Antonie van Leeuwenhoek 81: 537547, 2002. 2002 Kluwer Academic Publishers. Printed in the Netherlands.

537

Antibiotic production by bacterial biocontrol agents

Jos M. Raaijmakers , Maria Vlami & Jorge T. de Souza

Department of Plant Sciences, Laboratory of Phytopathology, Wageningen University, Binnenhaven 5, 6709 PG Wageningen, The Netherlands ( Author for correspondence)

Abstract Interest in biological control of plant pathogens has been stimulated in recent years by trends in agriculture towards greater sustainability and public concern about the use of hazardous pesticides. There is now unequivocal evidence that antibiotics play a key role in the suppression of various soilborne plant pathogens by antagonistic microorganisms. The signikcance of antibiotics in biocontrol, and more generally in microbial interactions, often has been questioned because of the indirect nature of the supporting evidence and the perceived constraints to antibiotic production in rhizosphere environments. Reporter gene systems and bio-analytical techniques have clearly demonstrated that antibiotics are produced in the spermosphere and rhizosphere of a variety of host plants. Several abiotic factors such as oxygen, temperature, specikc carbon and nitrogen sources, and microelements have been identiked to inkuence antibiotic production by bacteria biocontrol agents. Among the biotic factors that may play a determinative role in antibiotic production are the plant host, the pathogen, the indigenous microkora, and the cell density of the producing strain. This review presents recent advances in our understanding of antibiotic production by bacterial biocontrol agents and their role in microbial interactions.

Introduction For many decades, bacteria have been introduced into soil or on seeds, roots, bulbs or other planting material to improve plant growth and health (Weller 1988; Whipps 1997). The major objectives of bacterization include enhancement of symbiotic or associative nitrogen kxation, degradation of xenobiotic compounds, plant growth promotion and biological control of plant pathogenic microorganisms (Van Elsas & Heijnen 1990; Whipps 2001). To date, many bacterial genera are being used and tested in bacterization, including Acinetobacter, Agrobacterium, Alcaligenes, Arthrobacter, Azospirillum, Azotobacter, Bacillus, Bradyrhizobium, Frankia, Pantoea, Pseudomonas, Rhizobium, Serratia, Stenotrophomonas, Streptomyces, and Thiobacillus (Weller 1988; Van Elsas & Heijnen 1990; Whipps 2001). Recent advances in microbiological and molecular techniques have signikcantly contributed to new insights in the underlying mechanisms by which introduced bacteria function in natural environments and coordinately have led to an increase in studies on the diversity, physiology, and

activity of specikc bacterial genera and bacterial communities (Akkermans et al. 1995; Loper & Lindow, 1995). Interest in biological control of plant pathogens has increased considerably over the past years, partly as a response to public concern about the use of hazardous chemical pesticides, but also because it may provide control of diseases that can not, or only partially, be managed by other control strategies (Cook 1993). To date, many studies on biological control of plant pathogens by antagonistic bacteria focus on the suppressive effects of single strains introduced repeatedly into soil or on planting material at relatively high densities. In contrast to this inundative approach, management and manipulation of natural communities of antagonistic microorganisms through crop rotations and organic amendments have received relatively less attention, in spite of the fact that these strategies have resulted in highly effective forms of biological control (Hoitink & Boehm 1999). Because there are numerous detailed reviews on biological control of plant pathogens (Fravel 1988; Handelsman & Stabb 1996; Thomashow & Weller

538 1996; Weller 1988; Whipps 2001), the purpose of this review is to focus on new insights and concepts in biological control of plant pathogens by bacteria that produce antibiotics (excluding metal chelators and enzymes). We will highlight recent progress in identikcation of factors that affect antibiotic production by antagonistic bacteria and discuss strategies that provide a basis to improve the efkcacy of antibioticproducing bacteria. common inhabitants of rhizosphere and phyllosphere environments, are isolated easily from natural environments, utilize a wide range of substrates, are easy to culture and manipulate genetically, making them more amendable to experimentation. Nevertheless, increasingly more papers are being published on antibiotics produced by bacterial biocontrol agents other than Pseudomonas spp. (Table 1). For most of the antibiotics listed in Table 1, the chemical structures have been elucidated. Collectively, these and other studies have shown that bacterial biocontrol strains not only exhibit a wide range of diversity in the type but also in the number of antibiotics produced. For example, Bacillus cereus strain UW85 (Handelsman & Stabb 1996) and P. kuorescens strains CHA0 and Pf5 (Keel et al. 1996; Bender et al. 1999) produce multiple antibiotics with overlapping or different degrees of activity against specikc pathogenic fungi. These observations illustrate that for at least some biocontrol agents several antibiotics may account for the suppression of specikc or multiple plant diseases. Several studies have demonstrated that many of the antibiotics produced by bacterial biocontrol agents have a broad-spectrum activity. For example, the broad-spectrum activity of pyrrolnitrin, produced by Pseudomonas and Burkholderia species, was already noticed in the 1960s by Japanese scientists (Nishida et al. 1965) who tested and further developed this antibiotic for therapeutic purposes against human pathogenic bacteria and fungi. With respect to plant pathogenic fungi, pyrrolnitrin has shown activity against a wide range of Basidiomycetes, Deuteromycetes, and Ascomycetes, including several economically important pathogens like Rhizoctonia solani, Botrytis cinerea, Verticillium dahliae, and Sclerotinia sclerotiorum (Ligon et al. 2000). Furthermore, pyrrolnitrin was also reported to be active against several Gram-positive bacteria and in particular Streptomyces species (El-Banna & Winkelmann 1998). Similarly, DAPG, produced by several strains of Pseudomonas kuorescens (Table 1), not only has activity against a wide range of plant pathogenic fungi but also has antibacterial, anthelminthic and phytotoxic properties (Keel et al. 1992; Thomashow & Weller 1996). Work by Cronin et al. (1997) showed that puriked DAPG increased hatching of cysts of the nematode Globodera rostochiensis and signikcantly reduced juvenile mobility. Also zwittermycin A, an antibiotic produced by Bacillus cereus and Bacillus thuringiensis adversely affects the growth and activity of a wide range of microorganisms, including several plant pathogenic

Biological control by bacteria Some of the earliest studies on mechanisms of pathogen suppression by root-colonizing bacteria focused on their ability to produce specikc compounds (siderophores) that efkciently sequester iron, thereby depriving the pathogen from this essential element during its deleterious activities in the rhizosphere (Kloepper et al. 1980). Over the past 20 years, numerous studies have demonstrated unequivocally that several other metabolites including antibiotics, enzymes, and volatiles produced by antagonistic bacteria play key roles in the control of various plant pathogens (Weller 1988; Whipps 1997). Bacteria also can be benekcial to the host plant directly through the production of metabolites that either stimulate root development and plant growth or trigger the induction of systemic acquired resistance (Van Loon et al. 1998). Although most knowledge on mechanisms and metabolites involved in biological control by bacteria are based on studies with kuorescent Pseudomonas spp., information on antibiotics produced by representatives of other bacterial genera will be discussed whenever possible.

Structure and broad-spectrum activity of antibiotics produced by bacterial biocontrol agents Antibiotics encompass a chemically heterogeneous group of organic, low-molecular weight compounds produced by microorganisms. At low concentrations, antibiotics are deleterious to the growth or metabolic activities of other microorganisms (Fravel 1988; Thomashow et al. 1997). In the past decades, numerous antibiotics have been isolated from various biocontrol strains representing different bacterial genera (Table 1). According to Whipps (1997), there are several reasons for the abundance of studies on antibiotics produced by Pseudomonas spp.: they are

539
Table 1. Selected examples of antibiotics produced by bacterial biocontrol agents Antibiotic1 DAPG Species/strain Pseudomonas spp. Q2-87 CHAO F113 PFM2 Pf-5 Q8r1-96 Pseudomonas spp. 2-79RN10 30-84 PGS12 In-b-109 PCL1391 PNA1 P. kuorescens Hv37a P. kuorescens Pf-5 CHA0 P. kuorescens BL915 B. cepacia B37w Enterobacter agglomerans Serratia spp. P. borealis MA342 P. kuorescens DR54 P. aureofaciens 63-28 Pseudomonas sp. AB2 S. violaceusniger YCED-9 P. agglomerans EH318 Stenotrophomonas SB-K88 B. cepacia BC11 B. cereus UW85 B. cereus UW85 Target pathogen2 Origin/Host Reference

Ggt Ggt, Tb, Pu Pu St Pu, Rs Ggt Ggt Ggt Fo Rs, Gg Fo Fo, Ps Pu Pu, Rs Tb, Pu Rs Fs Bc, Rs Vd, Rs, Ss Pt, Tc Rs, Pu Pu, Pc Pu, Rs, Bc Pu Eh Pu Rs Pm Pm

Wheat, WA, USA Tobacco, Switzerland Sugar beet, Ireland Wheat, OK, USA Cotton, Texas, USA Wheat, WA, USA Wheat, WA, USA Wheat Kansas, USA Corn, Belgium Rice, Philippines Tomato, Spain Chickpea, India Barley, USA Cotton, Texas, USA Tobacco, Switzerland Cotton, USA Potato Grape, Uzbekistan Oilseed rape Wheat, barley Sugar beet, Denmark Canola, Canada Soil, Korea Lettuce Apple, USA Sugar beet, Japan Soil, cotton Alfalfa Alfalfa

Vincent et al. 1991 Keel et al. 1992 Shanahan et al. 1992 Levy et al. 1992 Howell and Stipanovic 1979 Raaijmakers and Weller 2001 Weller 1983 Pierson & Thomashow 1992 Georgakopoulos et al. 1994 Rosales et al. 1995 Chin-A-Woeng et al. 1998 Anjaiah et al. 1998 Gutterson et al. 1986 Howell & Stipanovic 1979 Keel et al. 1992 Ligon et al. 2000 Burkhead et al. 1994 Chernin et al. 1996 Kalbe et al. 1996 Hokeberg et al. 1998 Nielsen et al. 1998 Gamard et al. 1997 Ki Kim et al. 2000 Trejo-Estrada et al. 1998 Wright et al. 2001 Nakayama et al. 1999 Kang et al. 1998 Milner et al. 1996 Silo-Suh et al. 1994

Phenazines

Oomycin A Pyoluteorin

Pyrrolnitrin

DDR Viscosinamide Butyrolactones N-BBS AFA Pantocin A and B Xanthobaccins AFC-BC11 Kanosamine Zwittermycin A

1 DAPG 2,4-diacetylphloroglucinol; DDR 2,3-de-epoxy-2,3-didehydro-rhizoxin; N-BBS N-Butylbenzenesulphonamide. 2 At Agrobacterium tumfaciens ;Ggt Gaeumannomyces graminis var tritici; Tb Thielaviopsis basicola ;Pu, Pythium ultimum ;St

Septoria tritici ;Rs Rhizoctonia solani ;Fo F. oxysporum ;Gg G. graminis ;Ps Pythium splendens ;Fs Fusarium sambucinum ;Pt Pyrenophora teres ;Tc Tilletia caries ;Pc Phytophthora cryptogea ;Bc Botrytis cinerea ;Eh Erwinia herbicola ;Pm Phytophthora medicaginis ;Vd Verticillium dahliae ;Ss Sclerotinia sclerotiorum.

fungi and in particular Phytophthora and Pythium species (Silo-Suh et al. 1994, 1998). Interestingly, zwittermycin A appeared to interact synergistically with the Bt toxin produced Bacillus thuringiensis thereby enhancing its insecticidal activity (Broderick et al. 2000).

Role of antibiotics in biological control Most of the data on broad-spectrum activity of antibiotics produced by bacterial biocontrol agents are derived from assays performed in vitro. Several lines of evidence, however, have more conclusively demonstrated the role and function of antibiotics in in situ interactions between antagonistic bacteria and plant pathogens. The krst line of evidence was the observation that culture kltrates or puriked antibiotics provided similar levels of control as achieved by the

540 producing wild-type strain (Howell and Stipanovic 1979; Kang et al. 1998; Nakayama et al. 1999). Second, inactivation of antibiotic production by mutagenesis resulted, in many cases, in a reduced ability of the antagonistic bacteria to control the pathogen. Mutagenesis has been successfully used to demonstrate that antibiotics produced by Pseudomonas species (Thomashow & Weller 1988; Keel et al. 1990; Vincent et al. 1991; Cronin et al. 1997; Anjaiah et al. 1998; Chin-A-Woeng et al. 1998; Hokeberg et al. 1998), Burkholderia cepacia (Kang et al. 1998; Heungens & Parke 2001), and Bacillus cereus (SiloSuh et al. 1994) play an important role in the biological control of plant diseases. In several of these studies, complemented mutants with restored phenotypes and restored biocontrol activity were included (Thomashow et al. 1997). The third line of evidence is provided by enhancement of antibiotic production in the producing wild-type strains via introduction or modikcation of antibiotic biosynthetic or regulatory genes. Suppression of Pythium root rot of cucumber was signikcantly improved by enhancement of the production of DAPG and pyoluteorin in P. kuorescens strain CHA0 (Maurhofer et al. 1992). Ligon et al. (2000) introduced multiple copies of the regulatory gacA gene into P. kuorescens strain BL915 resulting in a 2.5-fold increase in pyrrolnitrin production. Similar increases in pyrrolnitrin production in strain BL915 were obtained by PCR-based modikcation of the initiation codon of the chromosomal gacA gene or by replacing the native promoter of this regulatory gene with the strong P tac promotor from E. coli. Relative to the wild-type strain, the genetically modiked strains showed, in most cases, an increase in biocontrol activity against Rhizoctonia solani (Ligon et al. 2000). The fourth line of evidence is provided by introduction of antibiotic biosynthetic genes in heterologous, non-producing strains and subsequent evaluation of their ability to control plant diseases. For example, Fenton et al. (1992) introduced a 6-kb fragment from DAPG-producing P. kuorescens strain F113 into DAPG-nonproducing Pseudomonas strain M114; the derivative produced DAPG and was signikcantly more effective than its parental strain in controlling Pythium ultimum on sugar beet. Most studies on heterologous expression of antibiotic biosynthetic genes describe the use of multi-copy plasmid vectors. Recently, Timms-Wilson et al. (2000) introduced a disabled Tn 5 vector harboring the genes for phenazine-1-carboxylic acid production into the chromosome of PCA-nonproducing P. kuorescens strain SBW25. PCA-producing derivatives were signikcantly more protective than their parental strain against Pythium ultimum on pea seedlings. The authors suggested that single copy chromosomal insertions are less likely to affect the ktness of the heterologous strain than plasmid-based modikcations. The latter two genetic strategies not only provide evidence for a role of antibiotics in biological control, but also clearly illustrate that knowledge about the genetics, regulation and biochemistry of antibiotic biosynthesis provides powerful tools to improve the efkcacy of bacterial biocontrol agents.

In situ antibiotic production A more direct line of evidence for the role of antibiotics in disease suppression by biocontrol agents is provided by detection of antibiotic production in situ, a strategy that complements indirect evidence provided by the above mentioned genetic approaches. The signikcance of antibiotics in biological control, and more generally in microbial antagonism, often has been questioned because of the perceived constraints to antibiotic production in natural environments (Gotlieb 1976; Williams & Vickers 1986). Because of the biotic and abiotic complexity of soils and plantassociated environments, there are several inherent difkculties in detecting antibiotics produced by microorganisms in situ. Recovery and detection may be hampered by chemical instability of the compound, irreversible binding to soil colloids or organic matter, or microbial decomposition (Thomashow et al. 1997). Sensitive methods have been developed by which antibiotic production by biocontrol agents can be measured in natural environments. One approach involves the use of reporter gene systems (Lindow 1995; Chin-A-Woeng et al. 1998). Reporter gene systems are widely used as a marker to monitor populations of introduced strains but also may provide information on the transcriptional activity of specikc antibiotic biosynthetic genes (Loper & Lindow 1997). Unfortunately, reporter gene systems generally do not provide an accurate measure of the amount of the antibiotic produced in situ.Bioanalytical techniques like thin layer and high-pressure liquid chromatography, respectively TLC and HPLC, are now being used to detect and quantify antibiotics produced by microorganisms in situ (Thomashow et al. 1997). The versatility, resolving capability, and quantitative accuracy of HPLC make it one of the best

541 direct methods to study the production of antibiotics in situ. In order to detect and quantify antibiotics by HPLC, however, the extracted and separated compounds need to be positively identiked. Identikcation of antibiotic compounds on the basis of their retention time is not sufkcient. Subsequent spectral analysis by photodiode array detectors is required to provide the necessary information about peak homogeneity and identity. In most cases, spectral analysis over a range of wavelengths will provide sufkcient information on the compound. However, also spectral analysis may have its limits. Determining the identity of compounds in overlapping peaks may be difkcult if the spectra of the compounds are similar and the peaks are poorly resolved (Dorschel 1997). Also, deviations in the spectral characteristics of the compound can be induced by volatilization of the solvents, and may occur after derivatization. In that case, HPLC linked to a mass spectrometer provides a means to conkrm the identity of antibiotics produced in situ (Raaijmakers et al. 1999). To date, HPLC has been successfully used for the in situ detection of a variety of antibiotics including phenazine-1-carboxylic acid, herbicolin A, pyrrolnitrin, pyoluteorin, surfactin, and DAPG (reviewed by Thomashowet al. 1997). In almost all cases, these data stem from studies in which specikc bacterial strains were introduced into soil or rhizosphere environments at relatively high densities. Recently, also antibiotic production levels by specikc indigenous bacterial populations were determined. In a soil, that is naturally suppressive to the soilborne pathogen Gaeumannomyces graminis var. tritici, the antibiotic DAPG was detected on roots of wheat at a concentration of approximately 20 ng per gram of root fresh weight (Raaijmakers et al. 1999). These results provided, for the krst time, biochemical support for the conclusion of other studies that DAPG-producing kuorescent Pseudomonas spp. are key components of the natural biological control that operates in several take-all suppressive soils in Washington State (USA) (Raaijmakers & Weller 1998). For a wide variety of other antibiotics, in situ production levels may range from 5 to 5000 ng per seed or gram of dry soil or root fresh weight (Thomashow et al. 1997). Although detection of antibiotics in situ has been described as a strategy that complements the indirect evidence provided by genetic approaches, it in fact only conkrms that antibiotic production has occurred. However, it does not address the question as to whether the amount of antibiotic is sufkcient to inhibit the growth or metabolic activity of the pathogen in situ. The answer to this latter issue is difkcult to give, because quantitative detection methods usually are not sensitive enough to determine spatial and temporal production patterns. The time and place of antibiotic production needs to be considered with respect to the efkcacy of biological control of several plant pathogens. For example, an antibiotic may reach threshold concentrations for activity within certain microsites while remaining below this threshold level at other sites where pathogen infection also occurs. This may offer an explanation for the observation that, in spite of a signikcant positive linear relationship between in situ DAPG production levels and rhizosphere population densities of P. kuorescens strain Q2-87, increases in the rhizosphere population density of Q2-87 did not signikcantly improve the level of suppression of takeall of wheat (Raaijmakers & Weller 1998; Raaijmakers et al. 1999). Recent advances in the use of different derivatives of the green kuorescent protein (Bloemberg et al. 2000) offer powerful tools to simultaneously visualize the pathogen and the biocontrol agent and, more specikcally, to determine whether spatial colonization patterns of the pathogen coincide with spatial colonization and antibiotic production patterns of the biocontrol agent. Also in situ PCR technologies (Hodson et al. 1995; Chen et al. 2000) provide powerful tools to detect single cells and target sequences in environmental samples and microsites on the plant root. These technologies not only allow detection of single cells but also enable visualization of gene expression inside the bacterial cells.

Factors affecting antibiotic production When evaluating the last two decades of research on biological control, it is clear that most bacterial biocontrol agents, including antibiotic-producing bacteria, are still too variable in their performance to be successfully used as a common practice in agriculture and horticulture. This inconsistency has been ascribed to a number of factors, including variable expression of genes involved in disease suppression and poor colonization of the host plant by the applied biocontrol agent. Consequently, research has focused on traits involved in the ecological competence of introduced strains and factors that affect antibiotic production. Antibiotic production in bacterial biocontrol agents, and in particular Pseudomonas spp., is modulated by a wide range of endogenous factors, including the two

542 component GacA/GacS regulatory system (Gaffney et al. 1994), cell-density dependent regulation via N-acyl homoserine lactones (Wood et al. 1997), and sigma factors (Sarniguet et al. 1995). According to Duffy & Defago (1999), identikcation of exogenous factors that affect antibiotic production and regulation has progressed comparatively slow. Here we will present some recent examples of abiotic and biotic factors that modulate antibiotic biosynthesis in bacterial biocontrol agents. For a more comprehensive review on this topic we refer to Duffy and Defago (1999). Some of the physical factors that have been reported to affect antibiotic production are temperature (Shanahan et al. 1992), soil moisture (Georgakopoulos et al. 1994), and pH (Ownley et al. 1992). Recent results also suggest that pH affects the activity of the antibiotic itself. Chin-A-Woeng et al. (1998) observed that at pH 5.7, the in vitro antifungal activity of phenazine-1-carboxamide was 10 times higher than that of phenazine-1-carboxylic acid (PCA). Given that PCA activity was completely abolished under less acidic conditions, the authors suggested that the nature of the phenazine derivatives may contribute to the differences in biocontrol activity between strains. Also the activity of DAPG is strongly inkuenced by pH; DAPG is considerably more active in vitro against various Pythium species at acidic than at neutral to basic pH levels (J.T. de Souza and J.M. Raaijmakers, unpublished data). Because microorganisms in the phytosphere depend on substrates liberated from the root or shoot for their growth, the host plant greatly inkuences the quantity and composition of indigenous microorganisms as well as the expression of antibiotic biosynthetic genes. This was nicely illustrated by Milner et al. (1996) who showed that production of the antibiotic kanosamine in Bacillus cereus was enhanced by more than 300% by the addition of alfalfa seedling exudates to the culture medium. Duffy and Defago (1999) tested the inkuence of a variety of carbon sources, inorganic phosphate and minerals on antibiotic production by P. kuorescens CHA0 and 41 other DAPG-producing Pseudomonas strains isolated from a wide range of geographical regions and representing different genotypic clusters. They found that in almost all strains glucose stimulated DAPG production, whereas stimulation of DAPG production by zinc occurred in a strain-specikc manner. Phosphate repressed DAPG production in a selection of nine strains out of a total of 41, a response that also was observed for kanosamine production in Bacillus cereus (Millner et al. 1996) and phenazine production in P. kuorescens (Slininger & Jackson 1992). Based on these kndings, Duffy & Defago (1999) questioned the potential adverse effects of phosphate fertilizers used in agriculture on the efkcacy of both introduced biocontrol agents and naturally occurring antagonistic microorganisms. Understanding the inkuence of several abiotic and biotic factors on expression of antibiotic biosynthesis opens up possibilities to manipulate the behavior and activity of biocontrol agents. The discovery of a genetic basis in tomato for support of growth and disease suppressive activity of Bacillus cereus (Smith et al. 1999) indicates that genetic variation in host species can be exploited to breed for cultivars that are highly compatible with particular biocontrol agents. Furthermore, AHL synthase genes involved in quorum sensing in pathogenic and antagonistic bacteria have been successfully expressed in transgenic tobacco plants to levels sufkcient to restore G. graminis growthinhibiting activity in mutants of P. aureofaciens 3084 defective in N -acylhomoserine lactone production (Fray et al. 1999).

Distribution and detection of antibiotic-producing bacteria Numerous bacterial strains that produce antibiotics in vitro have been isolated from different soils and plant hosts (Table 1). This apparent wide distribution suggests that antibiotic-producing bacteria are common constituents of the indigenous microkora in soil and plant-associated environments worldwide. To date, however, little is known about the frequency and ecology of indigenous antibiotic-producing bacteria. Knowledge about the ecology of naturally occurring strains that harbor specikc biocontrol traits will signikcantly contribute to improving the efkcacy of existing biocontrol agents and may help to identify new strains that are better adapted to specikc soils or host-pathogen systems. Moreover, knowledge about the distribution of antibiotic-biosynthetic genes in natural environments could lessen concerns about the environmental release of either non-indigenous strains containing these traits or transgenic biocontrol agents in which these traits have been introduced. Selection and identikcation of antibiotic-producing bacteria via random isolation from natural environments is very time-consuming and laborious. In fact, most of the model strains used to date were identiked via screening huge numbers of isolates obtained by

543 random procedures. The availability of cloned and sequenced antibiotic biosynthetic and regulatory genes, however, has facilitated the development of specikc primers and probes that can be used for targeted detection and isolation of specikc antibiotic-producing bacteria. For several antibiotics produced by strains of different bacterial genera, biosynthetic genes have been cloned and partially or fully sequenced. These include zwittermycin A produced by Bacillus cereus (Stohl et al. 1999), AFC-BC11 produced by B. cepacia (Kang et al. 1998) and the antibiotics DAPG, phenazines, pyrrolnitrin, and pyoluteorin produced by various Pseudomonas species (Pierson et al. 1995; Hammer et al. 1997; Mavrodi et al. 1998; Bangera & Thomashow 1999; Nowak-Thompson et al. 1999; Chin-A-Woeng 2000; Delaney et al. 2001). A prerequisite for DNA-based detection and isolation is that the genes of interest must be conserved among a wide range of bacterial strains harboring this trait. By Southern hybridization with a 4.8-kb chromosomal DNA fragment of P. kuorescens strain Q2-87, Keel et al. (1996) showed that the biosynthetic locus for DAPG production was highly conserved among 45 DAPG-producing Pseudomonas strains of worldwide origin. Subsequent development of primers and probes directed against phlD, a key gene in the biosynthesis of DAPG, allowed targeted detection, isolation and enumeration of DAPG-producing Pseudomonas spp. occurring naturally on roots of wheat (Raaijmakers et al. 1997). Also the genes or gene clusters involved in the biosynthesis of phenazines (Raaijmakers et al. 1997) and pyrrolnitrin (Hammer et al. 1999), respectively, appear to be conserved among different strains. In recent studies performed in our laboratory, PCR analysis, Southern hybridization and restriction fragment length polymorphisms (RFLP) analyses showed that specikc genes involved in pyrrolnitrin and pyoluteorin biosynthesis are conserved in a large collection of both Pseudomonas and Burkholderia species (J.T. de Souza and J.M. Raaijmakers, unpublished data). The use of probes and primers directed against genes involved in antibiotic biosynthesis has proven to be a powerful technique to study the distribution and function of indigenous antibiotic-producing Pseudomonas spp. Colony hybridization followed by PCR analysis showed that root-associated kuorescent Pseudomonas spp. producing the antibiotic DAPG were present on roots of wheat grown in several takeall suppressive soils at densities ranging from approximately 5 105 to 2 106 CFU per gram of root. In the complementary conducive soils, DAPG-producing pseudomonads were not detected or detected at densities at least 40-fold less than in the suppressive soils (Raaijmakers et al. 1997). Moreover, DAPGproducing Pseudomonas spp. were present on roots of wheat grown in at least three take-all suppressive soils at or above the threshold population density required for signikcant suppression of take-all of wheat. The specikc suppression that operates in take-all suppressive soils was lost when indigenous DAPG-producing kuorescent Pseudomonas spp. were eliminated by selective heat treatment, and conducive soils gained suppressiveness to take-all when indigenous DAPGproducing Pseudomonas strains were introduced via mixing in small amounts of raw take-all suppressive soil (Raaijmakers et al. 1998). Apart from genetic markers, also phenotypic markers may provide a source to rapidly isolate and identify bacteria with antagonistic potential. For example, Ellis et al. (2000) characterized a collection of 29 kuorescent pseudomonads with the intent to identify conserved phenotypic or genotypic traits in strains with activity against Pythium ultimum. They found a signikcant correlation between biological control activity and the accumulation of a specikc cyclopropane fatty acid (C17:O CFA) and hydrogen cyanide production. The authors postulated that screening isolates on the basis of elevated synthesis of C17:O CFA facilitates selection of highly effective biocontrol agents without prior knowledge of secondary metabolite synthesis. To further support their hypothesis, the authors identiked a highly active biocontrol strain by FAME analysis within a collection of at least 500 kuorescent Pseudomonas spp. The effective strain, referred to as P. chlororaphis S34/10, represented the only clonal group (out of a total of 13 groups) with a high relative C17:O CFA content. For detection of antibiotic-producing Bacillus strains, strategies similar to those described for Pseudomonas spp. have been used. One phenotypic strategy to detect zwittermycin A-producing strains of Bacillus cereus and Bacillus thuringiensis consisted of phage typing and growth inhibition of Erwinia herbicola (Stabb et al. 1994). Another strategy was based on FAME and PCR analysis with primers for the zwittermycin A-resistance gene zmaR (Raffel et al. 1996). These studies demonstrated that PCR was a more reliable method for identikcation of zwittermycin A-producers than FAME analysis. Furthermore, zwittermycin A-producing Bacillus strains were found in diverse soils from worldwide origin at densities of approximately 10 4 per gram and tended to suppress

544 damping-off disease of alfalfa more effectively than B. cereus isolates that do not produce zwittermycin A and/or kanosamine. In addition, Giacomodonato et al. (2001) developed a screening method for Bacillus isolates by using PCR-primers directed against conserved sequences in genes that are involved in the biosynthesis of a variety of peptide antibiotics. Among four Bacillus isolates that gave a positive signal in PCR, three had an inhibitory effect on Sclerotinia sclerotiorum, whereas two strains that failed to give an amplikcation product did not inhibit fungal growth. Although only a few isolates were included, this study illustrated that direct PCR on DNA extracted from the rhizosphere may provide a krst screening for the presence of indigenous strains with antagonistic potential. producing Pseudomonasspecies, occurring on roots of wheat grown in a soil naturally suppressive to take-all disease of wheat, 16 different groups were identiked by Random Ampliked Polymorphic DNA (RAPD) analysis with two 10-mer primers (Raaijmakers & Weller 2001). One RAPD-group made up 50% of the total population of DAPG-producing Pseudomonas spp. Subsequent root-colonization and biocontrol studies indicated that this dominant genotype, exempliked by P. kuorescens Q8r1-96, was highly adapted to the wheat rhizosphere and was very effective in suppression of take-all disease of wheat (Raaijmakers & Weller, 2001). The observation that strain Q8r1-96 showed the same population dynamics during successive growth cycles of wheat in two other soils, both of which had different physicalchemical properties than the Quincy virgin soil, suggested that its superior rhizosphere competence is not soil specikc. This was supported by results obtained in a study by McSpadden-Gardener et al. (2000), who found that nearly one-third of the DAPG-producing isolates obtained from soils of different wheat-growing areas in the United States were genotypically similar to strain Q8r1-96. Biochemical analyses indicated that the superior rhizosphere competence of Q8r1-96 was not related to elevated in situ DAPG production levels but possibly to its ability to utilize specikc substrates (Raaijmakers & Weller 2001). These data and results obtained by Sharik-Tehrani et al. (1998) illustrate that exploiting the diversity within a specikc group of antagonistic microorganisms has great potential for improving biological control. This approach capitalizes on existing knowledge concerning mechanisms, while exploiting differences among strains to face the biotic and abiotic complexity of natural environments. By matching bacterial genotypes with crops or varieties for which they have a preference, root colonization and biocontrol can be improved considerably. Acknowledgements This review has been made possible by a fellowship from the Royal Netherlands Academy of Arts and Sciences. References
Akkermans ADL, Van Elsas JD & de Bruijn FJ (1995) Molecular Microbial Ecology Manual. Kluwer Academic Publishers, Dordrecht.

Diversity of antibiotic-producing bacteria The genotypic and phenotypic diversity that occurs in natural populations of biocontrol agents provides an enormous resource for improving biological control of plant diseases (Handelsman & Stabb 1996; Thomashow & Weller 1996). This approach has been widely used to select for better biocontrol agents of insects, and to improve the use of microorganisms in the production of fermented foods and biodegradation of xenobiotic compounds (reviewed in Stabb et al. 1994). However, exploitation of genotypic diversity among bacterial biocontrol agents of plant pathogenic fungi, so far, has received much less attention. Knowledge of the diversity within a group of strains that share a common biocontrol trait may provide a new approach to identify biocontrol strains that are superior with respect to ecological competence and ability to suppress specikc plant diseases. Recent studies have shown that there is considerable genotypic diversity in populations of B. cepacia (DiCello et al. 1997; Bevivino et al. 1998; Parke & Gurian-Sherman 2001), Serratia plymuthica (Berg 2000), Bacillus cereus (Raffel et al. 1996), and Pseudomonas species (Keel et al. 1996; McSpaddenGardener et al. 2000). Different genotypes of DAPGproducing Pseudomonas spp. have been reported to differ in their ability to suppress Fusarium crown and root rot and Pythium root rot (Sharik-Tehrani et al. 1998), to produce other antibiotics in addition to DAPG (Keel et al. 1996), and to colonize roots of maize plants of different growth stages (Picard et al. 2000). Among 101 isolates of indigenous DAPG-

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