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Chapter 11 Plant Growth-Promoting Bacteria: Fundamentals and Exploitation Clara Pliego, Faina Kamilova, and Ben Lugtenberg 11.1 Introduction The rhizosphere was defined by Hiltner (1904) as “the soil compartment influenced by the roots of growing plants.” Scientists agree that this area is not more than a few millimeters thick. When bacteria are present on the root plane or near the plant root we call them rhizosphere bacteria or rhizobacteria. When present on the leaf or inside the plant tissues we speak about phyllosphere bacteria and endophytes, respectively. When they colonize the rhizosphere they can be present on the root surface (the “rhizoplane”) or close to the rhizosphere. The bacteria Rhizobium and Bradyrhizobium can be present in special organs, so-called root nodules (Spaink et al. 1998). In comparison to bulk soil, the rhizosphere is rich in nutrients because of rhizodeposition. Rhizodeposits consist of the total carbon transferred from the plant root to the soil. They consist of small molecules as well as of macromolecules including enzymes, lysates from dead cells, and mucilage. The pH of this exudate is around 5.5 (Kamilova et al. 2006b). The result of this richness in nutrients is that the C. Pliego Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora”, Universidad de Málaga Consejo Superior de Investigaciones Cientı́ficas (IHSM-UMA-CSIC), Área de Genética, Universidad de Málaga, Campus de Teatinos s/n, 29071 Málaga, Spain and Present address: Imperial College London. Div. of Biology, Department of Life Science, Imperial College Road, London SW7 2AZ, UK e-mail: [email protected], [email protected] F. Kamilova Koppert Biological Systems, Veilingweg 14, PO Box 155, 2650 AD Berkel en Rodenrijs, the Netherlands e-mail: [email protected] B. Lugtenberg (*) Leiden University, Institute of Biology, Sylvius Laboratory, Sylviusweg 72, PO Box 9505, 2300 RA Leiden, the Netherlands e-mail: [email protected] D.K. Maheshwari (ed.), Bacteria in Agrobiology: Crop Ecosystems, DOI 10.1007/978-3-642-18357-7_11, # Springer-Verlag Berlin Heidelberg 2011 295 296 C. Pliego et al. bacterial concentration in the rhizosphere is 100- to 1,000-fold higher than that in bulk soil. Bacteria that are introduced in the rhizosphere should be able to cope with an environment which contains other microbes (bacteria, fungi, and viruses) and predators such as nematodes and protozoa. Also, the amount of available water changes, especially in case of irrigation, rain, and drought. When studying the behavior of a microbe in the rhizosphere, one should realize that exudate collected from a gnotobiotic rhizosphere allows bacteria to grow to only a 100-fold lower cell concentration than a laboratory medium, meaning that microbes in the rhizosphere are often in a state of nutrient starvation. In horticulture, plants are not only grown in soil but also in various other substrates such as cocopeat, stonewool, perlite, and vermiculite. 11.2 Organisms in the Rhizosphere The rhizosphere represents a highly dynamic site for interactions between roots, pathogenic and beneficial soil microbes, invertebrates, and other competitors of root systems. Interactions between organisms on the root may be classified as positive, neutral, or negative associations. Positive interactions, which can contribute to plant growth promotion and biological control of plant diseases, include symbiotic associations with epiphytes, mycorrhizal fungi, and root colonization by biocontrol agents (BCAs). Negative interactions include competition or parasitism among plants, pathogenesis by bacteria or fungi, and by invertebrate herbivores. Plant–microbe interactions in the rhizosphere are responsible for a number of intrinsic processes such as carbon sequestration, ecosystem functioning, and nutrient cycling. The composition and quantity of microbes in the soil influence the ability of a plant to obtain nitrogen and other nutrients. On the other hand, plants can influence these net ecosystems changes through release of secondary metabolites that attract or inhibit the growth of specific microorganisms (Lugtenberg and Bloemberg 2004; Vivanco et al. 2004). In addition, the rhizosphere is subject to dramatic changes on a short temporal scale, which can be caused by fluctuations in salt concentration, pH, osmotic potential, water potential, and soil particle structure. Over longer temporal scales, the rhizosphere can change due to root growth and interactions with other soil biota. Various bacterial species have adapted to the ever-changing conditions of the rhizosphere and are capable of starting root colonization by multiplication and eventually by forming microcolonies on different parts of the roots, from tip to elongation zone. Such microcolonies eventually grow on roots to form mature biofilms (Bloemberg and Lugtenberg 2004; Rudrappa et al. 2008). Nonpathogenic plant growth-promoting rhizobacteria (PGPR) associated with plant root surfaces are known to contribute toward increases in plant quality and yield by mechanisms such as improved mineral uptake, phytohormone production (Biswas et al. 2000; Ryu et al. 2003; Ryu et al. 2004; Fujishige et al. 2006), competitive suppression of 11 Plant Growth-Promoting Bacteria: Fundamentals and Exploitation 297 pathogens by production of antibiotics (Chin-A-Woeng et al. 1998; Keel et al. 1992; Haas and Défago 2005), and induction of secondary metabolite-mediated systemic resistance (Ryu et al. 2004; Van Loon 2007). Besides, bacterial biofilms established on plant roots can protect the colonization sites and act as a sink for the nutrients in the rhizosphere, thereby reducing the availability of nutritional elements in root exudates required for pathogen stimulation or subsequent root colonization (Weller and Thomashow 1994; Kamilova et al. 2005). In addition to the interaction with the plant, beneficial microorganisms interact and compete with the endogenous microflora, consisting of other bacteria, fungi, and/or mycorrhizal fungi. It is the dynamic nature of the rhizosphere that makes it an interesting setting for the interactions that lead to plant growth promotion and biocontrol of the disease. Significant biological control most generally arises from manipulating mutualisms between microbes and their plant hosts or from manipulating antagonisms between beneficial microbes and pathogens. To do this successfully, we need to know the players and understand their interactions with each other and with the growth substrate. Moreover, the effect of abiotic factors should be taken into account. The analysis of these interactions requires a strong interdisciplinary approach and an extensive set of tools. 11.3 Nutrients in the Rhizosphere Compounds released by the plant root vary in relative and absolute amounts. The composition is influenced by the plant species, plant cultivar and age, and environmental properties such as the presence of microbes and some of their products, the growth substrate, and the levels of physical, biological, and chemical stresses. It should be realized that an exudate composition usually reflects the net result of secretion, conversion by enzymes, and uptake by microbes and the plant root. An excellent monograph on the rhizosphere was published recently (Pinton et al. 2007). The bulk of root products are carbon compounds derived from products of photosynthesis. An excellent overview of exudate compounds is written by Uren (2007). Organic compounds released by the root include organic acids, sugars and polysaccharides, amino acids, fatty acids, sterols, vitamins, enzymes, and nucleotides. Moreover, compounds as diverse as auxins, ethanol, flavonoids, and the osmoprotectant glycinebetaine are secreted. In many cases, compounds are only detected because one screens for their presence. Exudate compounds can also be detected accidentally. Kuiper et al. (2001a) studied a competitive colonization mutant and the identification of the knocked out gene suggested that putrescine could be an exudate compound. Subsequent tests confirmed this notion. The rhizosphere is also an important place for microbe–plant and microbe–microbe signaling (Perry et al. 2007). Rhizobacteria (Kamilova et al. 2006b), fungi (Meharg and Killham 1995), and rhizobacterial products such as phenazine, 2,4 diacetylphloroglucinol and zearalenone (Phillips et al. 2004), as well as the growth substrate (Kamilova et al. 2006a; Lipton et al. 1987), can influence the exudate composition. 298 C. Pliego et al. Extensive studies have been carried out on the exudate composition of gnotobiotically grown tomato, cucumber, and sweet pepper. For all three crops, the organic acid fraction was larger than the carbohydrate fraction which, in turn, was larger than the amino acid fraction (Kamilova et al. 2006a). The observation that a mutant of Pseudomonas unable to utilize organic acids is a poor colonizer (De Weert et al. 2007) but a mutant in sugar utilization can colonize the tomato root normally (Lugtenberg et al. 2001) is consistent with the notion that organic acids are a major carbon source. The presence of a disease-causing amount of the pathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici in the gnotobiotic tomato system resulted in a decrease of the amount of citric acid and an increase in the amount of succinic acid in such a way that the total amount of organic acid was not influenced. In contrast, when the Pseudomonas fluorescens biocontrol strain WCS365 was added to the gnotobiotic tomato system in amounts sufficient to cause biocontrol, a strong increase in the total amount of organic acid, especially of citric acid, but a dramatic decrease in the amount of another organic acid, succinic acid, was found. When both microbes were present under biocontrol conditions, the amounts of succinic acid decreased but the amount of citric acid was similar to that of the untreated control. Under all three conditions, the amounts of sugars were reduced by around 50% (Kamilova et al. 2006b). In contrast to the three dicots cucumber, sweet pepper, and tomato, the monocot grass produces equal amounts of organic acids and sugars in its root exudate (Kuiper et al. 2002). Whether this difference reflects a fundamental difference in exudate composition between monocots and dicots remains to be established. Although root exudates play a crucial role in plant growth and health, studies on composition, the influence of environmental factors on composition, and function of root exudates have hardly been performed in the past 4 decades. Such studies should be considered as a challenge for future research. 11.4 Root Colonization Root colonization is crucial for the application of beneficial bacteria. Recently, technologies for following root colonization have been developed and traits and genes involved in colonization have been identified. 11.4.1 Recently Developed Technologies for Studying Interactions in the Rhizosphere Until recently, most research on biological control has focused on the interactions between two trophic levels: the BCA and the pathogen. However, multitrophic interactions among organisms belonging to three or more trophic levels are increasingly gaining the attention of microbial ecologists and plant pathologists, driven by 11 Plant Growth-Promoting Bacteria: Fundamentals and Exploitation 299 the need for improving biological control during the management of crop diseases. As we have mentioned previously, successful biocontrol operates at the population level, not at the level of an individual organism. Consequently, a detailed understanding of species interactions across at least three levels, the plant, the pathogen, and the BCA, seems to be necessary (Pliego et al. 2008). During the last years, the development of technologies such as the bioreporter technology has dramatically influenced our knowledge of biocontrol in the rhizosphere. Bioreporters can be classified into three categories (Lugtenberg and Leveau 2007). One class of bioreporters are those that allow direct visualization of individual organisms in vivo. The most common reporter used for this purpose is the green fluorescent protein (GFP), which does not depend on cofactors or additional substrates for activity. In combination with a constitutive promoter, the gfp gene is expressed independently of environmental factors and accumulation of its product renders the cells green fluorescent and detectable by fluorescence microscopy or confocal laser-scanning microscopy (CLSM). This has been exploited to study the direct visualization of the interactions established among various organisms, i.e., BCAs and pathogenic fungi. The second class allows monitoring the general well-being or activity of BCAs in the rhizosphere. Some groups have used the lux genes, which only results in bioluminescence when the cells are metabolically active, to show that the metabolic activity of Pseudomonas inoculants is higher in rhizosphere soil than in bulk soil and that metabolic activity decreases over time after introduction of the inoculated BCA (Porteous et al. 2000). The third group of bioreporters is aimed at providing information on specific activities of BCAs in the rhizosphere. The expression of the reporter gene is driven by promoter sequences that are selected based on their biological function. Besides gfp and lux, other reporter genes can be used for this, including lacZ, xylE, gusA, and inaZ (Loper and Lindow 1997). Finally, Transposon-Based-Strategies (TSBs) and the development of functional genomic techniques such as In Vivo Expression Technology (IVET), Signature Tagged Mutagenesis (STM), and DNA microarrays allow the identification of microbial genes potentially involved in biocontrol. Application of these techniques is largely increasing our knowledge of the molecular mechanisms involved in the interactions established among different trophic levels. 11.4.2 Autofluorescent Proteins (AFPs) Microscopic visualization of AFP-tagged BCAs in their natural environment during their interaction with plants and/or with target pathogens provides crucial information about their functioning and about the possible successful application of commercial inoculants. The GFP is the most widely studied AFP but the number of other proteins and variants with different fluorescence characteristics has increased. This also applies for the development of improved variants of existing AFPs. Important GFP derivatives are Cyan Fluorescent Protein (CFP) and Yellow Fluorescent Protein (YFP) (Yang et al. 1998; Tsien 1998; Matus 1999; Ellenberg et al. 1999). 300 C. Pliego et al. A wide range of autofluorescent proteins with useful information is available on the Web site of the Clontech company (http://www.clontech.com). Taking into account that genes encoding AFPs have to be transformed into the organism of interest, the use of these markers is dependent on whether appropriate vectors and transformation protocols are available for a specific species or strain. In bacteria, AFP genes are usually delivered using plasmids or transposons (Bloemberg 2007). The advantage of a plasmid is that it can be present in multiple copies, which can improve the level of AFP molecules and does not disrupt hosts genes by chromosomal integration (Bloemberg 2007). In recent years, AFP technology in combination with CLSM has become an important tool for the analysis of processes such as microbe–plant interactions, biosensor design, biofilm formation, and horizontal gene transfer (Larrainzar et al. 2005). Besides, the ability to use AFPs with different fluorescent spectra allows the simultaneous visualization of different species and populations at the same time. The use of red fluorescent protein (RFP), isolated from Discosoma striata, in combination with eGFP, is very suitable as the excitation and emission spectra of these proteins are well separated (Matz et al. 1999). Because of the functional limitations of DsRed such as slow maturation and low photostability, new and improved variants have recently been produced, of which mCherry is one of the best (Shaner et al. 2004). For example, Lagendijk et al. (2010) were able to distinguish simultaneously in the tomato rhizosphere the presence of mixed microcolonies originated from two different populations of P. putida PCL1445 cells tagged with EGFP and mCherry. Most of the studies using AFP as markers for BCAs have been directed to their localization in the rhizosphere. These studies have shown that individual bacteria grow out to microcolonies and form biofilms, usually at junctions between epidermal root cells and at sites where side roots emerge. These are supposed to be microhabitats with enhanced exudation, humidity, and mucigel (Lugtenberg and Bloemberg 2004). Studies using mixed populations of two Pseudomonas species, P. chlororaphis PCL1391 and P. fluorescens WCS365, revealed that mixed colonies were formed and are present mostly on the upper part of the root and that P. fluorescens WCS365 was found preferentially on root hairs (Lagopodi et al. 2002). These studies revealed that different bacteria can have a preference for colonizing different parts of the roots. The most significant studies related with the use of AFPs to study the interaction of Pseudomonas spp. with plant roots are given in Table 11.1. Epifluorescence studies also showed the close interaction established between rhizosphere fungi and bacteria. Using this technique, bacterial cells have been shown to attach and colonize the hyphae of beneficial and phytopathogenic fungi. Colonization of fungal hyphae by antagonistic bacteria has also been postulated to enhance biocontrol in combination with the bacterial production of antifungal metabolites such as antibiotics, chitinases, or proteases (Hogan and Kolter 2002; Bolwerk et al. 2003; Lagopodi et al. 2002). For example, the biocontrol strains Collimonas fungivorans, P. fluorescens WCS365, and P. chlororaphis PCL1391 have not only been shown to be excellent root colonizers but appeared 11 Plant Growth-Promoting Bacteria: Fundamentals and Exploitation 301 Table 11.1 Microscopic visualization of AFP-tagged Pseudomonas strains in the plant rhizospherea Pseudomonas strain System P. fluorescens WCS365 Tomato Localization Promoter:: gfp-version (Plac: gfp) P. chlororaphis Lotus Localization (PpsbA::gfp) P. fluorescens DR54-BN14 P. fluorescens WC5365 Barley Localization and activity Localization P. fluorescens SBW25 Wheat (PA1/03/04: gfpmut3b) (Plac::rfp; Plac:: egfp; Plac::ecfp; Plac::eyfp) (PpsbA-gfp luxAB) P. putida Barley P. fluorescens F113 Alfalfa Tomato P. fluorescens WCS365 Tomato and P. chlororaphis PCL1391 P. brassicacearum Arabidopsis thaliana Analyzed trait Metabolic activity and localization Activity (PrrnBP1::gfp Localization [AGA]) (PA1/03/04:gfpmut3 Localization (Plac:gfpS65T) Localization (Plac::rfp) Distribution (PA1/03/04: RBSIIgfpmut3) (PA1/03/04:RBSIIdsred) (PA1/03/04:gfpmut3) (Plac:gfp) P. putida P. fluorescens 32 Tomato Wheat Localization Localization P. fluorescens CHA0 Set of plant species Avocado Gene expression Tomato Localization P. pseudoalcaligenes and P. alcaligenes P. putida PCL1445 Distribution (PphlA-gfp3) (PprnA-gfp3) (PA1/03/04:gfp) (Ptac:gfp; Ptac: mcherry) Reference Bloemberg et al. (1997) Tombolini et al. (1997) Normander et al. (1999) Bloemberg et al. (2000) Unge and Jansson (2001) Ramos et al. (2000); Boldt et al. (2004) Villacieros et al. (2003) Bolwerk et al. (2003) Achouak et al. (2004) G€ otz et al. 2006 Van Bruggen et al. (2007) De Werra et al. (2008) Pliego et al. (2008) Lagendijk et al. (2010) a PpsbA, Plac, Ptac, PA1/03/04 (a Plac-derivative) are constitutive promoters; PrrnBP1 is a ribosomal promoter; PphlA contains the promoter region of the genes encoding the antifungal metabolite 2,4-diacetylphloroglucinol; PprnA contains the promoter region of the genes encoding the antifungal metabolite pyrrolnitrin; gfp, egfp, gfpmut3b, gfpmut3, and gfpS65T are genes encoding stable versions of the GFP protein; gfp[AGA] is a gene encoding an unstable version of the GFP protein; dsred and rfp are genes encoding derivatives of the GFP protein emitting red fluorescence; yfp is a gene encoding a yellow fluorescent protein; luxAB genes encode luciferase from Vibrio fischeri also to be able to colonize the hyphae of the phytopathogenic fungus F. oxysporum f.sp. radicis lycopersici (Forl) (Lagopodi et al. 2002; Bolwerk et al. 2003; Kamilova et al. 2007). Furthermore, abundant colonization of hyphae of the fungus Rosellina necatrix, the causal agent of avocado white root rot, was observed for the biocontrol strain P. pseudoalcaligenes AVO110 (Pliego et al. 2008). Moreover, the latter strain also colonized profusely pyriform swellings, characteristic structures of R. necatrix hyphae that seem to be involved in chlamydospore formation (PérezJiménez et al. 2003). Since fungi are frequently part of the endogenous microflora and can be the direct target for the PGPR effect as in case of biological control, it is also of great 302 C. Pliego et al. relevance to tag fungi and study their interaction with the PGPR (Bloemberg 2007). Genetic transformation of filamentous fungi is usually more difficult and less developed than transformation of bacterial cells. However, GFP expression vectors have already been developed for all major classes of filamentous fungi: basidiomycetes, ascomycetes, and oomycetes. The vector to be used depends on the fungus to be transformed. Transformation of filamentous fungi has traditionally been performed using protoplasts, e.g., F. oxysporum f.sp. radicis lycopersici has been tagged with different autofluorescent proteins by co-transformation of two plasmids, one of which contained a hygromycin resistance gene and the second an afp gene (Lagopodi et al. 2002; Bolwerk et al. 2003). Until now, several root-infecting fungal pathogens have been tagged with GFP to study developmental processes occurring during root infection, for example, Magnaporte grisea (Sesma and Osbourn 2004), Forl (Lagopodi et al. 2002), Fusarium verticillioides (Oren et al. 2003), and Rosellinia necatrix (Pliego et al. 2009). These studies have made significant contributions to the understanding of how BCAs function during their beneficial interactions with plants. For example, Lagopodi et al. (2002) observed that the biocontrol bacteria P. fluorescens WCS365 and P. chlororaphis PCL1391, applied to seedlings, and the pathogenic fungus Forl, applied in the soil, occupy the same sites on the tomato root mostly in the junctions between cells. Furthermore, the observation that the biocontrol strain occupies these sites faster that the pathogenic fungus presumably underlies a crucial aspect of the bacterium’s biocontrol ability (Bolwerk et al. 2003). More recently, CLSM studies revealed that the biocontrol strain P. pseudoalcaligenes AVO110 is able to control the avocado white root rot disease caused by R. necatrix by colonizing profusely the same sites as used by the pathogen to penetrate the root such as root wounds and intercellular spaces (Pliego et al. 2008). 11.4.3 Colonization Genes and Traits One of the major challenges for researchers is to understand the role of bacteria in their natural environment. One way to do this is to identify genes that are specifically expressed in natural surroundings. As mentioned earlier, efficient protection of plant roots by BCAs firstly requires sufficient and competitive colonization of the rhizosphere. In this regard, most studies have been directed to the identification of bacterial genes involved in rhizosphere colonization. Two different approaches were used to identify traits involved in root colonization and rhizosphere competence for the model strain P. fluorescens WCS365, one of the best colonizers among biocontrol strains (Lugtenberg et al. 2001). These studies were facilitated by the development of a gnotobiotic system developed by Simons et al. (1996) which offers a sterile environment for comparing the wild type with various mutants. The first approach deals with bacterial genes predicted to have an effect on colonization. These genes can be disrupted by directed mutagenesis using homologous recombination. Prior to studying their root colonizing abilities, growth rate 11 Plant Growth-Promoting Bacteria: Fundamentals and Exploitation 303 and other differences between mutant and wild type should be established. Using this approach, the importance of, for example, motility and chemotaxis (Lugtenberg and Bloemberg 2004) for root colonization was demonstrated. In addition, using cheA mutants of various P. fluorescens strains, De Weert et al. (2002) showed that the presence of flagella is not sufficient for competitive colonization but that chemotaxis in addition is essential. Motility is required for the chemotactic response of microbes toward exudates compounds as a first step in the process of root colonization (De Weert et al. 2007). Major chemoattractants in tomato root exudates for P. fluorescens WCS365 are dicarboxylic acids and amino acids. Based on concentrations estimated to be present in the tomato rhizosphere (Simons et al. 1997; Kravchenko et al. 2003), malic acid and citric acid were concluded to be the major chemoattractants. As a second approach, random transposon mutagenesis can be performed using transposons such as Tn5lacZ (Lam et al. 1990) or Tn5luxAB (Wolk et al. 1991). Selected mutants can be studied individually for impaired root colonization (Simons et al. 1996). Applying of the transposon approach resulted in the discovery of relevant traits for rhizosphere colonization. An overview of these traits is presented in Table 11.2. Reviews of most of these traits can be found in Lugtenberg and Dekkers (1999), Lugtenberg et al. (2001), De Weert et al. (2007), and Lugtenberg and Kamilova (2009). Functional genomic techniques allow the simultaneous in vivo examination of the expression of many genes. These techniques can be divided into three broad Table 11.2 Functions of Pseudomonas genes required for root colonization Function Gene References Motility Involved in biosynthesis of flagella De Weger et al. (1987) Pilus retraction pilT Camacho Carvajal (2001) Chemotaxis cheA De Weert et al. (2002) Cell wall structure rhs De Weert et al. (2007) Synthesis of O-antigen Not identified Dekkers et al. (1998) of LPS Outer membrane integrity Homolog to hrtB Dekkers et al. (1998) Permeability of outer colR/colS required for expression De Weert et al. (2006) membrane of wapQ Increased pyrimidine pyrR Camacho Carvajal biosynthesis (2001) Utilization of exudate Homolog of mqo encoding malate Lugtenberg et al. (2001) compounds dehidrogenase De Weert and Homolog of cis-aconitate hydratase Bloemberg (2007) Downregulation of Binding site for regulator protein Kuiper et al. (2001a) putrescine uptake Phase variation xerC/sss Dekkers et al. (1998) Proton motive force nuo operon Camacho Carvajal et al. (2002) Secretion pathways secB, homolog of hrcC, hrcD encoding De Weert et al. (2007) components of the TTSS Adaptation to certain Homolog of mutY De Weert et al. (2004a) environments 304 C. Pliego et al. classes (1) IVET, (2) STM, and (3) transcriptomics, i.e., global gene expression analysis using DNA microarrays. IVET technology is a promoter-trap strategy designed to identify genes whose expression is induced in a specific environment, typically that encountered in a host. This technique was, for example, used to identify genes of P. fluorescens specifically expressed within the sugar beet rhizosphere (Rainey 1999; Gal et al. 2003) and to analyze gene expression in P. putida during colonization of the maize rhizosphere (Ramos-González et al. 2005). Rhizosphere-induced fusions identified genes with probable functions in nutrient scavenging, chemotaxis and motility, intracellular metabolism, regulation, secretion, cell envelope structure, nucleic acid metabolism and stress. One of the most important findings was the discovery of the hrC gene, encoding a component of the Type Three Secretion System (TTSS) of P. fluorescens (Rainey 1999), suggesting that the TTSS has functional significance in both pathogens and PGPR. Although TTSS in P. fluorescens was demonstrated to be functional, a hrcC mutation did not affect root colonization capacity (Preston et al. 2001). On the other hand, De Weert et al. (2007) compared the competitive root-colonizing abilities of both hrcD and hrcD genes of the TTSS of P. fluorescens SBW25. It appeared that both mutants were impaired in competitive tomato root tip colonization but were not impaired when analyzed alone on the root. They hypothesized that the mutants are using the TTSS for either involvement in attachment to seed and/or root or for feeding on the root surface cells by means of the TTSS. Because no difference was found in attachment to seeds or roots, in competition with the wild type, they suggested that the TTSS is not only used to inject proteins into plant cells but also to suck nutrients from the plant cell (De Weert et al. 2007). These results support the finding that biocontrol activity of P. putida against Pythium ultimum on cucumber was lower that that of the wild type when the hrcC gene was mutated (Rezzonico et al. 2004). Interestingly, application of IVET technology also led to the identification of the P. fluorescens wssE gene, being part of the wss operon involved in the synthesis of acetylated cellulose polymers (Rainey and Preston, 2000; Gal et al. 2003). The wssE gene encodes a cellulose synthase subunit. Acetylated cellulose polymers are known to play a role in colony development and bacterial cell–cell contact, which are known to be involved in the initial steps of biofilm development (Spiers et al. 2003). Mutational analysis of the wss operon has shown that it contributes to ecological fitness in the rhizosphere. Besides, an increased expression of oxidative stress genes (glutathione peroxidases and a paraquat inducible protein) was observed in P. fluorescens for protection against oxidative stress encountered in the sugar beet rhizosphere (Gal et al. 2003). In the functional genomic technique STM, the use of DNA signature tags facilitate functional screens by identifying mutants in mixed populations that have a reduced or increased adaptation to a particular environment. Besides, STM allows the identification of fitness or competition mutants, usually missed by standard mutagenesis approaches, which do not cause a total phenotype knockout. This technique was recently applied in Burkholderia vietnamiensis strain G4 to identify genetic determinants involved in colonization of the pea rhizosphere and in 11 Plant Growth-Promoting Bacteria: Fundamentals and Exploitation 305 phenol degradation. Seventy mutants involved in rhizosphere colonization were mapped to genes with the following putative functions: amino acid biosynthesis (36%), general metabolism (26%), transport and stress (2.8%), regulatory genes (5.7%), and hypothetical proteins (13%) (O’Sullivan et al. 2007). The majority of the mutants were associated with amino acid biosynthesis and metabolism. Amino acids are known to be present in root exudates but at limiting concentrations and not readily accessible (Lugtenberg et al. 2001). Several amino acid auxotrophs were also found for P. fluorescens WCS365 among competitive colonization mutants. Even when tested alone, these mutants were unable to colonize the root tip (Lugtenberg and Bloemberg 2004). One of the most interesting discoveries mediated by the rhizosphere-STM screen was the identification of three mutants, inactivated within a single virulenceassociated autotransporter adhesion gene (O’Sullivan et al. 2007). This mutation consistently produced a hypercolonization phenotype. They suggests that this massive surface-exposed protein (229–368 Da) masks part of the surface adhesins that are actually responsible for plant root cell binding, hence these protein adhesins would be more freely available for binding to plant cells in these three mutants. Alternatively, this adhesion could also play a role in the plant host response, in a way analogous to the way the known immunogenicity YadA homologs act in animal hosts (Cotter et al. 2005). Therefore, it may be possible that the plant is more tolerant to the adhesin mutants and allows them to colonize in greater numbers (O’Sullivan et al. 2007). Using a transposon mutant library of the efficient colonizer P. fluorescens WCS365 in combination with the enrichment procedure developed by Kuiper et al. (2001a), De Weert and co-workers (2004a) observed that competitive root tip colonization can be enhanced. The best colonizing mutant was shown to be mutated in a mutY homolog. This gene is involved in the repair of A:G mismatches caused by spontaneous oxidation of guanine. Since these mutants are defective in repairing their mismatches, it is hypothesized that such cells harbor an increased number of mutations and that some of these mutants, with a beneficial combination of mutations, can adapt faster to the environment of the root system. In order to investigate how Pseudomonas populations readjust their genetic program upon establishment of a mutualistic interaction with plants, Matilla et al. (2007) performed a genome-wide analysis of gene expression using microarrays of the root-colonizing bacterium P. putida KT2440. Genes involved in amino acid uptake and metabolism of aromatic compounds were found to be preferentially expressed in the rhizosphere (22%). It was also found that bacterial efflux pumps and enzymes related with glutathione metabolism were induced in the rhizosphere, indicating again that adaptation to adverse conditions and to oxidative stress is crucial for bacterial life in this environment. A gene encoding a protein containing GGDEF/EAL domains was identified among the induced genes. These domains are involved in c-di-GMP metabolism. Numerous studies have shown that c-di-GMP regulates biofilm formation and motility. Moreover, c-di-GMP also activates the production of extracellular polysaccharides (EPS), specifically those that serve as an extracellular matrix for formation and support of biofilm architecture 306 C. Pliego et al. (Tamayo et al. 2007). Results shown by Matilla et al. (2007) suggest a role for the turnover of c-di-GMP in root colonization. Besides, genes encoding flagellar proteins and chemotaxis proteins were also upregulated in the interaction of P. putida KT2440 with maize roots, confirming a role of chemotaxis in root colonization. 11.5 Bacteria Which Can Promote Plant Growth Directly Some beneficial bacteria can promote plant growth in the absence of a pathogen. They will be discussed here. 11.5.1 Introduction Some rhizobacteria are plant beneficial and can promote plant growth directly, e.g., by providing the plant with nutrients or growth hormones, whereas others can promote plant growth indirectly, e.g., by decreasing the growth of pathogens. Direct plant growth promotion will be discussed in this section whereas indirect plant growth promotion will be treated in the next section. Direct plant growth promoters can be divided into four classes: biofertilizers, rhizoremediators, phytostimulators or plant growth regulators, and stress controllers. 11.5.2 Biofertilizers Biofertilizers are best known for their ability to provide the plant root with nutrients such as nitrogen, phosphorous, and iron. Biological nitrogen fixation is an extremely energy intensive process. The reduction of one molecule of N2 catalyzed by the nitrogenase enzyme complex requires between 16 and 42 molecules of ATP. It is therefore not surprising that N2-fixing bacteria only reduce N2 when they are starved for nitrogen (De Bruijn et al. 1990). Rhizobium and Bradyrhizobium bacteria can induce special organs, so-called root nodules on the roots of leguminous plants in which they, in the form of modified bacteria called bacteroids, fix atmospheric nitrogen and convert it into ammonia which can be utilized by the plant as a nitrogen source. In return, the plant provides the bacterium with a carbon source, probably malate. Both rhizobia are economically important. Bradyrhizobium is important in agriculture as it can nodulate soybean. Rhizobium can nodulate crops such as pea, peanut, and alfalfa. Egamberdieva et al. (2010) recently showed that co-inoculation of fodder galega with Rhizobium and biocontrol pseudomonads improves shoot and root dry matter of the plant. One of these strains, the cellulose-producing P. trivialis strain 3Re27, 11 Plant Growth-Promoting Bacteria: Fundamentals and Exploitation 307 significantly increased nodule numbers and nitrogen content of the co-inoculated plant (Egamberdieva et al. 2010). The authors coined the term “rhizobium helper bacteria” for this biocontrol strain. Klebsiella pneumonia and Azospirillum are free-living nitrogen-fixing rhizosphere bacteria. In the past, the plant growth-promoting properties of Azospirillum were thought to be due to its N2-fixing property, but recent developments show that this property is mainly due to its ability to produce the root architecture influencing hormone auxin. Therefore, Azospirillum will be discussed under “Phytostimulators.” In some soils, phosphorous is the limiting factor for plant growth. Some bacteria are able to solubilize bound phosphorous from organic or inorganic molecules, thereby making it available for the plant (Lipton et al. 1987; Vassilev et al. 2006). Production of organic acids such as gluconic acid is a major factor in the release of phosphorous from mineral phosphate (Rodrı́guez et al. 2006). All organisms need iron ions for their metabolism. Iron in the form of ferric hydroxide is an abundant element on the earth crust but it is insoluble and therefore not suitable for uptake by living organisms. The concentration of Fe3+, the form of iron ions available for living organisms, is only 1018 M. Therefore, bacteria growing under low Fe3þ concentrations produce a variety of siderophores, which are Fe3þ chelators able to bind this ion with high affinity. Subsequently, the Fe3þ-siderophore complex is bound to specific iron starvation induced bacterial cell surface receptors and the Fe3þ ion is transported into the cell’s interior, where it, in the form of Fe2þ, is involved in metabolic processes (Neilands 1981; Leong 1986). Competition for ferric iron ions is a well-documented example of competition of biocontrol bacteria with pathogenic fungi for nutrients (Leong 1986; Lugtenberg and Bloemberg 2004). In the rhizosphere, there is often a limitation of solubilized Fe3þ which, in many microorganisms, is solved by the production of siderophores, i.e., Fe3þ-binding ligands, which have a high affinity to sequester iron from the microenvironment. The relevance of siderophore production as a mechanism of biological control of Erwinia carotovora by P. fluorescens strains was first described by Kloepper et al. (1980). As with antibiotics, mutants not producing siderophores such as pyoverdine showed a reduced capacity to suppress different plant pathogens (Keel et al. 1989; Loper and Buyer 1991; Duijff et al. 1994). This phenomenon is not only a form of plant fertilization but also of biocontrol (see Sect. 6.2.3). 11.5.3 Rhizoremediators Pollution of soil and water is an enormous problem worldwide. In the USA alone, the costs of restoration of all contaminated sites are estimated at 1.7 trillion USDs. The major approaches to solve these problems are incineration and removal of land. However, these approaches are not safe. Incineration results in air pollution and leaches from landfills can reach ground water and drinking water wells. Microbes at a site that is being polluted adapt to this situation and start degrading the pollutants. However, the degradation rate is very low. Scientists have isolated the microbes 308 C. Pliego et al. responsible for this process. For most contaminants it is possible to find microbes which can degrade them. The application of such microbes has been tested for decades. However, strains which were successful in the laboratory failed in soil. Due to lack of nutrients they go in a starvation mode soon after application and stop degrading the pollutants. Apparently, they are unable to multiply on the pollutants as their only carbon source. In order to degrade the pollutant molecules they have to co-metabolize it together with a regular C source (Cases and De Lorenzo 2005). With the notion, that it is possible to find bacteria able to solve almost any problem, Kuiper et al. (2001b) selected bacteria which combine two properties, namely (1) to utilize root exudate – the best carbon source available in soil and (2) are also able to degrade pollutants. Grass was used as the exudate source because the roots of some grasses can grow as deep as 5 m into the soil. The polyaromatic hydrocarbon compound naphthalene was chosen as the model pollutant. Starting with the crude rhizosphere of the roots, presumably containing a mixture of hundreds of thousands different rhizobacteria, the mixture was grown first on naphthalene as the only carbon source. The naphthalene users were subsequently coated on grass seeds, which were allowed to grow until the roots were 10 cm in length. Subsequently, the bacteria residing on the last centimeter of the root tip, i.e., the best root colonizers, were collected. This cycle was repeated twice (SeeFig. 11.1 for the principle of the enrichment procedure). In this way, strain P. putida PCL 1444 was isolated (Kuiper et al. 2001b). This strain combines the following properties (1) stable degradation of naphthalene; (2) efficient utilization of grass root exudate; (3) protection of coated grass seeds from poisoning by naphthalene, resulting in healthy plants. Mutants unable to degrade naphthalene are inactive in this respect; (4) roots are able to move bacteria through layers through which bacteria normally cannot penetrate (Kuiper et al. 2001b; Kuiper et al. 2002; Kuiper et al. 2004). In addition to rhizosphere bacteria, endophytes are often used in rhizoremediation. Reports on the ability of several endophytic bacteria to degrade some pollutants (i.e., explosives, herbicides, or hydrocarbons) have been published (Van Aken et al. 2004; Germaine et al. 2006; Phillips et al. 2008; Segura et al., 2009). Also, endophytic bacteria resistant to high concentrations of heavy metals, benzene, toluene, ethylbenzene and xylenes (BTEX), trichloroethylene (TCE), or polyaromatic hydrocarbons (PAHs) have been identified (Moore et al. 2006; Doty 2008). 11.5.4 Phytostimulators/Plant Growth Promoters The plant can produce phytohormones, i.e., compounds which can regulate plant growth and development. There are five classes of plant hormones, namely auxins, cytokinins, gibberellins (GAs), abscisic acid (ABA), and ethylene (ET). See Fig. 11.2 for structures. They are usually effective at concentrations lower than 1 mM (Garcı́a de Salome et al. 2006). In addition to plants, many rhizosphere 11 Plant Growth-Promoting Bacteria: Fundamentals and Exploitation 309 Fig. 11.1 Enrichment of bacteria which compete efficiently for nutrients and niches. The principle of the enrichment procedure was published by Kuiper et al. (2001b). Starting from a seed on which a crude mixture of rhizosphere bacteria has been applied, enhanced competitors are selected by repeatedly selecting those bacteria which reach the root tip first after application to a sterile seed. Strains isolated from the root tip after three cycles are subsequently tested on antibiotic activities toward pathogens, which results in (1) strains which are not antagonistic and easy to register as a product (Kamilova et al. 2005) or (2) in strains which have at least two mechanisms of biocontrol, namely CNN and antibiosis (Pliego et al. 2007; Pliego et al. 2008) bacteria can produce plant growth regulators. The first three classes of plant hormones will be discussed under “phytostimulators/plant growth promoters” whereas the last two classes will be discussed under “stress controllers.” For details on hormones produced by plants and rhizosphere bacteria, the reader is referred to excellent reviews by Spaepen et al. (2009) and Garcı́a de Salome et al. (2006). 310 C. Pliego et al. O COOH C NH2 OH N N N N N N CH3 Phenazine-1-carboxylate O Phenazine-1-carboxamide Pyocyanin O OH NH CH3 H3C HO OH Cl HO CL O OH NO2 HN Cl Cl 2,4-Diacetylphloroglucinol Pyrrolnitrin Pyoluteorin CH3 CH2 CH2 CH2 CH2 CH2 NH2 O HO O H N O HO N H N H N OH O O O HN NH O OH H H CH2 O HN O CH2 N H Viscosinamide OH O N H OH O CH3 2-hexyl-5- propyl resorcinol O O N H H AHL structure Fig. 11.2 (Continued) O H C N Hydrogen cyanide 11 Plant Growth-Promoting Bacteria: Fundamentals and Exploitation O 311 OH OH CO H CH2 HO O COOH N H CH3 Gibberellins (GA3) Auxins (IAA) HO H3C NH H3C N N N CH3 CH3 OH N H CH3 O Zeatin O OH Abscisic acid OH O OH OH OH H2C CH2 Ethylene OH OH D-gluconic acid Fig. 11.2 Structures of important metabolites which play a role in the interaction between plantbeneficial bacteria and plants 11.5.4.1 Auxins Auxins are involved in several processes such as establishment of polarity in embryogenesis, cell elongation, vascular differentiation, floral and fruit development, lateral root formation, and determination of root and shoot architecture. Indole-3-acetic acid (IAA) (Fig. 11.2) is the most abundant member of the auxin family. It has been estimated that as much as 80% of the rhizosphere bacteria can synthesize IAA (Khalid et al. 2004; Patten and Glick 1996). Rhizosphere bacteria use several different pathways for IAA biosynthesis. Most of them use tryptophan 312 C. Pliego et al. secreted in the rhizosphere as a precursor (Costacurta and Vanderleyden 1995; Spaepen et al. 2009). Indeed, Kamilova et al. (2006a) observed that P. fluorescens biocontrol strain WCS365, which can produce IAA in the presence of tryptophan, is able to stimulate root growth of radish, a plant which secretes high amounts of tryptophane, but not of tomato, sweet pepper, or cucumber, plants which secrete at least tenfold less tryptophan in the growth medium. Azospirillum brasilense is an N2 fixer, which promotes plant growth by increasing its root surface through shortening the root length and enhancing root hair formation. It has been thought for a long time that its plant growth-promoting ability was based on N2 fixation. However, the present notion is that auxin production is the major factor responsible for its root changes and therefore for its plant growth-promoting properties. This notion is based on the following observations (1) Dobbelaere et al. (1999) showed that the effect of the wild-type strain on the root can be mimicked by the addition of pure auxin. (2) A mutant strain strongly reduced in IAA production did not induce the root changes. (3) A strain constitutive for IAA production showed the same effect on the root changes as the wild-type strain but already at lower bacterial cell concentrations (Spaepen et al. 2008). Interestingly, when the amount of root exudate becomes limiting for growth, A. brasilense cells increase their IAA production, thereby triggering lateral root and root hair formation, which results in more exudation and therefore in further bacterial growth. In this way, a regulatory loop is created which connects plant root proliferation with bacterial growth stimulation (Spaepen et al. 2009). 11.5.4.2 Cytokinins Cytokinins consist of a group of molecules, of which zeatin is the major representative (Fig. 11.2). These compounds have the capacity to induce division of plant cells in the presence of auxin. Plants use ADP and ATP as substrates for cytokinin biosynthesis while bacteria use AMP. In all cases, an isopentenyl side chain is incorporated at the N6 position of the adenine ring. The balance between the amounts of auxin and cytokinin determines whether root or shoot differentiate from callus tissue. High auxin promotes root differentiation whereas high cytokinin promotes shoot morphogenesis. Equimolar concentrations induce cell proliferation. Cytokinins are synthesized in root tips and developing seeds. They are transported to the shoot where they regulate important processes such as chloroplast development, leaf expansion, and delay of senescence. Exogenous application of cytokinins to the plant can cause profuse lateral branching. Many rhizosphere bacteria can produce cytokinins, e.g. Agrobacterium, Arthrobacter, Bacillus, Burkholderia, Erwinia, Pantoea agglomerans, Pseudomonas, Rhodospirillum rubrum, Serratia, and Xanthomonas (Garcı́a de Salome et al. 2001). The spectrum of cytokinins produced by rhizobacteria is similar to that produced by the plant (Barea et al. 1976; Garcı́a de Salome et al. 2001; Frankenberger and Arshad 1995), of which isopentenyladenine, trans-zeatin, cis-zeatin, and their 11 Plant Growth-Promoting Bacteria: Fundamentals and Exploitation 313 ribosides are the most commonly found. Concerning the mechanism of action, one speculates that cytokinin produced by rhizosphere bacteria becomes part of the plant cytokinin pool, and thus influences plant growth and development. Evidence for a role of cytokinin of rhizosphere bacteria in plant growth promotion has been published. Garcı́a de Salome et al. (2001) have produced mutants of P. fluorescens strain G20–18 which produce reduced amounts of cytokinin and normal amounts of auxin. In contrast to the wild-type strain, the mutants are unable to promote growth of wheat and radish plants (Garcı́a de Salome 2000). Garcı́a de Salome and Nelson (2000) showed that cytokinin production is linked to callus growth of tobacco and suggested that this test can be used as a screening method for cytokinin-producing bacteria. For the pathogen Agrobacterium tumefaciens, the ability to produce auxins and cytokinins is a virulence factor. The strain produces crown galls. The genes for production of auxins and cytokinins are transferred to the plant and incorporated in its DNA (Spaink et al. 1998). Another bacterium from this genus, A. rhizogenes, modifies cytokinin metabolism causing the appearance of masses of roots instead of callus from infection site (Hamill 1993). 11.5.4.3 Gibberellins (GAs) These hormones consist of a group of terpenoids with 20 carbon atoms, although active GAs only have 19 carbon atoms (Fig. 11.2). This group of compounds consists of over 120 different molecules. GAs are mainly involved in cell division and cell elongation within the subapical meristem, thereby playing a key role in internode elongation. Other processes affected by these hormones are seed germination, pollen tube growth, and flowering in rosette plants. Like auxins and cytokinins, GAs mainly act in combination with other hormones. Hardly anything is known about gibberellin synthesis in rhizosphere bacteria. Bacteria that produce gibberellins, such as A. brasilense, A. lipoferum, Bacillus species, Bradyrhizobium japonicum, and Rhizobium phaseoli, secrete them in the rhizosphere (Frankenberger and Arshad 1995; Gutiérrez Manero et al. 2001; Rademacher 1994). The mechanism of plant growth stimulation by gibberellins is still obscure. Fulchieri et al. (1993) speculate that gibberellins increase root hair density in root zones involved in nutrient and water uptake. 11.5.5 Stress Controllers Plants can be subject to stress. Bacteria which can decrease plant stress are treated here. 314 11.5.5.1 C. Pliego et al. Abscisic Acid (ABA) ABA is a 15-carbon compound (Fig. 11.2) and, like ethylene, it is involved in plant responses to biotic and abiotic stresses. It inhibits seed germination and flowering. It is involved in protection against drought, salt stress, and toxic metals. It also induces stomatal closure. ABA can be produced by several bacteria such as A. brasilense (Cohen et al. 2008) and B. japonicum (Boiero et al. 2007). The effect of inoculation with ABAproducing bacteria on plant growth is experimentally poorly underpinned. Since ABA inhibits the synthesis of cytokinins (Miernyk 1979) it was speculated that ABA increases plant growth by interfering with the cytokinin pool (Spaepen et al. 2009). It could also alleviate plant stress by increasing the root/shoot ratio (Boiero et al. 2007). 11.5.5.2 Ethylene (ET) Ethylene, the gaseous hormone (Fig. 11.2), is best known for its ability to induce fruit ripening and flower senescence. It generally inhibits stem elongation in most dicots favoring lateral cell expansion and leading to swelling of hypocotyls. However, it promotes growth in submerged aquatic species. ET also breaks seed and bud dormancy. ET is synthesized under biotic stress conditions following infection by pathogens, as well as by abiotic stress conditions such as drought. It is therefore also known as the stress hormone. In the plant, ethylene is produced from S-adenosylmethionine (SAM) which is enzymatically converted to 1-aminocyclopropane-1-carboxylate (ACC) and 50 -deoxy-50 methylthioadenosine (MTA) by ACC synthase. The enzyme ACC deaminase is present in many rhizosphere bacteria. Such bacteria can take up ACC secreted by the plant root and convert it into a-ketobutyrate and ammonia. This results in the decrease of ACC levels, and therefore also of ethylene levels in the plant and in decreased plant stress. Inoculation of plants with ACC deaminase producing bacteria can protect plants against stress caused by flooding, salination, drought, heavy metals, toxic organic compounds, and pathogens (Glick 2005; Glick et al. 2007a; Glick et al. 2007b; Belimov et al. 2005). In addition to a direct role of ethylene on plant growth, this hormone can also act as a virulence factor and a signaling molecule in plant protection against pathogen attack. Ethylene production was reported to act as a virulence factor for bacterial pathogens, e.g., P. syringae (Weingart and Volksch 1997; Weingart et al. 2001). Furthermore, ethylene acts as a signaling compound in induced systemic resistance (ISR) caused by some rhizobacteria (Van Loon 2007). 11.6 Bacteria Which Can Control Plant Diseases Many plant diseases are caused by fungi. Some beneficial bacteria can reduce such diseases. 11 Plant Growth-Promoting Bacteria: Fundamentals and Exploitation 315 11.6.1 Introduction to Biocontrol Microbes The story of microbes which can protect plants against pathogens begins with the discovery by Schroth and Hancock (1982) of so-called suppressive soils. In these soils, pathogens can be present without causing diseases to the normally susceptible plants. These soils contain microbes which protect the plant from the pathogen. When the disease-suppressing microbe is absent we speak about “conducive soils” in which pathogens can cause diseases. A small amount of suppressive soil can, when mixed with conducive soil, make the latter suppressive. Many of the disease-suppressive microbes that have been isolated belong to the genera Pseudomonas and Bacillus. Fluorescent pseudomonads can easily be detected on Fe3þ poor medium because, under Fe3þ limiting conditions, e.g., in Kings medium B (King et al. 1954), they secrete a siderophore, i.e., a molecule which sequesters Fe3þ. In the case of fluorescent pseudomonads, (one of) the siderophore(s) is pyoverdin (Fig. 11.2). 11.6.2 Mechanisms of Biocontrol Researchers have focused on characterizing the mechanisms involved in biocontrol operating under different experimental situations. In all cases, pathogens are antagonized by the presence and activities of other organisms they encounter (Haas and Défago 2005; Pal and Gardener 2006; Alabouvette et al. 2006; De Weert and Bloemberg 2007). The most important pathogens are fungi, but there are also some pathogenic bacteria and nematodes. The modes of action of beneficial microorganisms can be based on either a direct or an indirect antagonism. However, both mechanisms are not mutually exclusive as they have been frequently described as co-occurring within the activity of the same BCA (Castoria and Wright 2009). Direct antagonism results from physical contact and/or from a high degree of selectivity of the mechanism(s) expressed by the BCA(s), in relation to the pathogen, i.e., parasitism and predation, production of antibiotics, and signal interference. In contrast, indirect antagonism results from activities that do not involve sensing or targeting of a pathogen by the BCA(s), i.e., competition for nutrients and niches, production of siderophores, and ISR. 11.6.2.1 Antibiosis Antibiotics produced by microorganisms have been shown to be particularly effective in suppressing plant pathogens and diseases. Most biocontrol strains of Pseudomonas spp. with a proven effect in plant bioassays produce one or several antibiotic compounds, e.g., P. fluorescens strains CHAO (Laville et al. 1998; Haas et al. 2000) and Pf-5 (Thompson et al. 1999) produce complex cocktails of 316 C. Pliego et al. these secondary metabolites. In vitro, these antibiotics inhibit the growth of the fungal pathogen, and for this reason, strains acting through antibiosis are usually identified by screening them for antagonistic activity on plates on which the target pathogen is also inoculated (Lugtenberg and Bloemberg 2004). The contribution of antibiotics to biological control of root disease is documented in five experimental steps (1) Purification and chemical identification of the antibiotic compound. (2) Detection and quantification in the rhizosphere of the secondary metabolite. (3) Identification and characterization of the structural and principal regulatory genes controlling the expression of the antibiotic compound. (4) Poor biocontrol strains can acquire biocontrol activity by the introduction of antibiotic biosynthetic genes not present in the original strain. (5) Detection of the expression of the antibiotic biosynthetic genes through the use of detectable reporter genes fused to structural genes for antibiotic biosynthesis (Haas and Keel 2003).Well-characterized antibiotics (Fig. 11.2) with biocontrol properties include phenazines (Phz), phloroglucinols (Phl), pyoluteorin, pyrrolnitrin, hydrogen cyanide (HCN), cyclic lipopeptides (Perneel et al. 2008; Keel et al. 1992; Thomashow and Weller 1988; Haas and Keel 2003; Raaijmakers et al. 2006), and the most recently discovered 2-hydroxymethyl-chroman-4-one (Kang et al. 2004), D-gluconic acid (Kaur et al. 2006), and 2-hexyl-5-propyl resorcinol (HPR) (Cazorla et al. 2006). Phenazines are analogs to flavin coenzymes, inhibiting electron transport, and are known to have various pharmacological effects on animal cells (Ran et al. 2003). 2-4-Diacetylphloroglucinol, the best known Phl compound, causes membrane damage to Pythium spp. and is particularly inhibitory to zoospores of this oomycete (De Souza et al. 2003). Gleeson et al. (2010) provided evidence that Phl acts through impairing the function of mitochondriae. Dikin et al. (2007) reported that pyrrolnitrin causes the loss of mitochondrial activity in the fungal cytoplasm, inhibiting succinate oxidase and NADH-cytochrome reductase. Pyrrolnitrin also interferes with cellular processes such as oxidative stress, blockage of electron transport as well as inhibition of DNA and RNA synthesis (Dikin et al. 2007). The cyanide ion derived from HCN is a potent inhibitor of many metalloenzymes, especially copper-containing cytochrome c oxidases (Blumer and Haas 2000). Finally, cyclic lipopeptides have surfactant properties and are able to insert themselves into membranes and perturb their function, resulting in broad antibacterial and antifungal activities (Haas and Défago 2005; Perneel et al. 2008). Recently, Mazzola et al. (2009) have shown that in the wheat rhizosphere the cyclic lipopeptides viscosin and massetolide not only protect the plant against fungi but also against protozoan predation. In fact, the protozoa Naegleria americana derepresses the syntheses of these antibiotics. To our knowledge, no mode of action has been described for pyoluteorin, HPR, and D-gluconic acid. The syntheses of antifungal metabolites (AFMs) are extremely sensitive to environmental conditions in the rhizosphere, e.g., soil mineral content, oxygen tension, osmotic conditions, carbon sources, as well as fungal, bacteria, and plant metabolites can all influence the expression of secondary metabolites (Haas and 11 Plant Growth-Promoting Bacteria: Fundamentals and Exploitation 317 Keel 2003; Lugtenberg and Bloemberg 2004; Duffy and Défago 1999; Van Rij et al. 2005). 11.6.2.2 Predation and Parasitism Soilborne bacteria and fungi are able to produce extracellular enzymes such as chitinases, ß-1-3 glucanases, lipases, cellulases, and proteases. These lytic enzymes can hydrolyze a wide variety of polymeric compounds, including chitin, proteins, cellulose, hemicelluloses, interfering with pathogen growth and/or activities. Production and secretion of these enzymes by different microbes can result in biocontrol abilities (Markowich and Kononova 2003). Ordentlich et al. (1998) showed that chitinase of Serratia marcescens is involved in the biocontrol of Sclerotium rolfssi. Beta-1,3-glucanase contributes significantly to the biocontrol activities of Lysobacter enzymogenes strain C3 against Bipolaris leaf spot caused by Phytium spp. (Palumbo et al. 2005). Chitinases of the mycoparasitic Trichoderma species have also been shown to play an important role in biocontrol and antagonistic activity against phytopathogens (Harman et al. 2004), including R. necatrix (Hoopen and Krauss 2006). Sometimes, these enzymes act synergistically with antibiotics playing an important role in the antagonistic effect on phytopathogenic fungi (Schirmb€ ock et al. 1994; Fogliano et al. 2002). Predation and parasitism are not always related to biocontrol, as it is the case for the fungus eater C. fungivorans, of which the major mechanism of action for controlling tomato foot and root rot (TFRR) probably is competition for space and nutrients and niches (Kamilova et al. 2007). 11.6.2.3 Competition for Nutrients and Niches To successfully colonize the rhizosphere, a microbe must effectively compete for the available nutrients. Competition for nutrients and niches (CNN) between pathogens and beneficials has been shown to be important for limiting disease incidence and severity (Kamilova et al. 2005). Enrichment for enhanced competitive tomato root tip colonizers was used to select for bacteria, not producing antibiotics, which control TFRR by CNN (Fig. 11.1) (Kamilova et al. 2005). The enhanced competitive root tip colonizers grow efficiently on root exudates. However, Kamilova et al. (2005) observed that these two characteristics were not sufficient for biocontrol, as one of the best competitive root-tip-colonizing strains did not control TFRR. A similar enrichment procedure was used by Pliego et al. (2007) for the isolation of competitive avocado root tip colonizers strains displaying biocontrol ability against R. necatrix. Two Pseudomonas sp. strains, P. pseudoalcaligenes AVO110 and P. alcaligenes AVO73, were selected for their efficient colonization abilities. However, only AVO110 demonstrated significant protection against avocado white root rot. Further analysis revealed that both strains colonize different sites on the 318 C. Pliego et al. root: biocontrol strain AVO110 was observed to colonize the root at preferential penetration sites for R. necatrix infection (intercellular crevices between neighboring plant root epidermial cells and root wounds) while P. alcaligenes AVO73 was predominantly found forming dispersed microcolonies over the root surface and in the proximity of lateral roots, areas not colonized by this pathogen (Pliego et al. 2008). These results strongly suggest that biocontrol bacteria acting through CNN must efficiently colonize the same mini-niche as the pathogen. Competition for ferric iron ions is a well-documented example of competition of biocontrol bacteria with pathogenic fungi for nutrients (Leong 1986, Lugtenberg and Bloemberg 2004). This topic has been treated under 5.2. 11.6.2.4 Induced Systemic Resistance Some PGPR have been identified as potential ISR elicitors, for their ability to induce resistance in plants toward pathogenic fungi, bacteria, and viruses (Van Loon et al. 1998; Van Loon 2007). The inducing rhizobacteria triggered a reaction in the plant roots that gave rise to a signal that spread systemically throughout the plant and enhanced the defensive capacity of distant tissues to subsequent infection by the pathogen (Van Loon 2000). ISR is distinct from systemic acquired resistance (SAR) in several key physiological and biochemical phenotypes that are best defined in A. thaliana (Van Wees et al. 1997). Studies with A. thaliana mutants indicated that the jasmonate/ethylene-inducible defense pathway is important for ISR. Many bacterial determinants induce ISR, i.e., flagella, siderophores (pycholin and pyocyanin), LPS, salicylic acid (Van Loon 2007), cyclic lipopeptides (Ongena et al. 2007), N-acyl homoserine lactone (AHL) molecules (Shuhegger et al. 2006), the bacterial volatile 2,3-butanediol produced by Bacillus spp. (Ryu et al. 2003), and antibiotics such as Phl (Lavicoli et al. 2003). In several ISR-competent strains of fluorescent pseudomonads, it has been difficult to identify specific ISR elicitors, possibly because a combination of siderophores, O-antigen, and flagella might account for the ISR effect (Bakker et al. 2003). It has also been shown that several Pseudomonas spp. are able to induce ISR in a wide range of plants toward different pathogens (Van Loon 2007). Generalization of the signal transduction pathways that are involved in ISR are further complicated by the fact that an ISR response to a given PGPR depends on the plant species and cultivar. For example, in Arabidopsis thaliana, the PGPR strain P. fluorescens WCS417r elicited ISR on all ecotypes examined, except ecotypes Wassilewskija and RLD (Van Wees et al. 1997). 11.6.2.5 Signal Interference In Gram-negative bacteria, one type of communication system functions via small, diffusible N-acyl homoserine lactone (AHL) signal molecules (Fig. 11.2). Such a regulatory system allows bacteria to sense the density of cells of their own kind and to express target genes in relation to their cell density. This cell–cell 11 Plant Growth-Promoting Bacteria: Fundamentals and Exploitation 319 communication mechanism regulates a variety of physiological processes, including swarming, swimming and twitching motilities, production of pathogenicity/virulence factors, and rhizosphere colonization (Gray and Garey 2001; Miller and Bassler 2001). Several groups of AHL-degradation enzymes have recently been identified in a range of organisms, including bacteria and eukaryotes. Expression of these enzymes was identified to interfere with the quorum-sensing system of pathogenic bacteria. E. carotovora produces and responds to AHL quorum-sensing signals to regulate antibiotic production and expression of virulence genes, whereas B. thuringiensis strains abolished the accumulation of the AHL signal by the expression of a AHL-lactonase, which is a potent AHL-degrading enzyme. In plants, B. thuringiensis significantly decreased the incidence of E. carotovora infection and symptoms development of potato soft rot caused by the pathogen (Dong et al. 2004). Discovery of these enzymes has not only provided a promising means to control bacterial infections, but also represents new challenges to investigate their roles in host organisms and their potential impacts on ecosystems (Dong et al. 2004). 11.6.3 Role of Root Colonization in Biocontrol Mechanisms Selection of bacterial biocontrol strains by their ability to inhibit fungal growth in vitro is not always correlated with efficient control of plant diseases. Different steps are known to be involved in root colonization (1) attachment to the plant surface, in which flagella and different types of pili can be involved (De Weert et al. 2004b) and (2) surface motility which confers potential benefits to bacteria in the rhizosphere, including increased efficiency in nutrient acquisition, avoidance of toxic substances, ability to translocation to preferred hosts, and ability to access optimal colonization niches (Andersen et al. 2003). Motility has been reported to depend on the soil type, the plant, and the bacterial strains used (Weller and Thomashow 1994). Pliego et al. (2008) reported that strains differing in surface motility can show differences in the architecture of the biofilms developed by these strains in the rhizosphere which, at the same time, can reflect differences in their root colonization patterns. As we explained earlier, in case of BCAs acting through CNN, colonization strategies of the biocontrol strain must be correlated with penetration sites used for the phytopathogenic fungi to infect the root. In the case of AFM production, efficient root colonization is essential for the delivery of the AFM along the root system at the right time and place (Lugtenberg et al. 2001; Haas and Keel 2003). Insertional mutagenesis studies demonstrated that colonization can be a limiting step in some biocontrol strains. P. chlororaphis PCL1391 controls TFRR through the production of the antifungal metabolite phenazine-1-carboxamide (PCN) (Fig. 11.2). Nonmotile mutants of this strain impaired in root colonization, but not in production of extracellular metabolites, did not show biocontrol activity (Chin-A-Woeng et al. 2000). 320 C. Pliego et al. In the case of microorganisms inducing the ISR response on the root, a decrease in root colonization does not clearly result in loss of biological control, i.e., P. fluorescens WCS365, which acts through ISR (Kamilova et al. 2005), colonization of seedlings for a brief period is sufficient to induce ISR in the whole plant (Dekkers et al. 2000). As is mentioned earlier, certain antifungal metabolites can induce ISR. This can explain the biocontrol activity of poorly colonizing PGPRs which produce antibiotics (Gilbert et al. 1994). 11.6.4 Mycelium Colonization by Bacteria As mentioned previously, biocontrol bacteria in the rhizosphere compete for nutrients and niches with endogenous microorganisms. Therefore, to gain a better understanding of how biological control functions in the rhizosphere, the interaction with other microbes, such as root pathogenic fungi, must be taken into account. It has become clear that biocontrol bacteria can interact with phytopathogenic fungi in a variety of ways, all converging on the same objective, i.e., to derive nutrition from these fungi (Leveau and Preston 2008). This interaction can be split into several stages including (1) detection of the fungal host, (2) attachment to the fungal cells, and (3) growth of bacteria on biotic fungal surfaces (Hogan et al. 2009). Microscopic visualization of bacterial–fungal interactions showed that at least some antagonistic bacteria exhibit chemotaxis toward fungal exudates allowing bacteria to congregate around populations of fungi in soils. For example, fusaric acid (Fig. 11.2), a secondary metabolite secreted by Fusarium hyphae, acts as a chemoattractant for cells of biocontrol strain P. fluorescens WCS365, which can actively move toward and colonize the surface of fungal hyphae (De Weert et al. 2004a). Bacteria showing chemotaxis to specific fungal compounds may use a variety of mechanisms to reach and attach to the fungus, including pili, exopolysaccharides, and biosurfactans. The biosurfactants reduce surface tension, enhance swarming motility, and facilitate contact between bacteria and fungal cells (Nielsen and Sorensen 1999; Braun et al. 2001). Once bacteria reach the fungus, they may scavenge nutrients from the fungal cell wall, consume products secreted by the fungus, or induce lysis of the fungal cells, thereby liberating the intracellular contents for consumption by the local bacterial population. Bacterial communities associated with fungi seem to be under selection to develop fungi-specific traits that confer a competitive advantage during colonization of fungal surfaces. Such traits could be the ability to detect fungus specific compounds, the utilization of nutrients that are specific to, or particularly abundant in, fungal exudates, or the ability to tolerate or suppress the production of antibacterial metabolites by fungal cells. Several studies support the idea that hyphal colonization by bacteria can play an important role in biocontrol activity (Bolwerk et al. 2003, Pliego et al. 2008). It has been shown that the ability of P. fluorescens WCS365 to inhibit pathogenic activity, survival, and germination of the pathogen significantly contributes to the reduction 11 Plant Growth-Promoting Bacteria: Fundamentals and Exploitation 321 of TFRR after introduction of this bacterium into the plant nutrient solution (Kamilova et al. 2008). Co-inoculation of the biocontrol strain P. pseudoalcaligenes AVO110 with R. necatrix (the causal agent of avocado white root rot) showed that AVO110 utilizes hyphal exudates more efficiently for proliferation than the nonbiocontrol strain P. alcaligenes AVO73 (Pliego et al. 2008). Although it is not always clear whether bacterial consumption of fungal exudates contributes to reduction of the disease (Kamilova et al. 2007), bacteria can inhibit the growth of harmful fungi by feeding on them, thus supporting their own growth, which in turn leads to a better biocontrol activity (Leveau and Preston 2008). While several studies have been focused on identifying bacterial genes involved in root colonization, little attention has been paid to genes involved in interactions with fungi. Bacterial genes involved in the interaction established between biocontrol strain P. putida 06909 and the phytophatogenic oomycete Phytophthora have been identified using IVET. Several P. putida promoters were induced during the growth of this biocontrol bacterium on the surface of the fungus, corresponding to genes involved in carbon catabolism, amino acid/nucleotide metabolism, and membrane transport processes (Lee and Cooksey 2000; Ahn et al. 2007) (Table 11.3). Separate studies showed that genes involved in trehalose utilization may be important for bacterial growth when associated with fungi. P. fluorescens sp. have been shown to induce genes involved in trehalose metabolisms when exposed to fungal culture supernatant, and the presence of this sugar enhances inhibition of Pythium debaryanum in a radial growth assay (Gaballa et al. 1997; Rincón et al. 2005). Recently, Pliego (2008) used STM, a powerful genomic mutagenesis technique which has been widely applied to bacterial pathogens (Hensel et al. 1995; Shea et al. 2000; Autret and Charbit 2005) to identify genes involved in growth and survival of a biocontrol Pseudomonas strain in fungal exudates. These genes were predicted to be involved in central metabolism, regulation of the second messenger cyclic (c)-di-GMP, and bacterial protection against fungal defenses. Table 11.3 Pseudomonas proteins upregulated during colonization of Phythophthora myceliaa Energy metabolism Nucleotide biosynthesis Membrane proteins/ transporters Nitrogen regulatory protein PII-2 Carbamoyl-phosphate synthase C4-dicarboxylate transport protein Flavin-dependent oxidoreductase Phosphoribosylaminoimidazol Arginine-/ornithine-binding (AIR) synthetase protein Malic enzyme Carbamoylphosphate Leucine-,isoleucine-, synthetase large subunit valine-binding protein Succinate-semialdehyde ABC transporter dehydrogenase Probable glyceraldehydeOuter membrane porins 3-phosphate dehydrogenase Ribulose-phosphate-3-epimerase Transaldolase a Adapted from Lee and Cooksey (2000) and Ahn et al. (2007) 322 C. Pliego et al. In summary, further studies must be performed to identify bacterial genes involved in fungal colonization or acquisition of nutrients from fungi. This will gain a broader view on genes and mechanisms involved in biological control and in future design of new and improved BCAs. One of the most exciting aspects is that genes similar to those involved in colonization of fungal hyphae were also identified in other IVET or STM assays involving phylogenetically diverse pathogenic and nonpathogenic microorganisms residing in diverse complex environments, in particular plant or animal host. These results suggest that similar or common mechanisms might be involved in processes as different as controlling and causing diseases. The identification of bacterial genes necessary for biocontrol of fungal pathogens will expand our ability to design new biocontrol strategies and, above all, feed our growing appreciation of the enormous diversity and adaptability of bacteria. 11.6.5 Resistance of the Pathogen Toward Biocontrol Agents Although there are hardly any reports about pathogenic fungi that have become resistant against BCAs, it is good to consider this possibility. It is clear that the mechanism of action used by the BCA determines the possible resistance strategies. Since little is known about the interaction between the pathogenic fungus and the plant when ISR is used as the biocontrol mechanism, it is difficult to predict possible defense strategies for this case. When CNN is used, it is hard to envisage resistance strategies of the pathogen. In contrast, for antibiosis as a mechanism of biocontrol several defense strategies can be envisaged. To do this, it is best to look at the much better studied resistance mechanisms that bacteria are using against antibiotics. Here, known resistance strategies can be based on (1) detoxification of the antibiotic, e.g., by degradation or modification, (2) modification of the target of the antibiotic, (3) making penetration to the target molecule more difficult, e.g., by making entry pores narrower or by shielding the surface with an additional less penetrable layer, and (4) actively pumping the antibiotic out of the cell. Duffy et al. (2003) published an excellent review about defense mechanisms used by pathogens against microbial antagonism. Detoxification of the antifungal compound 2,4-diacetylphloroglucinol produced by Pseudomonas species has been reported. Schouten et al. (2004) reported the screening of 76 plant-pathogenic and 41 saprophytic F. oxysporum strains for sensitivity to 2,4-diacetylphloriglucinol. They found that 18 and 25%, respectively, of these strains were tolerant to the antibiotic. Of course, an antibiotic-producing strain should protect itself against the antibiotic it produces. Indeed, Bacillus subtilis strain UW85 which produces the antibiotic zwittermycin A can inactivate the antibiotic by acetylation (Milner et al. 1996). Another mechanism that can be used by a pathogenic fungus is efflux of the antibiotic. Upon exposure to phenazines, Botrytis cinerea induces an efflux pump 11 Plant Growth-Promoting Bacteria: Fundamentals and Exploitation 323 for the antibiotic. Mutants lacking pump activity are more sensitive toward the antibiotic (Schoonbeek et al. 2002). A very interesting mechanism of defense is the ability of a pathogen to repress the biosynthesis of the antibiotic produced by the BCA. Duffy and Défago (1997) found that fusaric acid (Fig. 11.2), a small molecule secreted by many Forl strains, represses the synthesis of Phl in the biocontrol bacterium P. fluorescens CHAO by repression of the phlA promoter, a process for which an intact phlF gene is needed. Van Rij et al. (2004) observed that fusaric acid also suppresses the synthesis of another antibiotic, namely, PCN produced by P. chlororaphis strain PCL1391. This inhibition correlates with reduction of the level of the AHL autoinducer N-hexanoylL-homoserine lactone. The inhibition in P. chlororaphis strain PCL1391 takes place at or before the level of AHL production. It is striking to see that in two different Pseudomonas biocontrol strains inhibition of antibiotic production by fusaric acid has evolved, although at different molecular levels. Apparently, these mechanisms have evolved independently because of the need to protect against antibioticproducing bacteria. It should be noted that P. fluorescens strain CHAO does not produce AHL and therefore the mechanism to suppress Phl production at the level at or before AHL production could not be used in this strain. In practice, there are very few examples of resistance of pathogens against BCAs (Duffy et al. 2003). This may have several reasons, for example, that a BCA uses a mechanism to which no resistance exists or that it uses several different mechanisms to which no cross-resistance exists. The relevance of resistance in biocontrol was studied by Mazzola et al. (1995). They screened a collection of 66 isolates of the pathogen Gaeumannomyces graminis var. tritici against the antibiotics Phl and PCA, which are produced by many biocontrol strains which act through antibiosis. A large variation in sensitivity was found. Most importantly, it appeared that the Phl- or PCA-producing Pseudomonas strains could not control take-all caused by the Phl- or PCA-resistant G. graminis var. tritici isolates, respectively. 11.7 From Laboratory to Industrial Application It is important to realize that a beneficial bacterium, which is active under laboratory conditions, not necessarily is a good basis for a commercial product. Here, we discuss criteria that industrial products should fulfill. 11.7.1 Introduction The application of beneficial microbes for plant growth stimulation and biocontrol makes horticulture and agriculture sustainable and will, in contrast to many chemicals, not be a burden for the ecology and for the next generations of human beings. This notion, supported by public, politicians, and some supermarkets, is a major 324 C. Pliego et al. driving force for the agricultural and horticultural industry to use, where possible, environmentally friendly products for plant growth stimulation and crop protection. In this section we will discuss how, starting from promising beneficial microbes with known mechanisms of action, the industry will make the decision to carry out further research and which steps are required to develop a marketable product. Because this chapter focuses mainly on biocontrol microbes, we discuss only biocontrol products in this section. Furthermore, rules about registration as a product differ among countries. We will mainly focus on guidelines of the European Union (EU). 11.7.2 Evaluation of Opportunities How does the biocontrol industry come to the decision which strain should be used to make it a profitable product? What are the prerequisites for such a decision? What does the grower need? How does the development of a plant protection product occur? How can such a product be protected, registered, and commercialized? The biocontrol industry keeps constant contact with growers, the current users of their products. Collecting data on existing and emerging diseases of crops and search for solutions against these diseases is the starting point for decision making. The size of the market is another important factor. Therefore, the market potential for a new microbial product will be evaluated in terms of market size in relation to (1) the importance of the crop(s) and (2) the severity and geographical spread of the disease. What are the present biological and/or chemical products for disease prevention? Who are the competitors? How strong are they? Would the new product have a chance to successfully penetrate the market? 11.7.3 Industry Takes over In case the above-mentioned evaluation is positive, a good strain has to be found. If the biocontrol industry has no useful strains available, it can approach academic parties to evaluate whether they have promising strains that are effective against the target disease/pathogen combination. Academic research, as described in the previous paragraphs, is usually directed toward the discovery of the potential plantbeneficial strains by using approaches such as screening for antagonistic properties, studying root colonization, and identifying traits and metabolites involved in biocontrol and plant growth promotion. Most of this research is done in vitro or in planta under laboratory, growth chamber, or experimental greenhouse conditions. Academic scientists usually focus on one pathogen/crop model system as such experiments can easily be made suitable for publication. Industry should ensure that the selected strain is safe for environmental release and should come 11 Plant Growth-Promoting Bacteria: Fundamentals and Exploitation 325 to an agreement with the owner on intellectual property rights and the degree of exclusivity and/or licensing. 11.7.4 Industrial Research In contrast to the usually limited plant experiments done by academic researchers, industry would like to develop a product applicable on a broader range of crops and/ or pathogens. This implies further research as well as the development of a suitable formulation for the strain. After positive evaluation of marketability of the potential biocontrol product, the following considerations are taken into account: strain safety and efficacy, potential for upscale production and product development, and registration costs. 11.7.5 Strain Safety The beneficial strains have usually already been taxonomically identified in an early state of the research. According to guidelines (Anonymous 1998) the strain can then be classified in risk groups which predict their degree of safety. Before a BCA can be registered as a product, cost-intensive, time-consuming pathogenicity tests have to be carried out. There is a good reason to do this since several strains isolated as potential BCAs are potential human pathogens (Berg et al. 2005; Egamberdieva et al. 2008). Thus, strain identification is crucial for safe applications in agriculture and horticulture. According to European Commission Directive 2001/36/EC amending Council Directive 1991/414/EEC, concerning the introduction of plant protection products on the market, the best available technology should be used to identify and characterize the microorganism at the strain level. The use of molecular biological techniques, mostly at the DNA level, allows the identification of potential pathogens which subsequently will be excluded from further research. It should be noted that a promising new tool for future evaluation of the pathogenic potential of putative biocontrol strains could be the use of an assay with the nematode Caenorhabditis elegans (Zachow et al. 2009). Work will only be continued with strains from the risk group 1 (Anonymous 1998). In this stage also evaluation of the mode of action of the beneficial strains is very important, because it will give a clue to whether this would cause additional studies and, consequently extra costs, for registration of the biocontrol product. For example, for strains with mechanisms of actions based on production of new antibiotics and/or other toxic compounds, the regulatory authority will ask to provide very detailed investigations on metabolite production, toxicological and residue studies. 326 C. Pliego et al. 11.7.6 Large-Scale Production and Formulation Often the high cost of production for most of BCAs is a limiting factor for commercialization of a biocontrol product (Fravel 1998). High costs of the growth substrate, low productivity of the microbe, or limited economies of scale could be the reasons to stop development (Fravel et al., 2005). One of the goals of an industrial production unit is to minimize the fermentation costs and to produce the highest quantities of the microbe without negatively influencing the important traits involved in biocontrol such as (1) proliferation of spores in case of sporeforming bacteria and fungi, (2) ability to colonize rhizosphere or phyllosphere, or (3) competition with phytopathogenic organisms. Multiple tests on cell viability and plant root colonization should be performed in order to optimize a manufacturing process and to produce a good quality product. Beneficial microbes are formulated in order to preserve the BCA, to deliver the microbes to their targets and, after their delivery, improve their activity (Burges 1998). Formulation usually consists of the addition of a combination of various additives, such as carriers, preservatives, nutrients, etc. Furthermore, the choice of dry or liquid formulation to be used depends on the biological and physical properties of the microorganism, and on its abilities to survive the formulation process and to maintain the desired properties for a certain period of time (Burges 1998). Evidently, for producing powder and granular formulations, spore-forming strains such as Bacillus have an advantage over non-spore-forming strains such as Pseudomonas which have to be formulated as vegetative cells. Spores are more robust and resistant to elevated temperature and high concentrations of chemicals that are part of the spray-drying process. Moreover, the shelf life of biological products based on bacterial spores can be up to 1–3 years. A disadvantage of the use of spores is that, after application, they need time to return to the metabolic active stage of a vegetative cell. Vegetative cells of nonspore formers are more sensitive than spores toward dry formulation processes. This makes dry formulation of vegetative cells more expensive than formulation of spores. For example, it has been shown for Pseudomonas by Validov et al. (2007) that freeze-drying provides a more stable formulation product with a longer shelf life than the less expensive spray drying. A liquid formulation can be an option for vegetative cells. However, it can be a challenge to keep these bacterial cells in a low metabolic state in the presence of water for a substantial amount of time without losing their viability. For a seed company, seed coating with biocontrol strains is an interesting option. Coating means here the introduction of BCAs during seed priming and/or seed pelleting. In fact, this is a process for upgrading the value of seeds. An advantage for the user is that the seeds do not have to be treated with the bioproduct immediately prior to sowing by means of spraying or by soaking the seeds at site. The choice of the formulation type is dependent not only on the biological properties of the strains but also, in many cases, on the desired application methods and existing irrigation systems. For example, for a drench application, slurry, spray 11 Plant Growth-Promoting Bacteria: Fundamentals and Exploitation 327 or soaking treatment of seeds, liquid or wettable powder products are more suitable. A granular formulation would be appropriate for mixing with horticulture potting mix or for distribution in-furrow (Fravel 1998). 11.7.7 Registration in the EU: Legal Basis and Requirements Many biocontrol products have been registered. See Table 11.4 for examples. Active microorganisms used in the plant protection products in the EU are regulated according to the EU Council Directive 1991/414/EEC. This Directive provides definitions of the plant protection products and requirements for their authorization Table 11.4 Examples of active microorganisms registered as biopesticides in the EU and/or the USA (as at March 1, 2010) Active microorganism Formulation Pathogen Crop type Liquid, Fusarium, Rhizoctonia, Potato, vegetables, Bacillus subtilis FZB24b powder, Pythium ornamentals, turf granule Powder Botrytis, Sclerotinia, Vegetables, Bacillus subtilis Sphaerotheca macularis soft fruits QST 713b Powder Aspergillus, Pythium, Cotton, vegetables, Bacillus subtilis GB03b Rhizoctonia, Fusarium soybean Liquid Fusarium, Septoria, Cereals Pseudomonas Tilletia chlororaphis MA 342b Powder Fusarium Vegetables Streptomyces K61a,b Powder Rhizoctonia, Pythium, Gliocladium catenulatum Vegetables, Phytophthora, Fusarium, strain J1446a,b herbs, Didymella, Botrytis, ornamentals, Verticillium, Alternaria, trees, Cladosporium, shrubs, Helminthosporium, turf Penicillium, Plicaria Powder, Fusarium, Pythium, Vegetables, Trichoderma harzianum granule Rhizoctonia ornamentals, turf strain T-22a,b Powder Fusarium, Pythium, Vegetables, turf Trichoderma asperellum Rhizoctonia strain ICC012a,b Powder, Fusarium, Pythium, Vegetables, turf Trichoderma atroviride granule Rhizoctonia strain IMI206040a Fusarium, Pythium, Vegetables Trichoderma gamsii strain Powder Rhizoctonia, ICC080a,b Verticillium Trichoderma polysporum Powder Botrytis, Chondrostereum Soft fruits, strain IMI206039a ornamentals Liquid Alternaria, Botrytis, Canola, ornamentals, Pythium oligandruma,b Fusarium, Sclerotinia vegetables Liquid Ophiostoma nova-ulmi Elm Verticillium albo-atrum strain WCS850a,b a EU Annex I listed strains (http://ec.europa.eu/sanco_pesticides/public) b US-EPA registration (http://www.epa.gov/oppbppd1/biopesticides/ingredients) 328 C. Pliego et al. within the EU. Directive 1991/414/EEC was amended by Commission Directive 2001/36/EC regarding to the data requirements for the Annex I inclusion of microorganisms as active substances and national authorization of products (Annex II and III, respectively, of the directive). Council Directive 2005/25/EC lays down the Uniform Principles for evaluation and authorization of plant protection products containing microorganisms. Briefly, a registration dossier prepared in order to register a biopesticide in the European Union must contain all requested information on the active microorganism and the product, such as the biological properties of the microbe, the physical and chemical properties of the product, its safety for humans and for the environment, and its efficacy. In contrast to Europe, registration in the USA (http://www. epa.gov) does not require the efficacy data. In Table 11.4, we provide examples of microorganisms that have been registered as active substances in commercial biopesticides registered in the EU and/or the USA. Many physical and chemical studies of the product, the analysis of relevant metabolites and potential toxins in the preparations, the analysis of residues on treated plants and food, as well as toxicological and ecotoxicological studies should be performed under Good Laboratory Practice (GLP) conditions. In most of the cases, industry outsources these tasks to specialized GLP-certified laboratories and institutes. Toxicological studies include evaluation of acute toxicity studies performed on certain laboratory animals such as rats, rabbits, and guinea pigs. Genotoxicity testing includes both in vivo (carcinogenic) and in vitro (mutagenic) studies. Characteristics such as impact of the product on the environment, particularly its effects on birds, aquatic organisms, bees, and other nontarget arthropods, earthworms, and soil microorganisms, are analyzed by direct observation of the tested organisms under laboratory conditions. Information from the relevant literature on the ecology of biocontrol microorganisms as well as closely related strains can help to evaluate the impact of this strain on soil microflora by extrapolation. Providing this information therefore can somewhat reduce laboratory studies on this subject. For example, there is a number of useful publications about the analysis of microbial communities of various soils after introduction of different bacterial strains with antagonism against Rhizoctonia solani (Adesina et al. 2009; Garbeva et al. 2004; G€otz et al. 2006; Scherwinski et al. 2008), E. carotovora, and Verticillium dahliae (Lottmann et al. 2000). However, this reduction of actual laboratory studies would be rather an exception, since the vast majority of studies must be performed with the strain to be registered. The efficacy part of the dossier is based on data obtained in the trials, performed for registration purposes, as well as those carried out in the research laboratory. The latter ones are considered only as preliminary data and treated by the experts accordingly. In the EU, efficacy trials are required to be carried out by efficacy testing organizations officially recognized by the national authorities. Efficacy tests and analyses include field, glasshouse, or laboratory trials and tests to determine the effectiveness and crop safety of plant protection products. Official Recognition is also known as Good Experimental Practice (GEP). This depends on the national regulations. Efficacy tests can be required to be performed during a number of seasons and at different locations. 11 Plant Growth-Promoting Bacteria: Fundamentals and Exploitation 329 In the framework of the European project on regulation of biological control agents (REBECA, 2008), an analysis on registration issues of the most important products (Annex I – listed) was performed. Data indicates that the mean registration time for EU Annex I inclusion is 75 months. Country registrations vary widely and range from a few months up to over 100 months, averaging around 24–36 months. Overall registration cost for EU Annex I inclusion is estimated to be about 1,890,000 Euro; out of that 970,000 Euro are external costs (fees, etc.). The breakdown of total costs to different kinds of tests averaged as efficacy tests (21%), toxicological tests (43%), ecotoxicological studies (23%), and other required studies (13%) (REBECA 2008). In case of use of several microbial strains in one product (so-called microbial cocktails), registration costs will increase because an individual dossier for each active microorganism must be submitted. Registration strategy may include several options to minimize costs. For example, several companies that manufacture products with the same active microorganism(s) can unify into a task force team for inclusion of these organisms into Annex I list and share costs of this procedure. In this case member companies also share their studies and submit a joint dossier (Annex I) for the active microorganism. Individual access to certain protected data (mostly on acute toxicity and analysis of metabolites) belonging to other companies can be an option to save time for registration of some microorganisms, although financial considerations must be taken into account. 11.7.8 Intellectual Property Rights and Patenting Usually intellectual property rights on biocontrol strains belong to universities and small companies in which the basic research was performed. If a biocontrol company decides to commercialize a strain, both parties should come to an agreement on how the strain owner should be reimbursed. Protection is a rather sensitive subject for the biocontrol industry. In Europe, patenting of biocontrol strains per se is not possible due to the fact that these strains are naturally occurring living organisms. This is why the subject of invention usually is a combination of the strain with the bioproduct formulation(s) containing the viable cells or of the strain with applications against certain pathogenic organisms on certain crops. Another possibility for patenting is a combination of the strain and its specific metabolites involved in biocontrol activity and being produced during the fermentation and incorporated into the bioproduct formulation. Patenting can take place in various stages of the product development but should certainly take place prior to registration. 11.7.9 Marketing Commercialization of the biocontrol products is not a simple task. First of all, demonstration trials at sites should convince farmers that the proposed bioproduct 330 C. Pliego et al. can provide a sound protection which is preferentially better than, or at least comparable with, other biological or chemical products (if these exist). Commercialization should also include a strong education element with emphasis on the preventive character of the biopesticide. Also, the new product should be easy to use. It would be ideal if the bioproduct can be integrated in grower’s routine schemes for the use of pesticides. Last but not least, the price of the new biocontrol product must be competitive and affordable for farmers without compromising the desired sales margins. 11.7.10 Integrated Pest Management (IPM) In 2005, biopesticides had a share of approximately US$ 300–600 million of the US $ 35 billion agrochemical market (Cherry, 2005). Over the next 10 years, the market for biopesticides is expected to grow to US$10 billion (GIA, 2008). This is mainly due to the great public awareness of the risks of chemical fungicides and the clear trend toward sustainable agriculture, both in the EU and the USA. A good example of an effect of the public opinion is an activity of many supermarket chains and retailers toward to pesticide poor or pesticide free food. Within GLOBALG.A.P., a private sector body, many food retailers and supermarket chains, farmers and crop protection specialists collaborate in this respect. This organization sets standards for the certification of agricultural products worldwide. The GLOBALG.A.P standard is primarily designed to reassure consumers about how food is produced on the farm by minimizing detrimental environmental impacts of farming operations, reducing the use of chemical inputs, and ensuring a responsible approach to worker health and safety as well as animal welfare (www.globalgap.com). Increasingly more attention is paid to IPM. This includes, among other means, a balanced use of chemicals and biologicals in order to provide healthy food without loosing or, preferentially, with even increasing productivity. Commercial biocontrol products must address not only efficacy and, broadly speaking, safety requirements, but also be compatible with existing agricultural practices, including the use of fertilizers and chemical plant protection products. The ultimate intention of the biocontrol industry is to minimize and eventually eliminate the use of chemicals by replacing them with environmentally friendly biocontrol products. 11.8 Lessons from the Past to Create a Shining Future In the past 3 decades, insight in fundamental aspects of microbe–plant interactions has enormously increased, especially at the molecular level. Understanding of these interactions at the molecular level is important for (1) improving the efficacy of the applied microbes, (2) enhancing the registration process, and (3) marketing microbes as reliable products to the users. In the recent past, mechanisms of biocontrol 11 Plant Growth-Promoting Bacteria: Fundamentals and Exploitation 331 through antibiosis have been elucidated, and the biocontrol mechanism CNN has been proven to exist. The most obscure biocontrol mechanism known so far, ISR, is presently being studied intensively and major breakthroughs may be expected in the next decade. Plant growth promotion through hormones needs further attention. In the past decade, endophytic microbes received a lot of interest, also for applications as beneficial microbes. The possible advantage of the use of endophytes is two-fold. (1) The plant’s interior may harbor a novel type of microbes. (2) Within the plants, beneficial bacteria are much better protected against competition by rhizosphere bacteria than when they are present in the rhizosphere. It should, however, be noted that a bacterium, which has been isolated as an endophyte, does not automatically resume its endophytic lifestyle when applied to the plant’s exterior. In addition, once inside the plant, it may have to compete with endogenous endophytes. A bottleneck in the understanding of microbe–plant interactions is our limited knowledge about the composition and function of root exudates. This requires a thorough study in which experts in the fields of microbiology, plant physiology, engineering, and soil science should work together. They should use the most modern technology combined with knowledge of the facts from the literature. In addition, a role of volatile organic compounds in biocontrol is becoming clear. The characterization of these compounds and the elucidation of their functions should become a new area for future research. Many studied and commercialized biocontrol strains have been isolated only on the basis of antibiotic activity whereas important traits such as root colonization and compatibility with formulation and industrial practice were not taken into account. Considering the complex and expensive process required to commercialize a plantbeneficial microbial product, it is crucial that, before large research efforts are undertaken, one evaluates all steps of the process, from isolation strategy and laboratory experiments to efficacy, registration, and marketing. Weak and expensive steps should be identified, evaluated and, if necessary, tested first. In this way a lot of time can be saved if one wants to commercialize a strain. A major driver for the application of microbes in the developed world is the fact that the public opinion is opposed against the use of agrichemicals. Several supermarket chains have adopted a policy of zero or very low tolerance against chemical pesticides. Also politicians support this policy, at least in theory. In practice the process to register biological products in the EU is difficult. Here, the products should be registered separately in every member country and this process lasts from several months to several years, depending on the country. Beneficial microbes often have a clearer effect on plants grown under suboptimal conditions than on plants growing under optimal conditions. Suboptimal conditions include limitation for nitrogen, phosphorous, or iron; elevated salinity; and water disbalance. Such conditions are often accompanied by plant diseases. An example is cucumber grown in salinated and naturally infested soil in Uzbekistan. Here, as many as 17% of the cucumber plants were diseased by the indigenous pathogen Fusarium solani. Several European biocontrol strains were very effective in reducing the percentage of diseased plants. 332 C. Pliego et al. The area of salinated soil will increase, for example, at places with intensive irrigation. Also the predicted climate change will lead to an increase of the area of salinated agricultural soil, especially in the poorer areas in the world. Here, the yields are often low and chemicals are therefore too expensive. It can be expected that the need for the application of the relatively cheap beneficial microbes will largely increase in these areas. Therefore, it is comforting to know that many beneficial microbe isolates from plants growing on nonsalinated soil are salt tolerant and that they can be successfully applied in salinated soil and also in a warm climate, despite the fact that they have been isolated from a cold climate and from other plants than cucumber (Egamberdieva et al., submitted). This strong beneficial effect of bacterization can be explained by the low level of endogenous microbes in this salinated soil (Egamberdieva et al. 2008), which therefore has a low buffering capacity against pathogens. Nowadays many African, Asian, and South American countries with limiting financial resources for chemical fertilizers and pesticides clearly demonstrate their determination to use more microbial products as alternatives. Development of “cottage” type small production units helps local farmers to improve plant’s health and productivity (Harman et al. 2010). This low tech approach, as well as the increase in traditional sales of high-tech microbial products, indicates that the use of beneficial microbes in agriculture will play a more prominent role in the near future. 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