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Antonie van Leeuwenhoek 81: 373–383, 2002. © 2002 Kluwer Academic Publishers. Printed in the Netherlands. 373 Microbe–plant interactions: principles and mechanisms Ben J.J. Lugtenberg∗ , Thomas F.C. Chin-A-Woeng & Guido V. Bloemberg Leiden University, Institute of Molecular Plant Sciences, Clusius Laboratory, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands (∗ Author for correspondence) Key words: anti-fungal metabolites, biofertilizers, biopesticides, colonization, genomics, induced systemic resistance, pathogens, phase variation, phytostimulators, quorum sensing, recognition, regulation, rhizoremediators, two-component systems, Type 4 pili, Type III secretion systems, Type IV secretion systems Abstract The present status of research on the molecular basis of microbe–plant interactions is discussed. Principles and mechanisms which play a role in the interactions of microbial pathogens, biofertilizers, phytostimulators, rhizoremediators and biocontrol agents with the plants are treated. Special emphasis is given to colonization, phase variation, two-component systems, quorum sensing, complex regulation of the syntheses of extracellular enzymes and secondary metabolites, Type 4 pili and Type III and Type IV secretion systems. Abbreviations: ACC – 1-aminocyclopropane-1-carboxylic acid; AFM – anti-fungal metabolite; AHL – Nacylhomoserine lactone; CLSM – confocal laser scanning microscopy; DFI – differential fluorescensce induction; EPS – extracellular polysaccharide; ISR – induced systemic resistance; IVET – in vivo expression technology; LPS – lipopolysaccharide; ORF – open reading frame; OT – optical trapping; QS – quorum sensing; PC – phosphatidyl choline; PCN – phenazine-1-carboxamide; Phl – 2,4-diacetyl phloroglucinol; SAR – systemic acquired resistance; TTSS – type III secretion system The challenge of microbiology in the beginning of the 21st century In the second half of the twentieth century a major focus of microbiology was on trying to understand the behaviour of model microbes under laboratory conditions. The challenges for the next decades include understanding of the behaviour of microbes in their natural and often complex habitats, such as the rhizosphere and the phyllosphere. A microbe seems largely to be driven by principles which also govern our own behaviour: a microbe wants to survive, also under hostile conditions, and if conditions are more favourable it will eat and proliferate. In order to understand a microbe’s behaviour in a certain habitat, it is essential to know which genes are expressed, what their functions are, how the gene products interact, how these processes depend on biotic and abiotic conditions, and towards which behaviour traits this leads. In theory, the use of the new technology often designated as ‘Genomics’ can answer these questions. However, before becoming over-optimistic it is good to realize that even from the best-known organism Escherichia coli K-12, biochemical function has not yet been established for one third of the 4288 ORFs. Presently, the DNA of several microbes and plants which are studied in microbe-plant model systems has been sequenced or will soon be sequenced. On the other hand, sequencing is still expensive and the sequences of many interesting organisms will not soon be available. For example, although the opportunistic human pathogen Pseudomonas aeruginosa PAO-1 has been sequenced (5570 ORFs; Stover et al. 2000), it is unlikely that the genomic sequences of the many Pseudomonas strains which are important plant pathogens, phytostimulators, bioremediators or biocontrol agents will soon be available. Based on comparative genomics between E. coli strains it can be predicted that their genomes not only differ in a few ‘islands’, but that two Pseudomonas strains will differ in hundreds of segments larger than 50 bp. Also the genomes of fungi and plants are being sequenced, a process 374 which delivers less functional information since only approx. 1.5% represents ORFs. Other novel technologies which will also be useful to study microbial behaviour are the use of autofluorescent proteins in combination with CLSM (confocal laser scanning microscopy), atomic force microscopy, single molecule detection, and DFI (differential fluorescence induction) combined with OT (optical trapping) (Allaway et al. 2001). Considering the massive amounts of information that will be gathered with the help of these expensive technologies, networking and cost-sharing will be key words in the next decades. Microbe–plant interactions: general considerations Based on their effects on the plant, microbes interacting with plants can be classified as pathogenic, saprophytic and beneficial. Pathogens can attack leaves, stems or roots. An interesting new research field is the interaction between water-borne microbes and underwater plants. Saprophytes, which live on dead plant material, will not be further treated in this overview although they play a crucial role in important processes such as the cycling of elements and composting. Beneficial microbes are often used as inoculants (Bloemberg & Lugtenberg 2001). They can be classified according to the goal of their application: biofertilizers (such as rhizobia, which have been applied commercially for over a century), phytostimulators (such as auxin-producing, root-elongating Azospirillum), rhizoremediators (pollutant degraders which use root exudate as their carbon source), and biopesticides. None of the numerous microbe-plant interactions is completely understood. Therefore, we have not chosen for summing up a huge number of facts per interesting microbe but we will restrict ourselves to a number of newly discovered principles and mechanisms, and discuss a limited number of interactions between microbes and plants. By now it is clear that microbes in their interactions with plants, no matter whether the microbe is beneficial or pathogenic, often use the same mechanisms, although in different combinations and for different purposes. Similarly, it is clear that microbes in their interaction with plants use similar strategies as in their interactions with other eukaryotes such as fungi and humans (Lugtenberg & Dekker 1999; Chin-A-Woeng et al. 2000; Lugtenberg et al. 2001). It is thus good to realise that, although one uses the term microbe–plant interactions, the real- ity is that in the rhizosphere and in the phyllosphere microbes also interact with each other. Plant recognition by microbes Recognition is considered to be the initial key event in the response of plants to microbes. Recognition can occur through physical interaction – such as through adhesins, fimbriae, flagella, and Type III and Type IV secretion systems – or through signalling by small molecules. It has been estimated that between 30 and 40 genes are involved in the biosynthesis and functioning of type 4 pili in P. aeruginosa (Darzins & Russell 1997). In this bacterium type 4 pili are involved in the initial contact between the bacteria and the epithelial cell surface, in a unique type of locomotion called twitching motility, and in biofilm formation (O’Toole & Kolter 1998). Although type 4 pili are present in various plant pathogens, their role in attachment to plant surfaces is poorly documented. In the case of Xanthomonas campestris pv. hyacinthi type 4 pili were shown to be involved in attachment to hyacinth leaves whereas P. syringae pv. phaseolicola attaches to stomata via its type 4 pili (Romantschuck & Bamford 1986). Camacho (2000) obtained evidence for a role of type 4 pili of P. fluorescens WCS365 in tomato root colonization. Dörr et al. (1998) have indicated a role for type 4 pili in attachment of Azoarcus to grass roots as wel as to fungal mycelium. The pilA promoter of Azoarcus is induced upon starvation in carbon-free medium (Plessl et al. 2000). One of the interesting and sophisticated mechanism used by bacterial pathogens to attack eukaryotic host cells is a specialized protein secretion system, the TTSS, that delivers bacterial virulence proteins (‘effector proteins’) directly into the host cell. TTSSs share four important features (He 1988; Galán & Collmer 1999). (i) They are widespread among plant and animal bacterial pathogens, and mutations affecting Type III protein secretion often eliminate bacterial virulence completely. More recent evidence suggests also roles of TTSSs in symbiosis and biological control (see further on). (ii) Many of their genes share sequence similarities with those of the flagellar assembly machiney and flagellum-like structures, leading to the speculation that TTSSs are evolutionary derived from the flagellar assembly apparatus. (iii) Type III secretion is activated in vivo upon contact with the host cell. (iv). The TTSS is Sec-independent. 375 The TTSS of Yersinia spp. is the best-studied example (Cornelis & Wolf-Watz 1997). Proteinaceous pertussus toxins are injected into human cells by a Type IV secretion system. Another member of this system is used for the translocation of single stranded T-DNA-protein complexes by Agrobacterium tumefaciens. The channel is composed of 11 VirB proteins and the VirD4 coupling factor. Transfer of the VirE2 and VirF proteins into plant cells can also occur independently of T-DNA transport (Vergunst et al. 2000). DNA of broad host range conjugative plasmids is also transferred through similar structures. A second Type IV secretion system has been detected (Wood et al. 2001) in the genome of A. tumefaciens, which is composed of 4 replicons: one linear chromosome, one circular chromosome, the tumor-inducing (Ti) plasmid, and a cryptic plasmid. Plant surface colonization by microbes Colonization of plant tissues can be visualized by using CLSM analysis of autofluorescent proteins expressed in the bacterial and/or fungal partners (Spellig et al. 1996; Bloemberg et al. 1997; Tombolini et al. 1999; Lagopodi et al. 2002). Using different forms of autofluorescent proteins, Bloemberg et al. (2000) were able to visualize three different bacterial populations simultaneously. Using two differently colored populations of the same P. fluorescens WCS 365 bacterium, it was concluded that, after inoculation of seedlings, occasionally single bacteria reach a position at a lower part of the root where they can divide without being frequently visited by a new invader (Bloemberg et al. 2000). It is generally assumed that the colonization of plant tissue is a crucial step in pathogenesis as well as for beneficial effects of microbes on the plant. The plant surface contains many microbes which, however, are not homogeneously distributed. Rather, deserted areas as well as areas with many microbes, often present as microcolonies or biofilms, are present. Such microcolonies are ideal places for bacteria to communicate with each other, e.g. by QS (quorum sensing). Many genes and traits are involved in colonization (for a review, see Lugtenberg et al. 2001). The results of colonization studies show that successful colonization strategies include: (i) good defense of the cell’s interior, (ii) efficient uptake of low concentrations of nutrients, including those provided by other organ- isms, and (iii) weakening or destruction of target and competing organisms. In this respect it should be noted that little research has been carried out on microbemicrobe interactions in the rhizosphere. Investigations towards the molecular basis of bacterium–fungus interactions have already started (Smith et al. 1999; Lee & Cooksey 2000). The recent discovery that Pseudomonas biocontrol bacteria not only colonize the plant root but also hyphae of the pathogenic fungus Fusarium oxysporum f.sp. radicis-lycopersici (Lagopodi et al. 2002) indicates that such interactions may be important. Interestingly, many colonization genes are shared by plant-beneficial bacteria and humanpathogenic bacteria, indicating that colonization of eukaryotic tissue by bacteria has started early in evolution (Lugtenberg & Dekkers 1999; Lugtenberg et al. 2001). Using colonization mutants, it was shown that colonization is crucial for biocontrol of tomato foot and root rot by the PCN (phenazine-1-carboxamide) producing P. chlororaphis strain PCL1391 (Chin-AWoeng et al. 2000) but not, or to a lower extent, for biocontrol of this disease by the ISR inducing P. fluorescens strain WCS365 (Dekkers et al. 2000). Interestingly, P. putida mutants impaired in seed colonization have recently been described (Espinosa-Urgel et al. 2000). In this respect it is interesting to note that, after seed inoculation, growth of B. cereus in the spermosphere of tomato depends on the tomato genotype (Smith et al. 1999; Simon et al. 2001). IVET (in vivo expression technology) is a powerful tool to identify promoters and genes that are expressed specifically under certain conditions, e.g. when a bacterium interacts with a plant (Rainey 1999) or a fungus (Lee & Cooksey 2000). Considering the high number of colonization genes, a reasonable understanding of the mechanisms of root, seed and leaf colonization can only be obtained if the number of bacterial genes to be tested for a role in colonization can be reduced by preselection. IVET and Genomics of microbe-plant interactions are promising tools for such a preselection. Genomics is also a good approach to unravel which plant genes play a role in colonization. Phase variation Phase variation results in subpopulations within a bacterial ‘pure culture’ which differ in the make up of their bacterial cell surface. This phenomenon occurs in many pathogens and is assumed to be a strategy to 376 escape the host’s immune system. It also appeared to play a role in root colonization (Dekkers et al. 1998). In a bacterial culture subpopulations with different degrees of rhizosphere competence can apparently exist next to each other. One of the mechanisms of phase variation is based on the action of a site-specific recombinase of the λ-integrase gene family which can either delete or reverse DNA fragments. Such DNA rearrangements will largely contribute to the flexibility of bacterial genomes (e.g. Flores et al. 2000). It is important to note that phase variation can affect important (putative) root colonization traits, such as Type 4 fimbriae, and flagellum-mediated swimming, swarming and twitching, as well as chemotaxis (Déziel et al. 2001). Synthesis of extracellular enzymes and secondary metabolites by microbes Two-component systems are present in many gramnegative bacteria. Usually, a sensor kinase in the cytoplasmic membrane autophosphorylates upon activation by an environmental stimulus and subsequently phosphorylates a response regulator protein in the cytosol which then acts as a transcription factor. The GacA/GacS system is the best studied example but its environmental stimulus has still not been identified. It should be noted that GacS was formerly known as LemA. Regulation in P. aeruginosa is extremely complex with an extraordinary number of putative two-component systems: 55 sensors, 89 response regulators and 14 sensor-response regulator hybrids, far more than found in other genomes (Stover et al. 2000). Some bacteria can communicate with cells of their own population and sometimes with other bacteria through exchange of soluble signal molecules. Of these, the N-acyl homoserine lactones (AHLs) are the best studied examples, but other types of intercellular signal compounds exist. These include cyclic dipeptides and quinolones in P. aeruginosa (Holden et al. 1999; Pesci et al. 1999), two unidentified low molecular weight compounds in Xanthomonas campestris (Poplawsky et al. 1998), and a volatile fatty acyl methyl ester in Ralstonia solanacearum (Flavier et al. 1997). In gram-positive bacteria γ butyrolactones are used for intercellular communication. Interestingly, one of the AHL QS molecules has previously been described as a bacteriocin (Schripsema et al. 1996). AHLs are involved in interactions between bacteria and eukaryotes such as conjugation between Agrobacterium and plant cells, and production of AFMs and other antibiotics and of extracellular enzymes, all of which are produced to attack competitors or foreign cells. Interestingly, also plants secrete substances that mimic bacterial AHL activities (Teplitski et al. 2000). It appeared that halogenated furanones produced by a marine red algae are able to disrupt AHLregulated behaviours (Givskov et al. 1996; Kjelleberg et al. 1997), presumably by competitive inhibition for the AHL-receptor protein (Manefield et al. 1999). Molecules which inhibit the action of AHLs, if applied to pathogens, have promise (as lead compounds) for disease control. Tobacco has been genetically modified to secrete AHLs, which appeared to affect bacteria present in its rhizosphere (Fray et al. 1999). Extracellular enzymes and secondary metabolites, such as AHLs and AFMs (anti-fungal metabolites), are very important for the interactions of bacteria in the rhizosphere. The regulation of the production of these molecules is extremely complex, in terms of regulatory genes as well as regulatory environmental factors. Regulatory components involve rpoS, the gacS/gacA two-component system, a regulatory (PrrB) RNA molecule (Aarons et al. 2000), as well as an RNA binding protein, RsmA, which is a repressor of secondary metabolism. The latter molecule is involved in a GacA-dependent switch from primary to secondary metabolism, acting at the level of translation (Blumer et al. 1999). Quorum sensing plays a role in the synthesis of several exoenzymes and AFMs such as phenazines (Pierson et al. 1994; Chin-A-Woeng et al. 2001). A two component gacA/gacS system has been described which interacts with the phzl/phzR quorum sensing system (Chancey et al. 1999; Chin-A-Woeng et al. 2001). The production of AFMs is often limited. The product of phlR acts as a suppressor of Phl (2,4 diacetyl phloroglucinol) production (Delany et al. 2000), whereas psrA limits PCN production (Chin-A-Woeng, unpublished), and synthesis of pyoluteorin is repressed by the serine protease Lon (Whistler et al. 2000). The production of Phl, like that of auxin in Azospirillum (Vande Broek et al. 1999) is subject to autoinduction. This autoinduction is countered by the plant metabolite salicylate, by the bacterial metabolite pyoluteorin and by fusaric acid, a toxin produced by the pathogenic fungus Fusarium (Schneider-Keel et al. 2000). 377 Environmental factors which influence the biosynthesis of secondary metabolites by pseudomonads include ions, sugars and glycerol (Duffy & Défago 1999; Chin-A-Woeng 2000). P. putida PCL1444 was selected from an environmental sample as the strain which had the best combination of naphthalene degradation and grass root colonization. Its behaviour appeared to be determined to a large extent by grass exudates components: it grew fastest on the major grass exudates components glucose and succinic acid and expression of its naphthalene degradation operons was highest in the presence of these carbon sources (Kuiper 2001). These results strongly indicate that by selecting for the traits fast growth and good colonization, one in fact selects for efficient utilization of exudate. Plant pathogens Fungi are the most important plant pathogens followed by viruses and bacteria. Ordinarily, the plant rejects the pathogen: most plants are not normally colonised and parasitized by the pathogen. They are ‘non-host plants’ for these pathogens. This phenomenon is called ‘basic resistance’ or ‘basic incompatibility’. Successful parasitism depends on the production of the right combination of pathogenicity factors to attack a particular pathogen. In such a case ‘basic compatibility’ exists between these partners. Basic compatibility is a highly specific phenomenon between a particular plant species and the appropriate pathogen species or forma specialis. An elicitor is a microbial signal which is recognized by a plant receptor. Subsequent transduction of the signal can result in a defense response by which the plant may ward off the attack. Most flagellin molecules e.g. from Bacillus, Escherichia and Pseudomonas, but not from Rhizobium and Agrobacterium, share a stretch of approximately 20 amino acids in their N-terminal domain which act as general elicitors (Felix et al. 1999). Other general elicitors are β-glucans, chitin fragments (including Nod factors!) and ergosterol. Plants recognise these substances with extremely high sensitivity and specificity (Felix et al. 1999). Many of these general elicitors occur in pathogens as well as in saprophytes and symbionts. It is therefore unlikely that recognition of these molecules induces an all-out defense in the plant. Rather, it has been speculated that these chemoperception systems might have an orienting role, allowing the plant to ‘smell’ the presence of microbes and of non-self organisms in its environments (Boller 1995). One of the defense strategies which the host plant can deploy is the ‘hypersensitive response’ (HR) or ‘hypersensitive cell death’. The injured plant cell dies very rapidly, causing necrosis of immediately adjacent tissue (Programmed Cell Death; Lam et al. 2001). This strategy cuts the pathogen off from its nutrients. It is unable to spread and multiply and will finally die. Phytopathogenic bacteria often contain a cluster of conserved genes, the hrp cluster, which is essential for the induction of the hypersensitive response in nonhost plants as well as for pathogenesis in susceptible host plants. The majority of these hrp genes encode elements of a TTSS for proteins (Lindgren 1997; Van Gijsegem et al. 1993) which may be evolutionary derived from the flagellar structure (Macmab 1999). Some hrp gene products, including a protein called harpin, are assumed to be secreted through the type III ‘syringe’, similar to how flagellin subunits are secreted through the hollow cylinder in the flagellum’s basal body and its filament. In the case of the TTSS these secreted hrp gene products, presently often designated as ‘effector molecules’, are assumed to be injected into the host cell. Delivery of the Avr (avirulence) gene product, followed by recognition in the plant cell, is essential for development of plant resistance (e.g. Bonas 2000). In the human pathogen Yersinia enterocolitica several virulence factors, including a virulence-associated phospholipase are known to be exported through the TTSS (Cornelis & Wolf-Watz 1997; Young et al. 1999). The tomato pathogen Cladosporium fulvum grows within the intracellular spaces of the leaves, where it secretes in susceptible cultivars, as well as during infection of resistant plants, a polypeptide product of the Avr9 gene which causes HR only in race-specific resistant cultivars which carry the cf9 resistance gene. The 28 amino acid polypeptide encoded by Avr9 acts as a race-specific elicitor. This and a similar peptide encoded by Avr4, are used for unravelling the mechanism of plant resistance (see article by P. de Wit and colleagues in this volume). Like many plant resistance genes, the Cf9 and Cf4 resistance genes contain many glycosylated leucine-rich repeats which are believed to interact, directly or indirectly, with other proteins, carbohydrates or ligands. Important bacterial virulence factors include exotoxins such as coronatine (Liyanage et al. 1995) and the biosurfactant toxin syringomycin (Hutchison & Gross 1997). The latter toxin seems to act through the 378 formation of ion channels in plant plasma membranes which initiate a cascade of intracellular signalling events. Pectate lyases are important virulence factors of soft-rotting erwinias (Keen 1996). Interestingly, P. aeruginosa strain PA14 is infectious both for the plant A. thaliana and for mouse. Several different mutations reduce pathogenicity in both hosts, indicating that they encode virulence factors for both plants and animals (Rahme et al. 1995). Biofertilizers Biofertilization accounts for approximately 65% of the nitrogen supply of crops worldwide. Rhizobiaceae and Mycorrhizae are the best-known examples of biofertilization. The former microbes are applied to increase crop yield from leguminous plants, such as soybean, alfalfa and clover grown in nitrogen-poor soil (Spaink et al. 1998). The Mycorrhizae are used for reforestation of fir plants and act by extending the rhizosphere, thereby enormously increasing the effective root system of the plant (Bonfante & Perotto 1995; Smith & Gianninazzi-Pearson 1998). In addition, rather recently several endophytic nitrogen fixers were discovered. Azoarcus is able to fix nitrogen in Kaller grass and rice (Reinhold-Hurek & Hurek 1998; Steenhoudt & Vanderleyden 2000) whereas Herbaspirillum and Acetobacter fix N2 in sugar cane (Okon et al. 1998). Roots of leguminous plants secrete flavonoids which induce Rhizobium bacteria to form nodules in a host-specific way. Some of these flavonoids are able to activate the NodD regulatory protein which results in activation of nod operon which encodes the biosynthesis of extremely host-specific Nod-metabolites (reviewed in Spaink et al. 2000). This host-specificity is an excellent example of evolutionary fine-tuning of interactions between microbes and plants. The bacteria enter the plant through infection threads, change into bacteroids in which form they convert atmospheric N2 into ammonia. This N-source can be used by the plant. To complete the symbiosis, the plant supplies the bacteroids with a dicarboxylic acid carbon source. Bacterial components which play a role in the recognition of the plant in various stages of the nodulation process include the Nod-factors, EPSs (extracellular polysaccharides), LPSs (lipopolysaccharides), K-antigens (which are structurally very distinct from LPSs) and periplasmatic cyclic glucans (Spaink et al. 2000). Some of these molecules are modified during the nodulation process (Goosen-de Roo et al. 1991; Lugtenberg 1998). The exact role of the PSs (polysaccharides) is hard to unravel since many mutants in the biosyntheses of these macromolecules are pleiotrophic whereas non-pleiotrophic mutations are sometimes complemented by other PSs. A typicality of the LPS of rhizobia is the presence of very long chain HO-fatty acids, such as 27-OH-C28:0 (Hollingsworth & Carlson 1989), which can also be present in Nod-factors (Spaink 2000). The membrane phospholipid PC (phosphatidylcholine) is a major lipid of eukaryotic membranes, but found in only few prokaryotes, such as Bradyrhizobium japonicum, in which 52% of the total phospholipid can consist of PC. A group lead by Otto Geiger and Isabel Lopez-Lara discovered a new PC biosynthetic pathway in which choline (present in the rhizosphere) and CDP-diacylglyceride are condensed (Sohlenkamp et al. 2000). B. japonicum mutants unable to synthesize PC are inefficient in their symbiosis with the soybean host plant (Minder et al. 2001). The genomes of two Rhizobiaceae have been sequenced. Interesting features are the following. (1) The presence of genes homologous to those encoding TTSSs (‘machines’) which are used by pathogenic bacteria to deliver virulence factors into host cells. Recent results show that the rhizobial TTSS secretes specific proteins and is involved in the establishment of the symbiosis. In some cases secreted proteins negatively affect the nodulation process, perhaps by suppressing plant defense responses (Viprey et al. 1998; Marie et al. 2001; Spaink et al. 2001). (2) The presence of a 502 kb chromosomally-integrated ‘symbiosis island’ in Mesorhizobium loti R7A. This island is integrated in a phe-tRNA gene in a process mediated by a P4 integrase of which the gene is located on one end of the island. The island can be transferred by conjugation to non-symbiotic mesorhizobia which converts them to Lotus symbionts. The island of strain R7A contains a Type IV secretion system similar to the VirB/VirD4 pilus from A. tumefaciens whereas a similar M. loti symbiosis island encodes a TTSS (Ronson et al. 2001). Natera et al. (2000) analysed proteomes of the symbiosis between Sinorhizobium meliloti strain 1021 and the legume Melilotus alba (white sweetclover) with the aim of characterizing symbiosis proteins. They reported that hundreds of proteins were present in altered amounts in the symbiotic vs. the nonsymbiotic situation. 379 Vesicular-arbuscular Mycorrhizae occur in over 80% of all terrestrial plant species (Bonfante & Perotto 1995). These fungi are obligate symbionts and colonize the cortex of the root in order to obtain carbon from their host plant, while assisting the plant with the uptake of mineral nutrients, predominantly phosphate, from the soil. In many soils the bioavailable phosphate is present in low concentrations which may be limiting for growth (Harrison et al. 1996). The arbuscular mycorrhizal fungus Gigaspora margarita harbors a resident population of as many as 250 000 endosymbiotic Burkholderia cells in the cytoplasm of one cell (Ruiz-Lozano & Bonfante 2000). Phytostimulators Some bacteria belonging to the genus Azospirillum promote plant growth as free-living organisms. A polar flagellum, of which the flagellin was shown to be a glycoprotein, is involved in attachment of Azospirillum cells to plant roots. Azospirillum fixes atmospheric nitrogen but the observed growth promotion may rather be related to plant growth promoters produced by the bacterium than by its nitrogen fixing capacity. Indeed, three types of plant growth promoting substances have been detected in the supernatant fluids of Azospirillum cultures, namely auxins, cytokinins and gibberellines. Of these, the auxin IAA (indole-3-acetic acid) is quantitatively the most important one. Three pathways are known to convert tryptophan into IAA. Inoculation experiments with Azospirillum mutants altered in IAA production support the view that after Azospirillum inoculation IAA causes increased rooting, which in turn enhances mineral uptake (Steenhoudt & Vanderleyden 2000). Similar results were obtained with a plant growth promoting P. fluorescens strain, which converts exogenous tryptophan into IAA. The strain promotes maturation of radish in greenhouse experiments, most likely due to the high concentration of tryptophan measured in radish exudate (G. Bloemberg, N. Makarova, T. Azarova, C. Mulders, L. Kravchenko, I.A. Tikhonovich and B. Lugtenberg, unpublished). Rhizoremediators Most environmental pollutants can be degraded by selected microbes. The process stops when the microbe is starved of food. In order to ascertain that such microbes can have access to the best food source available in soil, namely root exudates, Kuiper et al. (2001) have described an enrichment method for the isolation of microbes which combine the properties of: (i) degradation of a selected pollutant, and (ii) excellent root colonization. They have termed this process ‘rhizoremediation’ instead of phytoremediation to emphasize the roles of the root exudates and the rhizosphere competent microbe. Biocontrol agents or biopesticides Preparations of many microbes are commercially available as biological control agents to protect plants against fungi. The major applied organisms are the bacteria Bacillus, Pseudomonas and Streptomyces and the fungi Trichoderma, Gliocladium and Fusarium. The mechanisms used by these biocontrol agents include the following. (i). Niche exclusion [e.g. by the frost damage causing leaf colonizing P. syringae (Lindow 1995)]. (ii). Competition for nutrients [e.g. for carbon by Fusarium (Alabouvette 1986) and for Fe3+ siderophore complexes by P. putida strain WCS358 (Bakker et al. 1990)]. (iii). Production of chitinase by Serratia marcescens (Ordentlich et al. 1987) and by Trichoderma (Sivan & Chet 1989). (iv). Production of AFMs (antifungal metabolites), such as Phl (2,4-diacetyl phloroglucinol) [e.g. by pseudomonads (Keel et al. 1990; Bangera & Tomashow 1996), phenazine derivatives (Tomashow & Weller 1988; Pierson et al. 1995; Chin-A-Woeng et al. 1998; Delany et al. 2001), zwittermycin (Stohl et al. 1996), pyoluteorin (Nowak-Thompson et al. 1999), and pyrrolnitrin (Kirnes et al. 1998). Recently it has been discovered that certain biosurfactants also act as AFMs (Stanghellini & Miller 1997; Nielsen et al. 2000; Thrane et al. 2000). (v). ISR (Induced Systemic Resistance) is triggered by certain nonpathogenic Pseudomonas rhizobacteria which make the plant extremely reactive towards a range of pathogens, including leaf pathogens (van Peer et al. 1991; M’piga et al. 1997). Flagella, LPS (lipopolysaccharide) and siderophores have been implied as bacterial components involved in causing ISR (van Loon et al. 1998). ISR differs from SAR (systemic acquired resistance), which is induced upon pathogen infection. Simultaneous activation of SAR and ISR resulted in an additive effect on the level of induced protection of Arabidopsis thaliana against P. syringae pv. tomato 380 (van Wees et al. 2000). Ethylene signalling plays a role in P. fluorescens WCS417r mediated ISR functions. Introduction of the ACC deaminase gene, encoding the cytoplasmic enzyme ACC deaminase, which converts ACC (1-aminocyclopropane-1-carboxylic acid, a precursor of ethylene) to ammonia and α-ketobutyrate, into P. fluorescens strain CHAO improved its ability to protect cucumber against Pythium damping off. It was proposed that ACC deaminase decreases the level of pathogen-induced plant ethylene and therefore eliminates the pathogen-induced inhibitory effect of ethylene on root elongation (Wang et al. 2000). 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