed fungicides diethofencarb and carbendazim in artiWcial inoculation experiments of Bo. cinerea as well as in natural disease occurrence experiments. N1E dilutions of 400- or 800-fold did not result in eVective disease control, but the low cost of formulation of N1E may allow the 100-fold dilution to be developed as a biofungicide. The spray treatments of N1E under production conditions were performed at around the beginning of the Bo. cinerea infection of tomato plants. The results showed that N1E treatment has a strong disease preventive eVect. Since our biological control activity experiments in plastic houses were performed by fungal pathogen application with 30% tomato juice, the biological control activity of B. licheniformis N1 may be dependent on the presence of nutrient. However, eVective suppression of tomato gray mold by the N1E application under production conditions suggested that the activity of B. licheniformis N1 was not dependent on the nutrient from tomato juice. Since our N1E formulation contained bacterial cells as well as culture Xuids, the remarkable disease control activity by N1E treatment was probably due to the strong antagonistic activity of B. licheniformis N1 cells and antifungal compounds against the gray mold pathogen. In fact, a SL formulation N1W1 with culture supernatant only was more eVective than N1W2 with bacterial cells only (Fig. 1). This result suggested that antifungal compounds are present in the culture supernatant of B. licheniformis N1. Although we have not tested unformulated bacterial culture supernatant for biological control activity, strong antifungal activity from crude extract of bacterial culture broth indicated the potential of antifungal compounds in biological control activity

(data not shown). Cultivation of tomato plants in a plastic house may also contribute to increased biological control activity by preventing the removal of the biofungicide from the plant surface by rainfall. Our overall experimental results suggest that the spray schedule of the biofungicide N1E should commence before fungal infection of the tomato plants, with at least three sprayings at 1-week intervals. N1E treatment of tomato plants protected tomato Xowers from infection, resulting in an increased number of fertilized tomato fruits per plant. The higher number of fruits per tomato plant may be due to the eVective control of gray mold on Xowers. In addition, the general status of tomato plants treated with the N1E biofungicide was better than that seen with the other treatments. This result suggested that N1E has growth promotion activity on tomato plants in addition to disease control activity. It will be interesting to test for whether the growth promotion eVect is speciWc to tomato plants. Since the N1E formulation was eVective against Bo. cinerea infection of tomato plants, experiments should be performed to determine if the biofungicide could be applied to other crops to control gray mold disease by Bo. cinerea. Acknowledgments This study was supported by the Technology Development Program for Agriculture from the Agricultural R&D Promotion Center of the Ministry of Agriculture and Forestry of the Republic of Korea (2991073). References
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