S.S. Shaukat and I.A. Siddiqui Soil Biology and Ecology Laboratory, Department of Botany, University of Karachi, Karachi, Pakistan

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1 Letters in Applied Microbiology 2003, 36, The influence of mineral and carbon sources on biological control of charcoal rot fungus, Macrophomina phaseolina by fluorescent pseudomonads in tomato S.S. Shaukat and I.A. Siddiqui Soil Biology and Ecology Laboratory, Department of Botany, University of Karachi, Karachi, Pakistan 2003/0050: received 20 January 2003, revised 12 February 2003 and accepted 18 February 2003 ABSTRACT S.S. S H A U K A T A N D I.A. S I D D I Q U I Aims: To determine the influence of various trace minerals and carbon source on the biocontrol performance of Pseudomonas aeruginosa strain IE-6S + and P. fluorescens strain CHA0 against Macrophomina phaseolina. Methods and Results: In dual culture plate assay, P. aeruginosa IE-6S + and P. fluorescens CHA0 inhibited radial growth of M. phaseolina producing zones of inhibition. Czapek s dox agar medium amended with both zinc and glucose remarkably improved antifungal activities of the bacterial inoculants. Under glasshouse conditions, soil amendment with zinc and/or glucose alone did not reduce M. phaseolina infection in tomato roots but did reduce significantly when used in combination with IE-6S + or CHA0. Soil amendments with zinc and/or glucose increased fresh shoot weights but zinc amendment greatly reduced bacterial populations in the rhizosphere. Conclusions: Mineral and carbon amendments enhance the biocontrol performance of fluorescent pseudomonads against M. phaseolina. Significance and Impact of the Study: Identification of mineral and carbon amendments that favour biocontrol of certain bacterial strains may provide clues to soil factors or components of nutrient solutions in hydroponic culture that will improve the level and reliability of control. Keywords: Biocontrol, inconsistency, Macrophomina phaseolina, mineral nutrition, Pseudomonas aeruginosa, Pseudomonas fluorescens INTRODUCTION Certain bacteria have been used for the control of crop diseases, and their use in agriculture is expected to increase (Cook 1993). This is related, in part, to the fact that the pesticide residues remaining in soil and groundwater are a cause of concern to human beings and the livestock. The specific strains of bacteria have demonstrated biological control against most soil-borne pathogenic fungi. Mechanisms of biological control by many strains of antagonistic bacteria involve the production of bacterial metabolites that adversely affect the pathogen. These metabolites include antibiotics, iron chelators, cell wall degrading enzymes and Correspondence to: S. Shahid Shaukat, Soil Biology and Ecology Laboratory, Department of Botany, University of Karachi, Karachi-75270, Pakistan ( imran_75850@yahoo.com). hydrogen cyanide (Benizri et al. 2001). An alternative mechanism is induced systemic resistance when biocontrol bacteria stimulate the host plant defences (Siddiqui and Shaukat 2002a). In commercial settings and field trials, however, inconsistent performance from site to site and from year to year tends to be a disappointing reality (Thomashow and Weller 1996). This variability in performance is the major cause of impediment of large-scale application of bacteria-based biocontrol products. Recently, there has been a growing interest in combining microbial biocontrol agents with other chemical components to enhance their activity against certain soil-borne plant pathogens (Siddiqui et al. 2001). The application of antagonist preparations supplemented with low doses of synthetic fungicides often offers a level of control equivalent to that of full-rate of synthetic fungicides (Chand-Goyal and Spotts ª 2003 The Society for Applied Microbiology

2 BIOCONTROL OF PSEUDOMONADS AGAINST MACROPHOMINA PHASEOLINA ). However, non-chemical fungicides including specific biocontrol agents are preferred because these pose minimal risk to health and can be used in the control of a particular plant pathogen. The aim of the present investigation was to determine the role of mineral and carbon amendments on the biological control performance of Psuedomonas aeruginosa strain IE-6S + and P. fluorescens CHA0 towards Macrophomina phaseolina, a charcoal-rot fungus. Psuedomonas aeruginosa IE-6S + and P. fluorescens CHA0 were selected because these agents have shown substantial control of a variety of soil-borne plant pathogens including M. phaseolina in tomato (Siddiqui and Shaukat 2002b). The attention was focused on mineral and carbon sources for several reasons: (i) they have long been known to influence the activity of phytopathogenic micro-organisms (Engelhard 1989), (ii) they contribute to the variability of biocontrol in different soils and on host crops that differ in root exudate composition (Latour et al. 1996), and (iii) they have been reported to influence production of other antibiotics in biocontrol strains (Gutterson 1990; Milner et al. 1995; Duffy and Défago 1999). MATERIALS AND METHODS Micro-organisms and cultural conditions Pseudomonas aeruginosa strain IE-6S + (a streptomycinresistant derivative IE-6) and P. fluorescens strain Tet- CHA0 (a tetracycline-resistant derivative of CHA0) were grown overnight at 27 C with continuous shaking in King s B (King et al. 1954) broth containing 100 lg ml )1 streptomycin and tetracycline, respectively, for P. aeruginosa and P. fluorescens. The bacterial culture was centrifuged at 2800 g for 20 min. The supernatant was discarded and the pellet resuspended in nutrient broth supplemented with 0Æ5% yeast extract. The cultures were allowed to grow for 48 h before further tests. Macrophomina phaseolina was isolated from infected roots of tomato growing in a cultivated field at Gharo, 45 miles south-east of Karachi. The fungus was cultured on potato dextrose agar (PDA) medium at 28 C. Effect of various minerals and carbon source on antifungal activity by IE-6S + and CHA0 Autoclaved Czapek s dox agar (CDA) medium was amended with filter-sterilized mineral solutions to give 1 mm: CaCl 2 Æ2H 2 O, FeSO 4 Æ7H 2 O, LiCl, MgSO 4 Æ7H 2 O, MnCl 2 Æ 4H 2 O, NaCl; 0Æ7 mm: CuSO 4, ZnSO 4 Æ7H 2 O; 0Æ5 mm: Mo 7 (NH 4 ) 6 O 24 Æ4H 2 O or 0Æ1 mm: CoCl 2 Æ6H 2 O and with autoclaved stock solutions of carbon sources (none, glycerol and glucose) to give 1% (w/v). Treatments were arranged as a10 3 factorial design with six replications. To determine the antifungal activity by pseudomonads, a dual culture plate method was employed. Bacterial strains were streaked on one side of a Petri plate (9 cm diameter) containing CDA. On other side of the Petri plate, a 5 mm diameter agar plug with actively growing M. phaseolina was inoculated. The plates were incubated at 28 C and the inhibition zone (if any) was recorded after 1 week. Effect of various concentrations of zinc sulphate on antifungal activity of IE-6S + and CHA0 In a separate experiment, the effect of a range of zinc sulphate concentrations, from 0 to 1Æ50 mm, on the antifungal activity of IE-6S + and CHA0 was evaluated on CDA. Each treatment and control was replicated six times and Petri plates were incubated at 28 C. Inhibition zone was recorded after an incubation period of 1 week. The influence of zinc and/or carbon sources on antifungal activity by IE-6S + and CHA0 Autoclaved CDA was amended with a mineral mixture of 0Æ75 mm zinc sulphate. Zinc sulphate amendments were tested alone and with carbon sources at 1% (w/v). Treatments were arranged as a 2 8 factorial each with six replicates. The factors included two zinc amendments (plus or minus zinc) and eight carbon source amendments (none, glucose, fructose, galactose, glycerol, lactate, citrate and mannitol). Antifungal activity of the biocontrol inoculants was assessed as mentioned earlier. Effect of zinc and/or glucose on the efficacy of IE-6S + and CHA0 against M. phaseolina in tomato roots Plastic pots (8 cm diameter) were each filled with 350 g of sandy loam soil (ph 7Æ9, moisture holding capacity of 37%) mixed with 0Æ9 mg zinc per kilogram of soil and/or 1% glucose (w/w). The soil was naturally infested with 15±1Æ8 sclerotia per gram of soil of M. phaseolina as assessed by wet sieving and dilution technique described by Sheikh and Ghaffar (1975). After amendments, a 35-ml aqueous cell suspension of IE-6S + (2Æ CFU ml )1 ) or CHA0 (2Æ CFU ml )1 ) was poured into each pot. A set of bacteria-treated plants without zinc or glucose was also kept for comparison. Soil drenched with 35-ml saline served as control. After treatments, 3-week-old tomato seedlings that had been raised in sterilized soil were planted at three seedlings per pot. Treatments and controls were replicated four times in the first experiment while five times in the repeated trial. The pots were randomized on a glasshouse bench. The experiment was terminated 30 days after seedling transplant and growth parameters including plant height and

3 394 S.S. SHAUKAT AND I.A. SIDDIQUI fresh shoot weight recorded. The root systems of two randomly chosen plants from a pot were cut into small (5 mm long) segments after thorough washing with running tap water. After surface sterilization with 1% Ca(OCl) 2 for 3 min, the root segments were rinsed several times with distilled water and plated (five segments) onto PDA plates supplemented with appropriate quantities of penicillin and sterptomycin sulphate to avoid bacterial contamination. The plates were incubated at room temperature (28 C) for 1 week and infection percentage by M. phaseolina calculated as follows: ð%þ Infection ¼ (No. of plants infected by the fungus/ total no. of plants) 100 Rhizosphere population of IE-6S + and CHA0 were isolated by placing a root sample of the remaining plant in the pot with adhering soil in a 250 ml Erlenmeyer flask containing 10 ml of 0Æ1 M MgSO 4 solution (ph 6Æ5) and 0Æ02% Tween-20. Tenfold serial dilutions of the suspension were prepared and 100-ll aliquots from the appropriate dilutions were plated onto King s medium B (KMB) supplemented with streptomycin and tetracycline, respectively, for IE-6S + and CHA0. The plates were incubated at room temperature (28 C) for 48 h and the number of CFU recorded. Statistical analysis Data sets were subjected to one-way ANOVA or factorial analysis of variance (FANOVA) depending on the experimental design using STATISTICA ver. 5Æ0 software (Statsoft, Inc., Tulsa, OK, USA). As a follow-up of ANOVA or FANOVA, Fisher s least significant difference (LSD) test was used for separation of mean values. RESULTS Effect of various minerals and carbon source on antifungal activity by IE-6S + and CHA0 In the first experiment, 10 minerals were added to CDA medium alone or in combination with a carbon source amended with 1% (w/v) glucose or glycerol to determine their influence on in vitro antagonism by P. aeruginosa strain IE-6S + and P. fluorescens strain CHA0 against M. phaseolina after 1 week of growth. Mineral amendment with or without glycerol or glucose had a differential (P < 0Æ001) influence on the inhibition of fungus producing zones of inhibition of varying extent (Table 1). In general, regardless of the mineral and carbon amendments, CHA0 caused greater suppression (P <0Æ01) producing larger inhibition zones compared with IE-6S +. When compared with glycerol, regardless of the bacterial strains and mineral amendments, glucose produced larger (P < 0Æ01) inhibition zones. Among the minerals tested, zinc in combination with glucose enhanced the bacterial performance resulting in a greater inhibition of the fungus. Amendment of FeSO 4 to CDA without any carbon source enhanced (P < 0Æ05) antifungal Table 1 Influence of minerals and carbon source amendments towards Macrophomina phaseolina by Pseudomonas aeruginosa IE-6S + and P. fluorescens CHA0. Values are mean (± one standard error of mean) of six replicates Zone of inhibition (mm) IE-6S + with (carbon source added) CHA0 with (carbon source added) Treatments None Glycerol Glucose None Glycerol Glucose CDA without amendment 3 ± 0Æ7 4 ± 0Æ7 7 ± 0Æ6 12 ± 0Æ9 14 ± 0Æ9 16 ± 1Æ3 CDA amended with CaCl 2 Æ2H 2 O 3 ± 0Æ5 3 ± 0Æ4 5 ± 0Æ4 10 ± 0Æ7 13 ± 0Æ9 13 ± 1 FeSO 4 Æ7H 2 O 5 ± 0Æ6 2 ± 0Æ3 6 ± 0Æ5 10 ± 0Æ8 9 ± 0Æ7 161Æ2 LiCl 3 ± 0Æ4 4 ± 0Æ7 3 ± 0Æ4 10 ± 0Æ7 10 ± 0Æ8 12 ± 0Æ9 MgSO 4 Æ7H 2 O 7 ± 0Æ6 6 ± 0Æ6 10 ± 0Æ8 13 ± 0Æ9 14 ± 0Æ9 17 ± 1 Mo 7 (NH 4 ) 6 O 24 Æ4H 2 O 6 ± 0Æ8 6 ± 0Æ5 8 ± 0Æ6 15 ± 1Æ2 17 ± 1Æ2 17 ± 0Æ9 MnCl 2 Æ4H 2 O 4 ± 0Æ5 6 ± 0Æ6 9 ± 0Æ7 14 ± 0Æ9 12 ± 0Æ8 15 ± 1 NaCl 6 ± 0Æ7 7 ± 0Æ6 10 ± 0Æ7 14 ± 1Æ1 14±1 19±1Æ3 CuSO 4 6±0Æ6 7 ± 0Æ5 9 ± 0Æ6 15±1 13±0Æ9 18 ± 1Æ2 ZnSO 4 Æ7H 2 O 7 ± 0Æ8 10 ± 0Æ8 13 ± 1 17 ± 1Æ3 19 ± 1Æ2 22 ± 1Æ4 CoCl 2 Æ6H 2 O 4 ± 0Æ6 6 ± 0Æ5 8 ± 0Æ7 12 ± 0Æ8 16 ± 1Æ1 16 ± 1 LSD 0Æ05 Treatments (T) 1Æ4 1Æ5 C source (C ) 0Æ9 1Æ1 T C 2 2Æ1 CDA, Czapek s dox agar; LSD, least significant difference.

4 BIOCONTROL OF PSEUDOMONADS AGAINST MACROPHOMINA PHASEOLINA 395 Table 2 Effect of various concentration of zinc sulphate on the efficacy of Pseudomonas aeruginosa and P. fluorescens towards Macrophomina phaseolina in vitro Zone of inhibition (mm) Treatments IE-6S + CHA0 CDA amended with zinc at 0mM 4±0Æ5 18 ± 1Æ2 0Æ25 mm 5 ± 0Æ4 18 ± 0Æ9 0Æ50 mm 7 ± 0Æ7 17 ± 1Æ1 0Æ75 mm 7 ± 0Æ6 21 ± 1Æ3 1mM 8±0Æ6 20 ± 1Æ2 1Æ25 mm 7 ± 0Æ8 21 ± 1 1Æ50 mm 8 ± 0Æ7 21 ± 1Æ2 LSD 0Æ05 1Æ1 1Æ3 CDA, Czapek s dox agar; LSD, least significant difference. activity while glycerol reduced (P < 0Æ05) biocontrol of the strain IE-6S +. Likewise, FeSO 4 without carbon source or with glycerol significantly (P <0Æ05) reduced antifungal activities of the strain CHA0. LiCl with glucose and LiCl with or without carbon source, respectively, reduced the efficacy of IE-6S + and CHA0 compared with the medium without LiCl. Effect of various concentrations of zinc sulphate on antifungal activity of IE-6S + and CHA0 With an increase in zinc sulphate concentration, in vitro antagonism of the two bacteria against M. phaseolina increased (Table 2). Antagonistic effects of the two bacteria were more pronounced when CDA was amended with ZnSO 4 ; fungal inhibition initially increased but levelled off at ca. 0Æ75 mm. The influence of zinc with or without carbon sources on antifungal activity by IE-6S + and CHA0 When various carbon sources were tested in CDA amended with or without zinc, the addition of 1% (w/v) glucose, fructose, galactose, glycerol or lactate increased (P < 0Æ05) in vitro antagonism of the two bacteria against M. phaseolina compared with the no carbon source control (Table 3). Across all carbon sources, CDA amended with zinc sulphate significantly (P <0Æ05) enhanced antagonistic activity of both the bacteria producing larger inhibition zone compared with those produced in the absence of zinc sulphate amendment. Among the carbon sources tested, glucose alone or in combination with zinc sulphate gave the highest antagonistic activity of P. fluorescens strain CHA0 while fructose alone or in conjunction with zinc sulphate gave the highest antagonistic activity of P. aeruginosa strain IE-6S +. Effect of zinc and/or glucose on the efficacy of IE-6S + and CHA0 against M. phaseolina in tomato roots Soil amendment with zinc sulphate or glucose without the bacterial inoculants did not affect root infection caused by M. phaseolina but when amendments were used with the bacteria did reduce pathogen infection (P < 0Æ05) (Table 4). Zinc and glucose applied together produced better biocontrol effects of P. aeruginosa strain IE-6S + compared with the amendment of either nutrient alone while soil application with glucose alone gave the best control of the pathogen by Table 3 Influence of zinc and various carbon source amendments on antifungal activity as measured by zones of inhibition produced by Pseudomonas aeruginosa IE-6S + and P. fluorescens CHA0 in vitro Zone of inhibition (mm) IE-6S + CHA0 Treatments Zn) Zn+ Zn) Zn+ CDA without amendments 5 ± 0Æ6 8 ± 0Æ7 15 ± 1 19 ± 1Æ1 CDA amended with Glucose 9 ± 0Æ8 11 ± 0Æ9 20 ± 1Æ3 24 ± 1Æ2 Fructose 13 ± 0Æ7 15 ± 0Æ7 17 ± 0Æ9 20 ± 1 Galactose 7 ± 0Æ6 9 ± 0Æ8 17 ± 1Æ1 20 ± 1Æ3 Glycerol 7 ± 0Æ7 9 ± 0Æ7 16 ± 0Æ9 20 ± 1Æ2 Lactate 7 ± 0Æ5 7 ± 0Æ6 17 ± 1Æ1 19 ± 1 Citrate 5 ± 0Æ4 9 ± 0Æ8 16±1 17±0Æ9 Mannitol 6 ± 0Æ8 10 ± 0Æ9 19 ± 1Æ1 21 ± 1Æ3 LSD 0Æ05 Treatments (T) 1Æ6 2 Zinc (Z) 1Æ3 1Æ6 T Z 2Æ4 3Æ3 Zn), without zinc; Zn+, with zinc; CDA, Czapek s dox agar; LSD, least significant difference.

5 396 S.S. SHAUKAT AND I.A. SIDDIQUI Table 4 Effect of zinc and/or glucose on the efficacy of Pseudomonas aeruginosa IE-6S + and P. fluorescens CHA0 on root infection caused by Macrophomina phaseolina, growth of tomato and colonization by IE-6S + and CHA0 in the rhizosphere and root of tomato. Each value is based on four replicates in experiment I and five replicates in the experiment II M. phaseolina infection (%) Shoot weight (g) Bacterial rhizosphere colonization Log 10 (x +1) Treatments Exp. I Exp. II Mean Exp. I Exp. II Mean Exp. I Exp. II Mean Control Æ7 1Æ4 1Æ Zinc Æ5 2Æ1 1Æ6 1Æ Glucose Æ7 1Æ6 1Æ Zinc + glucose Æ5 2 1Æ8 1Æ IE-6S Æ5 2Æ4 2Æ1 2Æ2 4Æ48 4Æ61 4Æ54 CHA Æ6 1Æ9 1Æ8 4Æ26 4Æ72 4Æ49 IE-6S + + zinc Æ6 2Æ2 2Æ4 4Æ27 4Æ05 4Æ16 IE-6S + + glucose Æ5 2Æ2 2Æ4 2Æ3 4Æ55 4Æ77 4Æ66 IE-6S + + zinc + glucose Æ5 2Æ8 2Æ4 2Æ6 4Æ37 4Æ53 4Æ45 CHA0 + zinc Æ7 1Æ5 1Æ6 4Æ11 4Æ48 4Æ29 CHA0 + glucose Æ8 2 1Æ9 4Æ38 4Æ68 4Æ53 CHA0 + zinc + glucose Æ5 2 2Æ2 2Æ1 4Æ28 4Æ75 4Æ51 LSD 0Æ05 16Æ8 0Æ6 0Æ13 Experiments I and II are two independent trials. P. fluorescens strain CHA0. Zinc and glucose without the bacterial inoculants also markedly reduced M. phaseolina infection in tomato roots. When the efficacy of the two bacteria was compared, P. aeruginosa IE-6S + caused greater suppression of M. phaseolina than did P. fluorescens CHA0. Soil amendments with zinc and/or glucose without the bacterial inoculants did not affect fresh shoot weights of tomato (Table 4). Pseudomonas aeruginosa IE-6S + used alone or concomitantly with zinc and/or glucose significantly (P < 0Æ05) enhanced fresh shoot weights compared with the controls. However, P. fluorescens strain CHA0 either used alone or in combination with zinc and/or glucose did not influence plant growth. Soil amendments with zinc significantly (P <0Æ01) reduced populations of both the biocontrol agents in the rhizosphere while glucose either applied alone or in combination with zinc did not alter bacterial populations in the rhizosphere. DISCUSSION The results show that mineral and/or carbon amendments improve biocontrol activity of fluorescent pseudomonads against M. phaseolina both under laboratory and glasshouse conditions. The amendments exert a differential influence on the biocontrol performance of the two bacteria. The bacterial inoculants produced larger zones of inhibition against M. phaseolina when growth medium was amended with zinc sulphate particularly with glucose. Furthermore, bacterial performance progressively improved when zinc concentration was elevated in the medium. The antifungal activity of the agents peaked when growth medium was amended with the highest concentration of zinc. The results obtained under laboratory conditions correlate well with those obtained under glasshouse conditions. Soil amendment with zinc alone did not inhibit M. phaseolina infection in tomato roots but did reduce significantly when used in combination with biocontrol bacteria indicating that indirectly zinc reduced disease through some influence on the interaction between the biocontrol agent and the fungus. Increased production of the antifungal compounds by P. aeruginosa and P. fluorescens is presumably the primary mechanism involved in the suppression of the pathogen. Alternatively, other mechanisms including (i) alteration in plant physiology following amendments with both zinc sulphate and biocontrol agent and (ii) the stimulation of soil micro-organism antagonistic to M. phaseolina, may also be operative singly or sequentially under natural conditions that in turn could reduce colonization potential of the fungal pathogen in the roots. Zinc and other trace elements are known to stimulate the in vitro production of 2,4-diacetylphloroglucinol, pyoluteorin (Duffy and Défago 1999), and phenazine-type antibiotics (Slininger and Jackson 1992) and stabilize regulatory genes critical for antibiotic production in fluorescent pseudomonads (Duffy and Défago 1995). From a practical perspective, mineral effects on the biocontrol effectiveness may explain the association between soil chemical and physical properties and the variable performance of biocontrol strains between field sites. Interestingly, following soil amendments with zinc sulphate, populations of both P. aeruginosa IE-6S + and

6 BIOCONTROL OF PSEUDOMONADS AGAINST MACROPHOMINA PHASEOLINA 397 P. fluorescens CHA0 in the rhizosphere declined sharply. However, regardless of the zinc application, soil amendment with glucose did not significantly alter bacterial populations in the rhizosphere. Zinc-deficient plants contain low levels of superoxidase dismutase and, therefore, high levels of superoxide radicals, leading to peroxidation of membrane lipids, loss of membrane integrity and increased membrane permeability (Cakmak and Marschner 1988). This results in the inhibition of protein synthesis and accumulation of free amino acids and amides in the exudates and as a consequence increased numbers of bacteria in the rhizosphere. Application of zinc prevents membrane leakage thereby reducing the quantity and also possibly the quality of organic materials that reduce microbial populations in the rhizosphere. In the present study, bacterial strains responded differentially to different carbon sources. For example, when various carbon sources were compared, fructose improved biocontrol potential of the strain IE-6S + while glucose enhanced antagonistic activity of the strain CHA0. These results are in large part related to the difference in carbon source utilization and metabolism among bacteria and explain why these bacterial agents exhibit variable performance on different hosts. Plant specificity of biocontrol strains has been demonstrated at both the species and cultivar level (Maurhofer et al. 1995). This has generally been attributed to differential utilization of various carbon and nitrogen compounds found in exudates and their effect on bacterial growth and their population structure (Westover et al. 1997). Duffy and Défago (1995) suggested that another factor in plant specificity might be the influence of root exudate components (C and N) on the biosynthesis of antimicrobial compounds. Pseudomonas aeruginosa IE-6S + and P. fluorescens CHA0 which utilize different carbon source indicate niche differentiation among bacteria and explain why in a previous study mixtures of both these biocontrol agents exhibited a general trend towards greater suppression of tomato root pathogens compared with their individual applications and coexistence in the rhizosphere (Siddiqui and Shaukat 2002b). Combining antagonists in the context of niche differentiation provides an effective means for improving biocontrol of soil-borne plant pathogens. Niche differentiation from in vitro carbon utilization profiles has been used as an indicator of ecological coexistence of micro-organisms on plant surfaces (Wilson and Lindow 1994). From a practical standpoint, trace mineral and carbon amendments offer an inexpensive way to improve the biocontrol activity of certain bacterial strains. 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