Appl Microbiol Biotechnol DOI 10.1007/s00253-014-5663-1
BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING
Mycosubtilin and surfactin are efficient, low ecotoxicity molecules for the biocontrol of lettuce downy mildew Jovana Deravel & Sébastien Lemière & François Coutte & François Krier & Nathalie Van Hese & Max Béchet & Nathanaëlle Sourdeau & Monica Höfte & Alain Leprêtre & Philippe Jacques
Received: 30 September 2013 / Revised: 26 February 2014 / Accepted: 4 March 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract The use of surfactin and mycosubtilin as an ecofriendly alternative to control lettuce downy mildew caused by the obligate pathogen Bremia lactucae was investigated. Preliminary ecotoxicity evaluations obtained from three different tests revealed the rather low toxicity of these lipopeptides separately or in combination. The EC50 (concentration estimated to cause a 50 % response by the exposed test organisms) was about 100 mg L−1 in Microtox assays and 6 mg L−1 in Daphnia magna immobilization tests for mycosubtilin and 125 mg L−1 and 25 mg L−1 for surfactin, respectively. The toxicity of the mixture mycosubtilin/surfactin (1:1, w/w) was close to that obtained with mycosubtilin alone. In addition, the very low phytotoxic effect of these lipopeptides has been observed on germination and root growth of garden cress Lepidium sativum L. While a surfactin treatment did not influence the development of B. lactucae on lettuce plantlets, treatment with 100 mg L−1 of mycosubtilin produced about seven times more healthy plantlets than the control samples, indicating that mycosubtilin strongly reduced the development of B. lactucae. The mixture mycosubtilin/ surfactin (50:50 mg L−1) gave the same result on B. lactucae J. Deravel : F. Coutte : F. Krier : M. Béchet : N. Sourdeau : P. Jacques (*) Laboratoire des Procédés Biologiques, Génie Enzymatique et Microbien, ProBioGEM, UPRES-EA 1026, Polytech’Lille/IUT A, Université Lille Nord de France, Lille1, Av. Paul Langevin, 59655 Villeneuve d’Ascq Cedex, France e-mail: [email protected] S. Lemière : A. Leprêtre LGCgE, Université Lille Nord de France, UPRES EA 4515, Lille1, ENE, Cité Scientifique, SN3, 59655 Villeneuve d’Ascq Cedex, France N. Van Hese : N. Sourdeau : M. Höfte Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Coupure links 653, 9000 Ghent, Belgium
development as 100 mg L−1 of mycosubtilin. The results of ecotoxicity as well as those obtained in biocontrol experiments indicated that the presence of surfactin enhances the biological activities of mycosubtilin. Mycosubtilin and surfactin were thus found to be efficient compounds against lettuce downy mildew, with low toxicity compared to the toxicity values of chemical pesticides. This is the first time that Bacillus lipopeptides have been tested in vivo against an obligate pathogen and that ecotoxic values have been given for surfactin and mycosubtilin. Keywords Bacillus subtilis . Mycosubtilin . Surfactin . Lipopeptides . Ecotoxicity . Bremia lactucae
Introduction Agrochemicals are commonly used to enhance crop yields and combat pests. However, some of them display toxicity to nontarget organisms such as aquatic animals or auxiliary insects (Battaglin and Fairchild 2002; Johnson et al. 2013). Their safety is usually estimated by using a battery of biotests incorporating different trophic levels (Kungolos et al. 1999; Pandard et al. 2006). The garden cress Lepidium sativum L., the fluorescent bacterium Vibrio fischeri, and the microcrustacean Daphnia magna are used to evaluate this chemical toxicity in highly sensitive and standardized tests (Günther and Pestemer 1990; Kungolos et al. 1999; Pandard et al. 2006). The more toxic agrochemicals are classified among environmental contaminants (Battaglin and Fairchild 2002), and governmental policies encourage their reduction, replacement, and removal. One of the tools developed to reduce the quantities or even to replace completely the more toxic agrochemicals is the use of biopesticides, which are living organisms or products derived from such organisms, having the ability to suppress or to limit pests (Thakore 2006).
Appl Microbiol Biotechnol
Owing to their biological origin, these biopesticides are generally considered to be safer than their chemical counterparts. Indeed, molecules originating from living organisms are usually less persistent in soil than agrochemicals. Over the last decade, numerous Bacillus spp. strains have been shown to combat the development of fungal phytopathogens and thereby strongly reduce plant diseases, i.e., behaving as biopesticides (Bais et al. 2004; Touré et al. 2004 ; Ongena et al. 2005; Romero et al. 2007; Arguelles-Arias et al. 2009; Chen et al. 2009; Snook et al. 2009; Alvarez et al. 2012). These “plant-growth-promoting rhizobacteria” (PGPR) are able indirectly to promote plant growth by reducing the impact of diseases using three main mechanisms: the competition for nutrients and space, the antagonism toward infectious microbes, and the elicitation of plant defense reactions by the phenomenon called “induced systemic resistance” (ISR) (Van Loon and Bakker 2005; Lugtenberg and Kamilova 2009). The lipopeptides produced by these Bacillus strains are considered as the main compounds involved in the biocontrol effect of these strains (Ongena and Jacques 2008). Four different families of lipopeptides are synthesized by Bacillus spp., i.e., the fengycins/plipastatins, the iturins (bacillomycins, iturins, mycosubtilin), the kurstakins, and the surfactins, some of them displaying antifungal activities (Leclère et al. 2005; Fickers et al. 2009; Coutte et al. 2010a; Jacques 2011; Béchet et al. 2012, 2013). These compounds are synthesized by the nonribosomal peptide synthetases (NRPS) and are composed of cyclic peptides containing either seven or ten L- and Damino acids, linked to β-amino or β-hydroxy-fatty acid moieties of various lengths and isomeries (Jacques 2011). Among all these lipopeptides, mycosubtilin is considered as the most powerful antifungal compound (Leclère et al. 2005; Fickers et al. 2009; Fickers 2012; Béchet et al. 2013). Surfactin is known for its remarkable surfactant properties and for its ability to elicit the defense responses of some plants such as bean, tomato, or melon (Ongena et al. 2007; García-Gutiérrez et al. 2013). The main objective of this work was to assess the potential use of mycosubtilin and surfactin as an eco-friendly alternative to reduce lettuce downy mildew. Among all the diseases threatening lettuce production, downy mildew caused by the oomycete Bremia lactucae is the most serious (Lebeda et al. 2008; Cohen et al. 2010). It reduces yields and the quality of the crop thereby rendering the lettuce heads unmarketable. B. lactucae can be controlled by chemicals (Brown et al. 2004). However, isolates insensitive to pesticides are frequent as this oomycete is a rapidly adapting pathogen (Brown et al. 2004; Lebeda et al. 2008). In order to demonstrate the eco-friendly properties of mycosubtilin and surfactin, purified and characterized samples of these lipopeptides were first evaluated for their toxicity alone or in combination toward the fluorescent bacterium V. fischeri, the microcrustacian D. magna, and the garden cress
L. sativum L. The same samples were then characterized for their ability to protect lettuce against B. lactucae under controlled conditions.
Materials and methods Production and purification of mycosubtilin and surfactin The strains Bacillus subtilis BBG131 and BBG125 used in this study are constitutive monoproducers of surfactin and mycosubtilin, respectively. These strains were obtained by several genetic optimizations from the wild-type strains 168 and American Type Culture Collection (ATCC) 6633, respectively (Coutte et al. 2010a; Béchet et al. 2013). Surfactin production and purification were carried out through an integrated process in a bubbleless membrane bioreactor (BMBR), as published recently (Coutte et al. 2010b, 2013). Mycosubtilin was produced by the strain B. subtilis BBG125 using an overflowing fed-batch process (OFBC) as described previously (Chenikher et al. 2010). Purification of these compounds required several supplementary steps of ultrafiltration/diafiltration/evaporation and freeze-dried methods according to Coutte et al. (2010b). After freezedrying, powders of surfactin and mycosubtilin were obtained. Purification yield was 82.3±6.2 % for surfactin and 84.4± 16.1 % for mycosubtilin. The degree of purity and the characterization of the homologous compound pattern of the purified surfactin and mycosubtilin samples were determined by high-pressure liquid chromatography (HPLC) and mass spectrometry using the procedure described by Hamley et al. (2013). Purified surfactin and mycosubtilin, supplied by Lipofabrik (Villeneuve d’Ascq, France), were used as standards. The purity reached 94.5±6.9 % for surfactin and 78.3± 12.6 % for mycosubtilin. The composition and ratio of the different homologous compounds of surfactin A and mycosubtilin agreed with those recently presented (Béchet et al. 2013; Hamley et al. 2013). The mixture of surfactin and mycosubtilin (50:50 mg L−1) was made up taking into account the purity of each compound. The powder of these two lipopeptides was solubilized at 20 °C in a 0.1 % dimethylsulfoxide (DMSO) stirred solution at concentrations of 0.1, 0.5, and 1 g L−1. Solubility analyses were performed by HPLC using a sample dissolved in MeOH 100 % as the reference. Results from dissolution assays in DMSO 0.1 % showed that dissolution of the surfactin and mycosubtilin was not affected by a concentration below 1 g L−1. At a concentration of 1 g L−1, only 93 % of surfactin and 83 % of mycosubtilin are dissolved in DMSO 0.1 %. The same samples of mycosubtilin and surfactin were used for the different ecotoxicity and biocontrol experiments.
Appl Microbiol Biotechnol
Preliminary ecotoxicity evaluation Microtox test The Microtox test is generally used to assess direct acute toxicity of various molecules and extracts, water, soil, or sediment samples. The principle is to measure the reduction in light emission of the marine bioluminescent bacterium, V. fischeri. The acute toxicity tests were performed according to the Microtox procedure manual using the Microtox Analyser 500. Microtox is the registered trademark of Azur Environmental (formerly Microbics Corp., Carlsbad, CA, USA). Briefly, freeze-dried luminescent V. fischeri were reconstituted and exposed to one saline water control, one vehicle solvent control, and a series of DMSO dilutions of the tested molecule osmotically adjusted with salt content of 2 %. In all tests, the DMSO never significantly affected light emission of the strain. The resulting decrease in bioluminescence was measured after 5, 15, and 30 min at a constant temperature of 15 °C. Toxicity was measured as percentage of inhibition of light emission from an exposed aliquot, corrected with loss of light in the control. All Microtox data were recorded and analyzed by dedicated analysis software. To ensure reliability of the Microtox method and reagents, a toxicity test using aqueous solutions of phenol or zinc sulfate was done every day prior to beginning the sample toxicity tests, and this positive check was then compared with the Microtox quality assurance guidelines as recommended in the manufacturer’s procedure manual or presented in Johnson (2005). Results are expressed as the effective concentrations (EC) estimated to cause respectively a 20 % (EC20) and a 50 % (EC50) response by the exposed test organisms. The most used was the median effective concentrations (EC50). D. magna (freshwater crustacean) immobilization test Bioassays using the freshwater microcrustacean D. magna are presently applied worldwide to screen samples to give a preliminary indication of toxicity. The D. magna immobilization assays were performed following the methodology prescribed in the Standard Operational Procedure for the Daphtoxkit F magna, which adhered to the Organisation for Economic Co-operation and Development (OECD) Guideline no. 202 (Cartwright 2007). Daphtoxkit is a registered trademark of MicroBioTests Inc. (Mariakerke-Gent, Belgium) and was purchased from R-Biopharm (Saint-Didier au Mont D’Or, France). Five daphnid neonates, hatched from ephippia, were used per well of the test plate, for each molecule concentration and control testing. Prior to the testing, the hatched daphnids were fed during 2 h with unicellular alga Spirulina sp. also provided in the kit. Test plates were incubated at 20 °C, in darkness, for 24 and 48 h. Then immobility of D. magna was recorded. Quality criteria of the tests were
fulfilled as in the control, immobilization of daphnids did not exceed 10 %, and tests with the reference chemical potassium dichromate (positive control) were carried out to evaluate the reliability of the test procedures. Plant germination inhibition test The influence of the lipopeptides on germination and seedling development of higher plants was tested using an in vitro test with garden cress seeds adapted from the work of Günther and Pestemer (1990) based on the pioneer study of Moewus (1949). Two sheets of filter paper, covering the bottom of a Petri dish (9 cm of diameter), were moistened with 5 mL of distilled water, containing the appropriate amount of the tested compound. Twenty-five untreated cress seeds were placed on the paper, and closed dishes were stored in the dark in a growth chamber at 24 °C and 90 % relative humidity. Distilled water was used as a control as well as distilled water with 0.1 % DMSO (vehicle solvent). Each experiment had two pseudoreplications (i.e., two Petri dishes with the same dilution). After 24, 48, and 72 h, as well as any phytotoxic reactions, the percentage of germination was recorded. A visible root was used as the operational definition of seed germination. At the end of exposure, the radicle length of 20 randomly selected seeds per Petri dish was measured. The percentages of relative seed germination (RSG), after 24, 48, and 72 h, and the relative root growth (RRG) and germination index (GI) after 72 h of exposure to mycosubtilin, surfactin, and the mixture (mycosubtilin/surfactin) solutions were then calculated as follows: number of seeds germinated in the tested concentration number of seeds germinated in the control mean root length in the tested concentration RRG ¼ mean root length in the control RSG ð%Þ RRG ð%Þ and % Adverse effects ¼ 100 %–GI GI ð%Þ ¼ 100
RSG ¼
Quality criteria of the tests were fulfilled as in the control, RSG exceeded 85 % after 72 h, and tests with the reference chemical potassium dichromate (positive control) were carried out to check the reliability of our test procedures. Effects of lipopeptides on the phytopathogen B. lactucae Pathosystem and lettuce growth The International Bremia Evaluation Board (IBEB) (http://www.worldseed.org/isf/ibeb.html) is a joint initiative of lettuce breeding companies in France and in the Netherlands, the Dutch Inspection Service (Naktuinbouw), and the French National Seed Station (GEVES). The IBEB carried out a campaign of the evaluation of all the B. lactucae races listed in 2006 and 2007. During this campaign, a new
Appl Microbiol Biotechnol
race of B. lactucae with an inter-area significance has been identified and named B. lactucae Bl: 26. This race, used in this study, was found 31 times across European countries such as Belgium, France, England, Ireland, and the Netherlands. The department in Fijnaart of Rijk Zwaan, one of the breeding companies of the Netherlands, part of the IBEB, provided us the isolate Bl: 26. The sensitive lettuce (Lactuca sativa L.) cultivar Cobham green was used to study the effects of mycosubtilin, surfactin, and the mixture (mycosubtilin/ surfactin) on the oomycete B. lactucae Bl: 26. Ethanol 70 % was sprayed into plastic boxes (25×15× 5 cm). Once the plastic boxes dried, a layer of sterilized cotton was placed in the boxes and then soaked with 200 mL of sterilized bidistilled water. A sterilized filter paper was put on the cotton layer. About 50 lettuce seeds were sown evenly on the filter paper. Boxes were covered with a plastic sheet, and seeds were allowed to germinate for 8 days in a growth chamber (80 % relative humidity, 18 °C, 16 h light/8 h darkness). Cultivated under these conditions, the lettuce plantlets were used to prepare the starting inocula of B. lactucae (8 days of growth) as well as to study the lipopeptide effects (10 days of growth).
mL−1 and 10 mL were sprayed onto lettuce plantlets of each box. Subsequently, plantlet boxes were put in a dark plastic bag and placed in the growth chamber for 24 h. Then, the plastic bag was removed and the boxes of treated lettuces were left in the growth chamber for 8 days. Disease assessment In all samples, each lettuce plantlet was observed for disease symptoms to evaluate the number of healthy plantlets. The data are expressed in mean percentage of healthy plantlets. Treatments were compared by the nonparametric KruskalWallis test (P
BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING
Mycosubtilin and surfactin are efficient, low ecotoxicity molecules for the biocontrol of lettuce downy mildew Jovana Deravel & Sébastien Lemière & François Coutte & François Krier & Nathalie Van Hese & Max Béchet & Nathanaëlle Sourdeau & Monica Höfte & Alain Leprêtre & Philippe Jacques
Received: 30 September 2013 / Revised: 26 February 2014 / Accepted: 4 March 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract The use of surfactin and mycosubtilin as an ecofriendly alternative to control lettuce downy mildew caused by the obligate pathogen Bremia lactucae was investigated. Preliminary ecotoxicity evaluations obtained from three different tests revealed the rather low toxicity of these lipopeptides separately or in combination. The EC50 (concentration estimated to cause a 50 % response by the exposed test organisms) was about 100 mg L−1 in Microtox assays and 6 mg L−1 in Daphnia magna immobilization tests for mycosubtilin and 125 mg L−1 and 25 mg L−1 for surfactin, respectively. The toxicity of the mixture mycosubtilin/surfactin (1:1, w/w) was close to that obtained with mycosubtilin alone. In addition, the very low phytotoxic effect of these lipopeptides has been observed on germination and root growth of garden cress Lepidium sativum L. While a surfactin treatment did not influence the development of B. lactucae on lettuce plantlets, treatment with 100 mg L−1 of mycosubtilin produced about seven times more healthy plantlets than the control samples, indicating that mycosubtilin strongly reduced the development of B. lactucae. The mixture mycosubtilin/ surfactin (50:50 mg L−1) gave the same result on B. lactucae J. Deravel : F. Coutte : F. Krier : M. Béchet : N. Sourdeau : P. Jacques (*) Laboratoire des Procédés Biologiques, Génie Enzymatique et Microbien, ProBioGEM, UPRES-EA 1026, Polytech’Lille/IUT A, Université Lille Nord de France, Lille1, Av. Paul Langevin, 59655 Villeneuve d’Ascq Cedex, France e-mail: [email protected] S. Lemière : A. Leprêtre LGCgE, Université Lille Nord de France, UPRES EA 4515, Lille1, ENE, Cité Scientifique, SN3, 59655 Villeneuve d’Ascq Cedex, France N. Van Hese : N. Sourdeau : M. Höfte Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Coupure links 653, 9000 Ghent, Belgium
development as 100 mg L−1 of mycosubtilin. The results of ecotoxicity as well as those obtained in biocontrol experiments indicated that the presence of surfactin enhances the biological activities of mycosubtilin. Mycosubtilin and surfactin were thus found to be efficient compounds against lettuce downy mildew, with low toxicity compared to the toxicity values of chemical pesticides. This is the first time that Bacillus lipopeptides have been tested in vivo against an obligate pathogen and that ecotoxic values have been given for surfactin and mycosubtilin. Keywords Bacillus subtilis . Mycosubtilin . Surfactin . Lipopeptides . Ecotoxicity . Bremia lactucae
Introduction Agrochemicals are commonly used to enhance crop yields and combat pests. However, some of them display toxicity to nontarget organisms such as aquatic animals or auxiliary insects (Battaglin and Fairchild 2002; Johnson et al. 2013). Their safety is usually estimated by using a battery of biotests incorporating different trophic levels (Kungolos et al. 1999; Pandard et al. 2006). The garden cress Lepidium sativum L., the fluorescent bacterium Vibrio fischeri, and the microcrustacean Daphnia magna are used to evaluate this chemical toxicity in highly sensitive and standardized tests (Günther and Pestemer 1990; Kungolos et al. 1999; Pandard et al. 2006). The more toxic agrochemicals are classified among environmental contaminants (Battaglin and Fairchild 2002), and governmental policies encourage their reduction, replacement, and removal. One of the tools developed to reduce the quantities or even to replace completely the more toxic agrochemicals is the use of biopesticides, which are living organisms or products derived from such organisms, having the ability to suppress or to limit pests (Thakore 2006).
Appl Microbiol Biotechnol
Owing to their biological origin, these biopesticides are generally considered to be safer than their chemical counterparts. Indeed, molecules originating from living organisms are usually less persistent in soil than agrochemicals. Over the last decade, numerous Bacillus spp. strains have been shown to combat the development of fungal phytopathogens and thereby strongly reduce plant diseases, i.e., behaving as biopesticides (Bais et al. 2004; Touré et al. 2004 ; Ongena et al. 2005; Romero et al. 2007; Arguelles-Arias et al. 2009; Chen et al. 2009; Snook et al. 2009; Alvarez et al. 2012). These “plant-growth-promoting rhizobacteria” (PGPR) are able indirectly to promote plant growth by reducing the impact of diseases using three main mechanisms: the competition for nutrients and space, the antagonism toward infectious microbes, and the elicitation of plant defense reactions by the phenomenon called “induced systemic resistance” (ISR) (Van Loon and Bakker 2005; Lugtenberg and Kamilova 2009). The lipopeptides produced by these Bacillus strains are considered as the main compounds involved in the biocontrol effect of these strains (Ongena and Jacques 2008). Four different families of lipopeptides are synthesized by Bacillus spp., i.e., the fengycins/plipastatins, the iturins (bacillomycins, iturins, mycosubtilin), the kurstakins, and the surfactins, some of them displaying antifungal activities (Leclère et al. 2005; Fickers et al. 2009; Coutte et al. 2010a; Jacques 2011; Béchet et al. 2012, 2013). These compounds are synthesized by the nonribosomal peptide synthetases (NRPS) and are composed of cyclic peptides containing either seven or ten L- and Damino acids, linked to β-amino or β-hydroxy-fatty acid moieties of various lengths and isomeries (Jacques 2011). Among all these lipopeptides, mycosubtilin is considered as the most powerful antifungal compound (Leclère et al. 2005; Fickers et al. 2009; Fickers 2012; Béchet et al. 2013). Surfactin is known for its remarkable surfactant properties and for its ability to elicit the defense responses of some plants such as bean, tomato, or melon (Ongena et al. 2007; García-Gutiérrez et al. 2013). The main objective of this work was to assess the potential use of mycosubtilin and surfactin as an eco-friendly alternative to reduce lettuce downy mildew. Among all the diseases threatening lettuce production, downy mildew caused by the oomycete Bremia lactucae is the most serious (Lebeda et al. 2008; Cohen et al. 2010). It reduces yields and the quality of the crop thereby rendering the lettuce heads unmarketable. B. lactucae can be controlled by chemicals (Brown et al. 2004). However, isolates insensitive to pesticides are frequent as this oomycete is a rapidly adapting pathogen (Brown et al. 2004; Lebeda et al. 2008). In order to demonstrate the eco-friendly properties of mycosubtilin and surfactin, purified and characterized samples of these lipopeptides were first evaluated for their toxicity alone or in combination toward the fluorescent bacterium V. fischeri, the microcrustacian D. magna, and the garden cress
L. sativum L. The same samples were then characterized for their ability to protect lettuce against B. lactucae under controlled conditions.
Materials and methods Production and purification of mycosubtilin and surfactin The strains Bacillus subtilis BBG131 and BBG125 used in this study are constitutive monoproducers of surfactin and mycosubtilin, respectively. These strains were obtained by several genetic optimizations from the wild-type strains 168 and American Type Culture Collection (ATCC) 6633, respectively (Coutte et al. 2010a; Béchet et al. 2013). Surfactin production and purification were carried out through an integrated process in a bubbleless membrane bioreactor (BMBR), as published recently (Coutte et al. 2010b, 2013). Mycosubtilin was produced by the strain B. subtilis BBG125 using an overflowing fed-batch process (OFBC) as described previously (Chenikher et al. 2010). Purification of these compounds required several supplementary steps of ultrafiltration/diafiltration/evaporation and freeze-dried methods according to Coutte et al. (2010b). After freezedrying, powders of surfactin and mycosubtilin were obtained. Purification yield was 82.3±6.2 % for surfactin and 84.4± 16.1 % for mycosubtilin. The degree of purity and the characterization of the homologous compound pattern of the purified surfactin and mycosubtilin samples were determined by high-pressure liquid chromatography (HPLC) and mass spectrometry using the procedure described by Hamley et al. (2013). Purified surfactin and mycosubtilin, supplied by Lipofabrik (Villeneuve d’Ascq, France), were used as standards. The purity reached 94.5±6.9 % for surfactin and 78.3± 12.6 % for mycosubtilin. The composition and ratio of the different homologous compounds of surfactin A and mycosubtilin agreed with those recently presented (Béchet et al. 2013; Hamley et al. 2013). The mixture of surfactin and mycosubtilin (50:50 mg L−1) was made up taking into account the purity of each compound. The powder of these two lipopeptides was solubilized at 20 °C in a 0.1 % dimethylsulfoxide (DMSO) stirred solution at concentrations of 0.1, 0.5, and 1 g L−1. Solubility analyses were performed by HPLC using a sample dissolved in MeOH 100 % as the reference. Results from dissolution assays in DMSO 0.1 % showed that dissolution of the surfactin and mycosubtilin was not affected by a concentration below 1 g L−1. At a concentration of 1 g L−1, only 93 % of surfactin and 83 % of mycosubtilin are dissolved in DMSO 0.1 %. The same samples of mycosubtilin and surfactin were used for the different ecotoxicity and biocontrol experiments.
Appl Microbiol Biotechnol
Preliminary ecotoxicity evaluation Microtox test The Microtox test is generally used to assess direct acute toxicity of various molecules and extracts, water, soil, or sediment samples. The principle is to measure the reduction in light emission of the marine bioluminescent bacterium, V. fischeri. The acute toxicity tests were performed according to the Microtox procedure manual using the Microtox Analyser 500. Microtox is the registered trademark of Azur Environmental (formerly Microbics Corp., Carlsbad, CA, USA). Briefly, freeze-dried luminescent V. fischeri were reconstituted and exposed to one saline water control, one vehicle solvent control, and a series of DMSO dilutions of the tested molecule osmotically adjusted with salt content of 2 %. In all tests, the DMSO never significantly affected light emission of the strain. The resulting decrease in bioluminescence was measured after 5, 15, and 30 min at a constant temperature of 15 °C. Toxicity was measured as percentage of inhibition of light emission from an exposed aliquot, corrected with loss of light in the control. All Microtox data were recorded and analyzed by dedicated analysis software. To ensure reliability of the Microtox method and reagents, a toxicity test using aqueous solutions of phenol or zinc sulfate was done every day prior to beginning the sample toxicity tests, and this positive check was then compared with the Microtox quality assurance guidelines as recommended in the manufacturer’s procedure manual or presented in Johnson (2005). Results are expressed as the effective concentrations (EC) estimated to cause respectively a 20 % (EC20) and a 50 % (EC50) response by the exposed test organisms. The most used was the median effective concentrations (EC50). D. magna (freshwater crustacean) immobilization test Bioassays using the freshwater microcrustacean D. magna are presently applied worldwide to screen samples to give a preliminary indication of toxicity. The D. magna immobilization assays were performed following the methodology prescribed in the Standard Operational Procedure for the Daphtoxkit F magna, which adhered to the Organisation for Economic Co-operation and Development (OECD) Guideline no. 202 (Cartwright 2007). Daphtoxkit is a registered trademark of MicroBioTests Inc. (Mariakerke-Gent, Belgium) and was purchased from R-Biopharm (Saint-Didier au Mont D’Or, France). Five daphnid neonates, hatched from ephippia, were used per well of the test plate, for each molecule concentration and control testing. Prior to the testing, the hatched daphnids were fed during 2 h with unicellular alga Spirulina sp. also provided in the kit. Test plates were incubated at 20 °C, in darkness, for 24 and 48 h. Then immobility of D. magna was recorded. Quality criteria of the tests were
fulfilled as in the control, immobilization of daphnids did not exceed 10 %, and tests with the reference chemical potassium dichromate (positive control) were carried out to evaluate the reliability of the test procedures. Plant germination inhibition test The influence of the lipopeptides on germination and seedling development of higher plants was tested using an in vitro test with garden cress seeds adapted from the work of Günther and Pestemer (1990) based on the pioneer study of Moewus (1949). Two sheets of filter paper, covering the bottom of a Petri dish (9 cm of diameter), were moistened with 5 mL of distilled water, containing the appropriate amount of the tested compound. Twenty-five untreated cress seeds were placed on the paper, and closed dishes were stored in the dark in a growth chamber at 24 °C and 90 % relative humidity. Distilled water was used as a control as well as distilled water with 0.1 % DMSO (vehicle solvent). Each experiment had two pseudoreplications (i.e., two Petri dishes with the same dilution). After 24, 48, and 72 h, as well as any phytotoxic reactions, the percentage of germination was recorded. A visible root was used as the operational definition of seed germination. At the end of exposure, the radicle length of 20 randomly selected seeds per Petri dish was measured. The percentages of relative seed germination (RSG), after 24, 48, and 72 h, and the relative root growth (RRG) and germination index (GI) after 72 h of exposure to mycosubtilin, surfactin, and the mixture (mycosubtilin/surfactin) solutions were then calculated as follows: number of seeds germinated in the tested concentration number of seeds germinated in the control mean root length in the tested concentration RRG ¼ mean root length in the control RSG ð%Þ RRG ð%Þ and % Adverse effects ¼ 100 %–GI GI ð%Þ ¼ 100
RSG ¼
Quality criteria of the tests were fulfilled as in the control, RSG exceeded 85 % after 72 h, and tests with the reference chemical potassium dichromate (positive control) were carried out to check the reliability of our test procedures. Effects of lipopeptides on the phytopathogen B. lactucae Pathosystem and lettuce growth The International Bremia Evaluation Board (IBEB) (http://www.worldseed.org/isf/ibeb.html) is a joint initiative of lettuce breeding companies in France and in the Netherlands, the Dutch Inspection Service (Naktuinbouw), and the French National Seed Station (GEVES). The IBEB carried out a campaign of the evaluation of all the B. lactucae races listed in 2006 and 2007. During this campaign, a new
Appl Microbiol Biotechnol
race of B. lactucae with an inter-area significance has been identified and named B. lactucae Bl: 26. This race, used in this study, was found 31 times across European countries such as Belgium, France, England, Ireland, and the Netherlands. The department in Fijnaart of Rijk Zwaan, one of the breeding companies of the Netherlands, part of the IBEB, provided us the isolate Bl: 26. The sensitive lettuce (Lactuca sativa L.) cultivar Cobham green was used to study the effects of mycosubtilin, surfactin, and the mixture (mycosubtilin/ surfactin) on the oomycete B. lactucae Bl: 26. Ethanol 70 % was sprayed into plastic boxes (25×15× 5 cm). Once the plastic boxes dried, a layer of sterilized cotton was placed in the boxes and then soaked with 200 mL of sterilized bidistilled water. A sterilized filter paper was put on the cotton layer. About 50 lettuce seeds were sown evenly on the filter paper. Boxes were covered with a plastic sheet, and seeds were allowed to germinate for 8 days in a growth chamber (80 % relative humidity, 18 °C, 16 h light/8 h darkness). Cultivated under these conditions, the lettuce plantlets were used to prepare the starting inocula of B. lactucae (8 days of growth) as well as to study the lipopeptide effects (10 days of growth).
mL−1 and 10 mL were sprayed onto lettuce plantlets of each box. Subsequently, plantlet boxes were put in a dark plastic bag and placed in the growth chamber for 24 h. Then, the plastic bag was removed and the boxes of treated lettuces were left in the growth chamber for 8 days. Disease assessment In all samples, each lettuce plantlet was observed for disease symptoms to evaluate the number of healthy plantlets. The data are expressed in mean percentage of healthy plantlets. Treatments were compared by the nonparametric KruskalWallis test (P
