Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
25 Jun 2001 10:48 AR AR138-18.tex AR138-18.SGM ARv2(2001/05/10) P1: GJB Annu. Rev. Phytopathol. 2001. 39:461–90 c 2001 by Annual Reviews. All rights reserved Copyright ° MOLECULAR DETERMINANTS OF RHIZOSPHERE COLONIZATION BY PSEUDOMONAS Ben J. J. Lugtenberg, Linda Dekkers, and Guido V. Bloemberg Leiden University, Institute of Molecular Plant Sciences, Clusius Laboratory Wassenaarseweg 64, 2333 AL Leiden, The Netherlands; e-mail: [email protected] Key Words Pseudomonas, root, rhizosphere competence, biocontrol, pathogenic fungi ■ Abstract Rhizosphere colonization is one of the first steps in the pathogenesis of soilborne microorganisms. It can also be crucial for the action of microbial inoculants used as biofertilizers, biopesticides, phytostimulators, and bioremediators. Pseudomonas, one of the best root colonizers, is therefore used as a model root colonizer. This review focuses on (a) the temporal-spatial description of root-colonizing bacteria as visualized by confocal laser scanning microscopal analysis of autofluorescent microorganisms, and (b) bacterial genes and traits involved in root colonization. The results show a strong parallel between traits used for the colonization of roots and of animal tissues, indicating the general importance of such a study. Finally, we identify several noteworthy areas for future research. CONTENTS INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ADVANTAGES OF A GNOTOBIOTIC SYSTEM FOR STUDYING RHIZOSPHERE COLONIZATION . . . . . . . . . . . . . . . . . . . . . . . . . . . SIMULTANEOUS IMAGING OF DIFFERENT POPULATIONS OF MICROBES IN THE RHIZOSPHERE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ISOLATION OF (COMPETITIVE) ROOT TIP COLONIZATION MUTANTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . FLAGELLA, MOTILITY, AND CHEMOTAXIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PILI AND TWITCHING MOTILITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . LIPOPOLYSACCHARIDE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . OTHER CELL SURFACE POLYSACCHARIDES . . . . . . . . . . . . . . . . . . . . . . . . . . . PROTOTROPHY FOR VITAMINS AND BUILDING BLOCKS . . . . . . . . . . . . . . . . NUTRITIONAL BASIS OF RHIZOSPHERE COLONIZATION . . . . . . . . . . . . . . . . INTERACTION OF P. FLUORESCENS WCS365 WITH PUTRESCINE IN EXUDATE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0066-4286/01/0901-0461$14.00 462 463 465 466 467 468 469 470 470 471 472 461 25 Jun 2001 10:48 462 AR AR138-18.tex LUGTENBERG ¥ AR138-18.SGM DEKKERS ¥ ARv2(2001/05/10) P1: GJB BLOEMBERG PROTEIN SECRETION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SITE-SPECIFIC RECOMBINATION AND COLONY PHASE VARIATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . OUTER MEMBRANE PERMEABILITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROLE OF NADH DEHYDROGENASES IN COMPETITIVE ROOT TIP COLONIZATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROLE OF RHIZOSPHERE COLONIZATION IN BIOCONTROL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . QUORUM SENSING AND MICROCOLONIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . COLONIZATION AND BIOFILM FORMATION . . . . . . . . . . . . . . . . . . . . . . . . . . . OTHER COLONIZATION MUTANTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PROMOTORS INDUCED BY EXUDATE AND IN THE RHIZOSPHERE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . COMPARISON OF GENES AND TRAITS INVOLVED IN COLONIZATION OF ROOTS AND OF ANIMAL TISSUES . . . . . . . . . . . . . . . . . CAN COLONIZATION BE IMPROVED? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . COLONIZATION AND FUNCTIONAL GENOMICS . . . . . . . . . . . . . . . . . . . . . . . . FUTURE PERSPECTIVES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473 473 475 475 476 477 477 478 478 478 479 480 481 INTRODUCTION Rhizosphere bacteria can have a profound effect on plant health. Rhizosphere colonization is important not only as the first step in pathogenesis of soilborne microorganisms, but also is crucial in the application of microorganisms for beneficial purposes. Most significant among these applications are biofertilization, phytostimulation, biocontrol, and phytoremediation (75). Colonizing microorganisms can be detected attached to the root, as free organisms in the rhizosphere (e.g., attracted to the root environment by nutrients present in exudate), or as endophytes. Endophytes, usually experimentally defined as not accessible to mild root surface sterilization, are not considered in this review. It has long been assumed that many microorganisms are attracted by nutrients exuded by plant roots. Indeed, this “rhizosphere effect” was first described in 1904 by Hiltner (58) who observed increased numbers and activity of microorganisms in the vicinity of plant roots. The methods chosen to study root colonization depend to a considerable degree on the particular problem to be studied. For example, determining which microorganisms enter the rhizosphere from the soil requires an experimental approach different from that needed to follow the fate of an inoculant bacterium after application on the seed for commercial purposes. For details on these strategies the reader is referred to a review by Kloepper & Beauchamp (65). Classical electron microscopy studies as well as the use of marked strains (33, 35, 118) have shown a nonuniform distribution of bacteria on the root: Some areas, including the extreme tip of the root, are practically free from bacteria whereas other areas can be heavily populated (10, 43, 45, 46, 87, 90, 110). With Pseudomonas the heavily populated 25 Jun 2001 10:48 AR AR138-18.tex AR138-18.SGM ARv2(2001/05/10) P1: GJB RHIZOSPHERE COLONIZATION BY PSEUDOMONAS 463 areas are usually found at junctions between epidermal root cells, indented parts of the epidermal surface, or sites of side root appearance (4, 21), all presumed sites of exudation. Rhizobium bacteria also attach to root hair tips (121, 122), presumably because of the presence of specific receptors (129). The observation that strain GB03 of Bacillus subtilis did not colonize the root (46) is consistent with results in our group showing that Bacillus is a poor colonizer (B. Lugtenberg, L. Dekkers & G.V. Bloemberg, unpublished data). Initially bacteria were not detected on the root because the root surface is covered by a distinct matrix, the mucigel (62). When this layer was disrupted during preparation for electron microscopy (42, 44) rhizosphere bacteria became visible. Using a modified method, Chin-A-Woeng et al (21) were able to make this layer semitransparant and visualize the bacteria underneath the mucigel layer by scanning electron microscopy (SEM). The mucigel layer does not prevent visualization of green fluorescent protein-(GFP) labeled bacteria using confocal laser scanning microscopy (CLSM) (4, 5, 27) or visualization of lacZ marked strains (8). Root colonization has been most intensively studied in Pseudomonas, the most effective root-colonizing bacterium. This review therefore mainly focuses on rootcolonization genes and traits of Pseudomonas. ADVANTAGES OF A GNOTOBIOTIC SYSTEM FOR STUDYING RHIZOSPHERE COLONIZATION The interactions between plants and microorganisms are immensely complex and very little is known about the sum of factors that lead to reliable biocontrol application (66). As one of our main purposes is to understand the mechanism of microbiological control of plant diseases caused by fungi at the molecular level, we have developed a less complex system devoid of field-soil variables, which therefore yields more reproducible results (120). In this system sterile germinated seedlings are grown after bacterization by incubating them in a suspension containing 107 to 108 CFUs/ml for 15 min. At 108 CFUs/ml the number of bacteria attaching to the seed is close to saturation (T.F.C. Chin-A-Woeng, unpublished data). After inoculation, the seedlings are aseptically placed 5 mm below the surface of a 10-cm long quartz sand column moisturized with a plant nutrient solution (PNS) without added carbon source. After growth for seven days the plant, including the sand particles adhering to the root, is removed. The root is divided into segments that are then shaken in phosphate buffered saline (PBS) to remove bacteria. The number of colony forming units (CFUs) is determined after plating (120). Colonization is evaluated by counting the numbers of released bacteria after shaking root segments for 20 min. in PBS. The high bacterial numbers at the root base (107 CFU/cm) rapidly decrease (to 103 to 104 CFU/cm at the root tip). The total number of wild-type P. fluorescens WCS365 bacteria on the root increases 16- to 11 Jul 2001 12:25 464 AR AR138-18.tex LUGTENBERG ¥ AR138-18.SGM DEKKERS ¥ ARv2(2001/05/10) P1: GJB BLOEMBERG 32-fold in 7 days. However, this does not necessarily mean that all applied bacteria make 4 to 5 divisions. The adhering sand contains few bacteria, and the shaking procedure removes practically all bacteria from the root (21). Since Loper et al (74) showed a log normal distribution of rhizosphere bacteria, the data are log10 (CFU + 1)/cm transformed before any statistical treatment. Most commercial inoculants are coated to the seed or are applied in the furrow where the bacteria can reach the young seedling. However, to increase reproducibility, bacterized seedlings are often used instead of seeds, because this allows the choice of a homogeneous set of seedlings at the start of the experiment. For potato, sterile stem cuttings are used after these have been allowed to root (31, 36). This gnotobiotic system functions well for tomato, radish, potato, cucumber, wheat, and grass. The advantages of the gnotobiotic system over the use of field soil are (a) better reproducibility due to the use of the plant growth medium; (b) higher numbers of the test bacterium on the root in the absence of competition from indigenous soil bacteria. For an efficient colonizer such as P. fluorescens WCS365 cell numbers are tenfold lower when quartz sand is replaced by potting soil; (c) simple testing of the effect of individual biotic factors, such as other bacteria (27), fungi (70), or protozoae; and (d ) easier screening of mutants for their colonizing ability (29, 119). Colonization is studied mainly in the context of biocontrol of tomato foot and root rot caused by Fusarium oxysporum f. sp. radicis lycopersici. Because this fungus can reside deep in the soil, the deeper root parts must also be protected by the biocontrol agent. Colonization is therefore defined as the ability of bacteria applied on the seed(ling) to reach the growing root tip. Using gfp-labeled microorganisms (see Figure 1), Lagopodi et al observed that the biocontrol bacteria P. fluorescens WCS365 and P. chlororaphis PCL1391 applied to the seedlings, and the pathogenic fungus Fusarium oxysporum f. sp. radicis lycopersici applied in the soil, occupy the same sites on the tomato root. The observation that the biocontrol bacterium occupies these sites faster than the phytopathogenic fungus presumably underlies a crucial aspect of the bacterium’s biocontrol ability (70). In the gnotobiotic system, all six tested single strains of wild-type pseudomonads, in the absence of competing organisms, reached the root tip. However, after inoculation of seedlings with a 1:1 mixture of two different wild-type strains, some strains (e.g. WCS315 and WCS358) performed poorly in competitive root tip colonization whereas WCS365 outcompeted the other five tested strains (120). That WCS365 was also the best strain of an inoculation mixture of 11 Pseudomonas strains in the colonization of a 100-cm long potato root system growing in soil (6) further supports the relevance of the gnotobiotic system to practical conditions. Additional support for choosing this strain as a model came from the observation that mutants, established to be defective in colonization in soil, also appeared to be defective in colonization in the gnotobiotic system (120). When wheat plants were grown gnotobiotically after inoculation with one of 150 single fluorescent Pseudomonas isolates from the wheat rhizosphere, 40% of 25 Jun 2001 10:48 AR AR138-18.tex AR138-18.SGM ARv2(2001/05/10) P1: GJB RHIZOSPHERE COLONIZATION BY PSEUDOMONAS 465 the strains inhibited plant growth, 20% had no effect, and 40% stimulated plant growth (9). Note that had this test been carried out in soil, the number of strains that affect plant growth would certainly have been smaller since soil contains high numbers of indigenous bacteria that would have competed with the inoculant strains for sites and nutrients from the rhizosphere. Consistent with this notion are the observations that barley plants grown under gnotobiotic conditions have increased susceptibility to two powdery mildew fungi (114) and that biocontrol Pseudomonas bacteria perform much better in suppressing tomato foot and root rot in a gnotobiotic system than in soil (A.L. Lagopodi, B. Lugtenberg & G. Bloemberg, unpublished). We have observed in gnotobiotic root colonization studies that the root tips of the monocots grass and wheat are colonized with ten times more Pseudomonas bacteria than are those of dicots such as potato, radish, and tomato. This observation correlates with a better growth yield in root exudates of monocots. SIMULTANEOUS IMAGING OF DIFFERENT POPULATIONS OF MICROBES IN THE RHIZOSPHERE Green fluorescent protein (GFP) (133) has been used as a marker for noninvasive observation of the fate of individual cells, and its gene has been used as a reporter for the activity of specific promoters (2) and to show that more than one rhizobial species can be present in the same nodule (128). The problem of GFP being too stable to study real-time gene expression has been overcome by constructing g fp mutants with a short half-life (107). This technology allows detailed studies of the behavior of individual cells in the rhizosphere (4, 144), e.g., with respect to ribosomal content and synthesis rates (40). WCS365 derivatives of P. fluorescens harboring genes encoding enhanced cyan, green, and yellow variants of GFP and encoding the red fluorescent protein (DsRed) were constructed (47) in the rhizosphere-stable cloning vector pME6010 (56). After seedling inoculation and gnotobiotic plant growth, the roots were isolated, rinsed briefly in PBS to remove most sand particles, then put on microscopic slides in PBS to prevent them from drying during microscopy. Using confocal laser microscopy, it was confirmed that Pseudomonas cells on the tomato root are present mainly as elongated stretches on indented areas, such as junctions between epidermal cells and the deeper parts of the epidermis on the root surface (4, 5), and on root hairs (27). Up to three differently labeled bacteria could be visualized simultaneously. Since the vector has a broad host range for Gram-negative bacteria, the constructed plasmids will be of great value in analyzing microbial communities in processes such as colonization, biofilm formation, competition for niches, and gene expression in the rhizosphere (5). Mixed inoculation of tomato seedlings with differently labeled WCS365 derivatives showed that mixed microcolonies are present predominantly on the upper part of the root whereas on the lower part of the root microcolonies consist of only one 25 Jun 2001 10:48 466 AR AR138-18.tex LUGTENBERG ¥ AR138-18.SGM DEKKERS ¥ ARv2(2001/05/10) P1: GJB BLOEMBERG population to which cells from another population are sometimes attached. This observation strongly suggests that (a) only infrequently does a single bacterium start forming a microcolony and (b) such a growing homogeneous microcolony is only infrequently reached by other cells (27). A low frequency of microcolony formation also explains the strong variation found among plants with respect to ratios of two strains found at the root tip after mixed inoculation. We find it surprising that growing microcolonies consist of one population for so long; rather we would expect many bacteria to use chemotaxis to reach a new site of exudation. The results suggest that a single bacterium rarely swims toward exudate. We propose that most bacteria are somehow contained, e.g., bound to the plant surface, caught under the mucigel layer, or aggregated to other bacteria. Aggregation brought about by PspB (12) may play an important role in this process. Mixed inoculation of tomato seedlings with two biocontrol pseudomonads, strain PCL1391 (acting through production of PCN) and strain WCS365 (acting through ISR) in equal numbers, showed that at the older part of the root the two cell types were present in larger microcolonies of either one or both cell types. Small microcolonies, usually of one cell type, and single cells were present at the middle part of the root. At the root tip, single cells or small groups of two to four cells, predominantly of one cell type, were observed. The observation that mixed microcolonies exist in the rhizosphere implies that two populations can influence each other extensively, through either direct contact or signal exchange. Direct contact could explain the observation (132, 140) that the frequency of recombination in the rhizosphere is high. Exchange of signal molecules by bacteria in the wheat rhizosphere has been reported by Pierson et al (98). Mixed inoculation with equal numbers of WCS365 and PCL1391 cells indicated that the two bacteria show some preference for different sites. On the middle part of the root, WCS365 cells were approximately five times more abundant, whereas root hairs were colonized almost exclusively by PCL1391 cells. Equal numbers of both cells were found at the root tip (27). Inoculation by a mixture of WCS365 and PCL1391 cells tended to improve biocontrol in comparison with cells of a single strain inoculation (27). We interpret this to be the result of a synergistic effect due to the use of two different mechanisms. This positive synergistic effect, however, is likely to be reduced by the twofold dilution of each strain (27). ISOLATION OF (COMPETITIVE) ROOT TIP COLONIZATION MUTANTS Two approaches have been used to isolate colonization mutants. In the targeted approach, mutants in traits possibly involved in colonization are tested against the parental strain for colonization ability. This approach showed the importance for colonization of the following traits: motility (36), production of the O-antigen of 25 Jun 2001 10:48 AR AR138-18.tex AR138-18.SGM ARv2(2001/05/10) P1: GJB RHIZOSPHERE COLONIZATION BY PSEUDOMONAS 467 lipopolysaccharide (LPS) (31), of cellulose (83), of thiamine (120), of amino acids (119), and of biotin (125), as well as the synthesis of an isoflavonoid-inducible efflux pump (93). In the second approach, based on the method developed by Lam et al (71), two mutants (labeled with Tn5 and Tn5lacZ, which allows their discrimination on X-gal plates) were screened in competition with each other on one tomato plant (29, 119), using the gnotobiotic system of Simons et al (120). After plant growth, bacteria were removed from the root tip and transfered to King’s medium B plates supplemented with X-gal. Putative colonization mutants were tested for motility and auxotrophy, already known as traits for colonization. After a second screening for competitive colonization on four plants, this time against the wild-type strain P. fluorescens WCS365 or its lacZ-marked derivative PCL1500, the remaining mutants were tested for their ability to synthesize the O-antigen of LPS. The remaining mutants were tested for competitive growth in liquid King’s medium B to avoid collecting mutants that are colonization defective because of aspecific poor growth. Mutants without any growth defect were considered as novel colonization mutants. The nature of their mutations is discussed later in this review. Starting with 1300 mutants, 15 appeared to be nonmotile, 13 auxotrophic for amino acids, 6 did not produce the O-antigen of LPS, 1 produced a shortened O-antigen side chain, and 11 mutants, including the 6 O-antigen deficient ones, had a 5% to 50% growth rate defect. Eight mutants remained and were analyzed to identify the novel genes and traits involved in colonization. When tested alone in the gnotobiotic system, these mutants all colonized the root system as well as their parental strain, although they were severely defective in competitive colonization, both in the gnotobiotic system (29) and in potting soil (28, 29; C. Mulders & B. Lugtenberg, unpublished). Furthermore, these mutants were all defective in competitive colonization on several crops, including the monocot wheat, indicating that they are impaired in colonization traits covering a broad host range (29). The temporal spatial competitive colonization behavior of wild-type WCS365 and colonization mutant PCL1233 was analyzed after inoculation of potato cuttings with a 1:1 mixture of these bacteria. The percentage of mutant cells gradually decreased along the root, and the percentage at the root tip decreased with time, i.e., with increasing root length (28). FLAGELLA, MOTILITY, AND CHEMOTAXIS Pseudomonas bacteria produce up to nine polar flagella per cell. Nonmotile mutants are easy to screen for since, unlike wild type, they do not swarm on medium containing 0.3% agar. The role of flagella in colonization has been disputed in the 1980s. Howie et al (60) and Scher et al (115) reported that nonmotile mutants of Pseudomonas are not impaired in root colonization of wheat and soybean, respectively. On the other hand, de Weger et al (36) found that 25 Jun 2001 10:48 468 AR AR138-18.tex LUGTENBERG ¥ AR138-18.SGM DEKKERS ¥ ARv2(2001/05/10) P1: GJB BLOEMBERG several different nonmotile mutants of P. fluorescens strain WCS374 were severely impaired in colonization of potato roots, with the defect largest at sites most distant from the site of inoculation. In later studies, using different parental Pseudomonas strains, soil systems, and plants (29), we observed that nonmotile mutants belong to the most defective competitive class of colonization mutants (19, 29, 120). To test whether the role of flagella in root colonization is based on random motility or on chemotaxis (e.g., to root exudate components), H. Vermeiren, J. Vanderleyden & R. de Mot, unpublished) isolated mutants in the general chemotaxis gene cheA of biocontrol strains WCS365 (27, 49), OE 28. 3 (30), SBW 25 (105), and F113 (117) of the European P. fluorescens. All mutants were as severely (100- to 1000-fold) defective in competitive tomato root tip colonization as nonmotile strains (C. Mulders, S. de Weert & B. Lugtenberg, unpublished), indicating that chemotaxis rather than random motility is involved in root colonization. Surprisingly, when nonmotile mutants or cheA mutants are tested alone in the gnotobiotic system, the ability to colonize the root tip is not significantly different from that of the wild type (S. de Weert & B. Lugtenberg, unpublished). This implies that other factors are also involved in transportation of bacteria to the root tip. Root growth is an obvious candidate. The realization that chemotaxis is important for colonization has been used to select bacterial strains that are good cereal root colonizers (67). PILI AND TWITCHING MOTILITY Pili or fimbriae are cell surface appendages involved mainly in attachment. The presence of pili has been described extensively for pathogens for which they are considered to be virulence factors (55, 127). In P. aeruginosa, type 4 pili mediate the initial contact between the bacteria and the epithelial cell surface (55, 97), are involved in a unique type of locomotion known as twitching motility (24), and have also been implicated in attaching to abiotic surfaces and forming biofilms (92). Roles for type 4 pili have also been described for plant pathogens (91, 109). Furthermore, type 4 pili are involved in colonization of both plant and fungal hosts of Azoarcus, an associative nitrogen-fixing bacterium (37). Although the WCS series of biocontrol pseudomonads had been isolated after thorough washing of the roots (49), and therefore could be assumed to be tightly bound to the plant root, several attempts by our group to visualize pili were unsuccessful. More recently, Camacho Carvajal (8) was able to visualize, after growth in vitro, an average of one to two type 4 pili per WCS365 cell, no pili on cells of a pilA mutant, and three to four pili on the surface of pilT mutant cells. pilA is the gene encoding prepilin, whereas pilT is involved in pilus retraction and is required for twitching motility (148). Furthermore, she has shown expression of the pilA promoter in the tomato rhizosphere and defects in competitive root tip colonization for the pilA mutant PCL1092 and for the pilT mutant PCL1093 (8). We conclude 25 Jun 2001 10:48 AR AR138-18.tex AR138-18.SGM ARv2(2001/05/10) P1: GJB RHIZOSPHERE COLONIZATION BY PSEUDOMONAS 469 that type 4 pili play a role in competitive tomato root tip colonization, presumably through twitching motility. We suggest that rhizosphere conditions could enhance the production and activity of type 4 pili. However, preliminary attempts to test the influence of exudate on pilA expression were not positive (M. Camacho Carvajal, G. Bloemberg & B. Lugtenberg, unpublished). LIPOPOLYSACCHARIDE Lipopolysaccharide (LPS) of Pseudomonas and of most other Gram-negative bacteria consists of lipidA, core, and O-antigen. The latter can consist of many repeating units. LPS of a pure culture is heterogenous with respect to the number of repeating units in its O-antigen. The result is that upon sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) a ladder pattern forms in which every band represents a different LPS molecule, with the O-antigen having a distinct number of repeating units. Short molecules run fastest (52, 94). Different Pseudomonas strains produce LPSs that differ in ladder pattern obtained after SDS-PAGE (34). Since LPS acts as the receptor for many bacteriophages, mutants in LPS structure can simply be obtained by isolating clones resistant to such phages (31). Mutants devoid of the LPS ladder pattern, and therefore defective in the O-antigenic side chain, appear to be impaired in potato root colonization, as judged from tests in clay soil (31) and in the gnotobiotic quartz sand system (120). As expected from these results, screening of large numbers of mutants for competitive root tip colonization in the gnotobiotic system also yielded several mutants with a defective O-antigen (29). Originally the interpretation of defects in root colonization by mutants missing their O-antigen was difficult because these mutants had serious growth rate defects in both laboratory medium (29) and in root exudate (C. Phoelich, L.C. Dekkers & B. Lugtenberg, unpublished). However, from the screening of 1300 mutants on competitive root colonization defects, the colonization mutant strain PCL1205 appeared to have a shortened LPS ladder, next to six mutants that were completely missing the ladder (29). In contrast to these six mutants, strain PCL1205 had a normal growth rate in laboratory medium and in exudate despite its colonization defect. Therefore, the simplest explanation of these results is that part of the O-antigen is required for competitive colonization but not for maximal growth rate. The mechanism by which this part of the O-antigen plays a role in colonization is not known. Mutant PCL1216 appeared to have a mutation in a gene homologous to htrB from Haemophilus influenzae (72). The LPS of both mutants migrates faster than that of their corresponding wild-type strain (29, 72). HtrB of Escherichia coli encodes a lauroyl transferase that uses (KDO)2 lipid IVA as the laurate receptor (22). The assumption that lipid A is underacylated explains the faster migration of the LPSs and also why PCL1216 cells lose competition, because of a defective outer membrane structure, in root exudate (77). 25 Jun 2001 10:48 470 AR AR138-18.tex LUGTENBERG ¥ AR138-18.SGM DEKKERS ¥ ARv2(2001/05/10) P1: GJB BLOEMBERG OTHER CELL SURFACE POLYSACCHARIDES Matthysse & McMahan used a root colonization assay that involved dipping roots in a suspension of Agrobacterium tumefaciens cells, followed by growth for 10 days. Bacterial numbers on the tomato root increased 10,000-fold during this period. A cellulose-minus mutant (84) colonized the tomato root 10,000-fold less and the Arabidopsis root 10- to 100-fold less (83). De Weger et al described a polysaccharide of P. putida WCS358 that shares characteristics with K-antigens of E. coli. A mutant lacking this polysaccharide, isolated using magnetic monoclonal antibodies, had wild-type colonizing ability on potato root (32). PROTOTROPHY FOR VITAMINS AND BUILDING BLOCKS As early as in 1961 (112), biotin was reported to be present in exudates of all ten plants tested in amounts sufficient to influence the growth of rhizosphere microorganisms (112). Indeed, Streit et al (125, 126) reported that 4 nM biotin promotes growth of strain 1021 of Rhizobium meliloti in laboratory medium. These authors reported that growth of the same strain in the rhizosphere is supported by the watersoluble vitamins biotin, thiamine, and riboflavin (96, 126). Mutants of R. meliloti unable to synthesize biotin are poor competitors in the alfalfa rhizosphere (126). Similarly, a thiamine auxotroph of P. fluorescens WCS365 appeared to be a poor competitor in the tomato rhizosphere (120). Apparently, the ability to synthesize these vitamins is an important colonization trait. The presence of amino acids in root exudates has been reported many times (48, 64, 96, 102, 134–139). In an analysis of tomato root exudate, we found that aspartic acid, glutamic acid, isoleucine, leucine, and lysine are the major amino acid components. To test whether the amino acid level in exudate is sufficient to support growth of auxotrophs, five different mutants of strain WCS365 unable to synthesize leucine, arginine, histidine, valine + isoleucine, and tryptophan, respectively, were isolated. None of these mutants was able to colonize the tomato root tip in the gnotobiotic system, either alone or after coinoculation with the wildtype strain. However, addition of the appropriate amino acid to the system restored colonization by the mutants, usually to wild-type levels (119). Glandorf (51) had reported previously that amino acid auxotrophs of WCS365 are impaired in potato root colonization in field soil. Apparently the ability to synthesize amino acids is also an important colonization trait. The competitive colonization mutant PCL1218 of P. fluorescens WCS365, which also loses competition in exudate, has a transposon insertion in two overlapping genes with different orientations, namely wbpN and tyrB. Gene wbpN plays an unknown role in lipooligosaccharide synthesis. Gene tyrB encodes an aromatic amino acid amino transferase. Since the addition of tyrosine, phenylalanine, aspartic acid, and leucine restores both competitive colonization and competitive 25 Jun 2001 10:48 AR AR138-18.tex AR138-18.SGM ARv2(2001/05/10) P1: GJB RHIZOSPHERE COLONIZATION BY PSEUDOMONAS 471 growth in exudate, it was concluded that the colonization defect is caused by the tyrB mutation (77). The mutant PCL1202 of P. fluorescens WCS365 is impaired in competitive tomato root tip colonization as well as in competitive growth in tomato root exudate. The transposon Tn5lacZ is inserted in the pyrR gene, which encodes a regulatory protein of the de novo pyrimidine biosynthetic pathway. Downstream of pyrR the pyrimidine biosynthetic genes pyrB and pyrC were identified. An independently constructed pyrR mutant also appeared to be defective in competitive colonization ability. Addition of exogenous uracil restored both competitive colonization as well as competitive growth on exudate, despite the observation that PCL1202 is not auxotrophic. Expression studies showed that PyrR has a positive effect on the transcription of the pyrB promoter. These results strongly suggest that this enhanced transcription is required for optimal competition in the rhizosphere. Although no function for PyrR of P. fluorescens was apparent under laboratory conditions, the tests under rhizosphere conditions described here revealed a function for this protein (8). NUTRITIONAL BASIS OF RHIZOSPHERE COLONIZATION Root exudates are important carbon and energy sources for colonizing microorganisms. Thirty percent of the total net photosynthesis in wheat seedlings is used for rhizosphere respiration by roots and associated microorganisms. Sixty percent of this amount was attributed to microbial respiration of exudates (14, 111). The presence of bacteria can alter the profile of exuded metabolites (145, 151). Extracellular metabolites of various microorganisms, such as Pseudomonas and Fusarium oxysporum, increase carbon exudation significantly (87). Generally, amino acids, monosaccharides, and organic acids are considered as the major exudate compounds, but other components can also be present (96). We have determined the amino acid (119), sugar (78), and organic acid (79) composition of tomato root exudate and used mutants to evaluate the importance of these components for proliferation and root colonization. Glucose, xylose, and fructose are the major monosaccharides in tomato root exudate (78). A mutant was isolated that was impaired in growth on the sugars glucose, fructose, sucrose, and xylose but which grew normally on organic acids such as succinate. The mutation in this mutant, strain PCL1083, was localized in zwf, which encodes glucose-6-phosphate dehydrogenase. The mutant was able to compete with the wild type for tomato root tip colonization, indicating that utilization of exudate sugars is not important for rhizosphere competence (78). The major organic acids in seedling exudate are citric, oxalic, pyruvic, and lactic acids, whereas citric, malic, lactic, and succinic acids are the major organic acids of root exudate. The total amount of organic acids in root exudate is five times higher than the amount of sugars (79). Mutant PCL1085 of P. fluorescens strain WCS365 was isolated and found to grow poorly in vitro on the organic acids malic 25 Jun 2001 10:48 472 AR AR138-18.tex LUGTENBERG ¥ AR138-18.SGM DEKKERS ¥ ARv2(2001/05/10) P1: GJB BLOEMBERG and succinic acid and moderately on citric acid. Consistent with this result, the mutation was found just upstream of the single gene mdh operon, which encodes malate dehydrogenase. Mutant PCL1085 appeared to be such a poor competitive colonizer on tomato root tips that it was completely outcompeted by its parental strain. These results are consistent with the composition of the tomato root exudate and also show that utilization of exudate organic acids is the nutritional basis of tomato root colonization by Pseudomonas. These data open opportunities for choosing sets of beneficial microbe-plant pairs based on exudate composition and high bacterial growth rate on the major exudate carbon sources (79). INTERACTION OF P. FLUORESCENS WCS365 WITH PUTRESCINE IN EXUDATE The competitive colonization mutant PCL1206 has a mutation immediately upstream of the pot operon, which encodes the putrescine uptake system and consists of two potF genes (potF encodes a putative putrescine binding protein), followed by potG, potH, and potI. This observation suggested the presence of the polyamine putrescine in exudate. Indeed, putrescine, but not the polyamines cadaverine, spermine, and spermidine, is present in tomato root exudate, its concentration being higher in exudate of 21-day-old plants than in that of 8-day-old plants. The mutant shows impaired growth on putrescine as the sole N-source. The obvious interpretation, namely that putrescine is an important N-source for the root colonizing strain WCS365, was unlikely since the plant nutrient solution present in the gnotobiotic system contains sufficient nitrate (5 mM). A new mutant, strain PCL1270, was constructed within the first potF gene. This strain is not impaired in colonization. Subsequent experiments on the effect of putrescine on growth, viability, and on the rate of 14C-putrescine uptake resulted in the following interpretation. Wild-type WCS365 takes up putrescine to a certain intracellular level that is determined by a regulator protein which can prevent transcription of the pot operon. In strain PCL1206 the binding site for this regulator protein is mutated. This causes excessive putrescine uptake in strain PCL1206, which results in transient bacteriostasis but does not cause cell death. This phenomenon is likely to be the cause of its colonization defect. The other mutant, strain PCL1270, is unable to take up putrescine owing to its potF mutation. It uses nitrate as the N-source in the rhizosphere when studied in the gnotobiotic system. In retrospect, mutant PCL1206 did not contribute to our knowledge of the colonization process per se. However, analysis of this mutant strain indicated that (a) putrescine is present in exudate; (b) Pseudomonas is able to protect itself against levels of putrescine that otherwise would cause slower growth; and (c) putrescine concentrations in the rhizosphere are bacteriostatic unless the uptake of this polyamine is carefully regulated (68). Interestingly, the pot operon of Agrobacterium tumefaciens has been implicated in attachment of bacterial cells to carrot suspension culture cells. The ability of 25 Jun 2001 10:48 AR AR138-18.tex AR138-18.SGM ARv2(2001/05/10) P1: GJB RHIZOSPHERE COLONIZATION BY PSEUDOMONAS 473 mutants to bind to host cells was restored by the addition of conditioned medium (85). The relationship of this finding with P. fluorescens mutant PCL1206 is not clear. PROTEIN SECRETION Mutant PCL1268 has a mutation in the single gene operon secB but no growth rate defect (I. Kuiper, G. Bloemberg & B. Lugtenberg, unpublished). In E. coli this gene encodes a chaperonin involved in secretion of proteins over the cytoplasmic membrane, such as periplasmic proteins, outer membrane proteins, and extracellular proteins. Since no extracellular proteins are known to be secreted by WCS365, we assume that the colonization defect caused by secB affects one or more periplasmic and/or outer membrane proteins involved in colonization. Rainey (104) described many genes of the plant-growth-promoting SBW25 strain of P. fluorescens that are specifically induced during rhizosphere colonization. About one third of these genes showed significant homology to sequences in GenBank that are involved in nutrient acquisition, stress response, or secretion. Interestingly, one gene is a homologue of hrcC, a type III secretion pathway, not previously described in nonparasitic bacteria (104). In a more recent study, it was shown that hrcC resides in a 20-kb gene cluster that encodes a functional secretion pathway capable of delivering ArrB from P. syringae to plant cells. hrp genes appeared to be widespread among Pseudomonas spp, suggesting that type III secretion has functional significance in both pathogens and plant growth promoting rhizobacteria (101). SITE-SPECIFIC RECOMBINATION AND COLONY PHASE VARIATION Mutant PCL1233 is at least 50-fold impaired in competitive root tip colonization in the gnotobiotic system on tomato, potato, and wheat. It is also colonization defective on tomato when tested in potting soil. The transposon in this mutant resides in the orf 235/233 homologue of a partly sequenced operon consisting of lppL, lysA, dapF, orf 235/233, xerC/sss, and the largely incomplete orf 238. Further studies have revealed that the xerC/sss homologue is crucial for colonization (28). xerC in E. coli and sss in P. aeruginosa belong to the λ-integrase family of site-specific recombinases, which play a role in phase variation caused by DNA rearrangements. These enzymes promote conservative reciprocal recombination, which does not require DNA synthesis, between two small (approximately 15-bp) homologous DNA sequences. The presence and orientation of two of these sequences will lead to inversion or excision of the DNA fragment situated between these small recognition sites (113). 25 Jun 2001 10:48 474 AR AR138-18.tex LUGTENBERG ¥ AR138-18.SGM DEKKERS ¥ ARv2(2001/05/10) P1: GJB BLOEMBERG Most DNA rearrangements involved in phenotypic switching regulate expression of phase-variable surface antigens, such as fimbriae, flagella, LPS, and lipoprotein. These events have been studied in detail in animal pathogens for which it makes sense to change the cell surface when another niche has to be occupied (e.g., within host cells the presence of flagella is probably a handicap) or to escape the animal’s (immune) defense system. For the biocontrol bacterium P. fluorescens WCS365, we favor Dybvig’s hypothesis (39) that subpopulations generated by DNA rearrangements enable a bacterial population to respond adequately at all times to environmental changes. According to this notion, WCS365 is present in at least two subpopulations and its sss mutant PCL1233 is locked in a genetic configuration that is not optimally rhizosphere competent (28). DNA rearrangements are often associated with colony sector formation. Indeed, it was found that 5-day-old colonies of P. fluorescens strain WCS365 contain morphologically distinct sectors. Sectoring was observed less frequently in sss mutant strain PCL1233 (28). Höfte et al (59) have described a sss mutant of P. aeruginosa. The major defect in this sss mutant was its inability to produce the siderophore pyoverdin in a certain medium. Furthermore, the mutant had slight defects in log phase growth and in colonization. This mutant, strain 7NSK2, is therefore different from PCL1233, which is unaffected in growth and siderophore synthesis but has a serious colonization defect (28). To identify genes encoding traits that are targets of sss, an sss mutant of strain PCL1391 of P. chlororaphis, which has many traits relevant for biocontrol (20, 21), was tested. The sss derivative PCL1126 appeared not to be altered in the production of PCN, chitinase, protease, and HCN. The observation that PCL1126 is defective in competitive tomato root tip colonization and biocontrol (19) shows that targets of sss other than the intact traits already noted are involved in colonization and biocontrol. After the discovery that colony phase variation plays a role in colonization, we investigated a series of 43 Pseudomonas strains from which colony phase variation was obvious from segmented colonies (usually opaque and translucent). From this work it appeared that production of antifungal activity (as judged from antagonism on plates), biosurfactant and chitinase are correlated with colony phase variation, whereas the production of lipase, protease, and siderophore is often, but not always, correlated with colony phase variation (D. van den Broek, T. F. C. Chin-A-Woeng, G. Bloemberg & B. Lugtenberg, unpublished). Vesper (143) described two colony types for the PCA (phenazine-1-carboxylic acid) producing biocontrol strain 2-79 of P. fluorescens (131). He observed that, in contrast to cells of mucoid colonies, those of the nonmucoid colonies were highly piliated and showed hydrophobic attachment to polystyrene, attachment to corn roots, and were active in hemagglutination (143). Recently, a new Pseudomonas species, Pseudomonas brassicacearum, was described as a root colonizer of Arabidopsis thaliana and Brassica napus (1). After growth for prolonged periods on rich media a new colony type appeared that, unlike the original “phase I” isolate, showed fluorescent pigmentation when grown 25 Jun 2001 10:48 AR AR138-18.tex AR138-18.SGM ARv2(2001/05/10) P1: GJB RHIZOSPHERE COLONIZATION BY PSEUDOMONAS 475 in iron-poor medium. Furthermore, this “phase II” variant differs from the original isolates with respect to nitrate reduction and does not hydrolyze gelatin and Tween 20 (1), indicative of extracellular protease and lipase, respectively. Using a DNA probe containing the alkaline protease gene aprA from P. aeruginosa, Chabeaud et al (12) identified hybridizing signals with DNA fragments of identical size in digests of both phases of P. brassicacearum. Further cloning resulted in the identification of a fragment containing a single operon containing seven ORFs of which the predicted proteins are homologous to alkaline protease (aprA), protease inhibitor (aprI), and the ABC transporter (aprDEF) of P. aeruginosa, and to the putative serine protease (pspB) and the lipase (lipA) of P. fluorescens. Since the operon was only expressed in phase I cells, its expression must be controlled by phase variation (12). No serine protease activity was detected when the serine protease gene was expressed in E. coli (12). Since many members of the autotransporter family function as adhesins (57), the notion that PspB might be involved in the adhesion of bacteria to plant roots was examined. E. coli cells harboring the pspB containing fragments did indeed form large aggregates (12). We suggest that autoaggregation could be very relevant for colonization and biocontrol, e.g., it could play a role in microcolony formation, which is likely to be important for biocontrol (21). OUTER MEMBRANE PERMEABILITY Mutant PCL1210 of P. fluorescens WCS365 is impaired in the two-component sensor/response regulator system colR/colS (25) downstream of which a putative orf222-inaA/wapQ operon was found (26). Since wapQ encodes a putative heptose kinase and since two-component systems often regulate a nearby operon, we tested whether the integrity of the mutant’s outer membrane was impaired because of a defective LPS. Indeed, mutant PCL1210 is more resistant to various chemically unrelated antibiotics and grows more slowly on some nutrients. The only single factor to explain these results is a less permeable outer membrane. Further evidence for an impaired outer membrane came from the finding that the mutant is more sensitive toward the antibiotic polymixin B, which attacks the outer membrane from the outside (76). We conclude that mutant PCL1210 has a less permeable outer membrane, presumably a disadvantage in the competitive uptake of exudate components on the root (26). ROLE OF NADH DEHYDROGENASES IN COMPETITIVE ROOT TIP COLONIZATION Dekkers et al (29) found that a mutant of P. fluorescens WCS365 in the nuoD gene is impaired in competitive root colonization. Two membrane-bound NADH dehydrogenases have been identified in E. coli: NADH: ubiquinone oxidoreductase or NADH dehydrogenase I (NDH-I), which is encoded by the 14-gene nuo 25 Jun 2001 10:48 476 AR AR138-18.tex LUGTENBERG ¥ AR138-18.SGM DEKKERS ¥ ARv2(2001/05/10) P1: GJB BLOEMBERG operon (147), and NDH-II, which is encoded by ndh (149, 150). Dekkers et al (29) observed that a competitive root colonization defective mutant of P. fluorescens WCS365 has its mutation in a nuoD homologue, suggesting a role for NDH-I in colonization (29). By constructing a second nuoD mutant and biochemically characterizing the enzymes, Camacho Caraval (8) demonstrated that the colonization defect is indeed due to the defective NDH-I. They showed further the presence of NDH-II encoded by ndh in P. fluorescens and also that a mutation in this gene has no effect on competitive colonization. Both the nuo and ndh promoters are expressed in the rhizosphere (8). NDH-I is involved in the generation of the proton motive force, which can be used for uptake of various nutrients, generation of ATP, and ATP-dependent rotation of flagella. The finding that NDH-I plays a role in colonization can be understood in this context. ROLE OF RHIZOSPHERE COLONIZATION IN BIOCONTROL The ability of P. putida to utilize heterologous siderophores can contribute to the bacterium’s competence in the rhizosphere (103). The production of phenazine antibiotics contributes to ecological fitness by competing with the resident microflora (86). Biocontrol mechanisms of bacteria, such as certain Pseudomonas strains, are usually based on secreted bioactive factors that attack the pathogen, e.g., antibiotics, exo-enzymes, or HCN (reviewed in 131). Colonization of large parts of the root system will obviously facilitate biocontrol since colonization can be expected to function as the delivery system for bacterial cells that act as factories of antifungal metabolites. Indeed, Schippers et al (116) showed that inadequate colonization leads to decreased biocontrol activity, and Bull et al (7) reported an inverse relation between the numbers of bacteria present on the wheat root and the number of take-all lesions seen on the plant. On the other hand, Roberts et al (108) claimed that colonization is not important for biocontrol. The availability of a series of different colonization genes made it possible to test thoroughly whether colonization is required for biocontrol (19). As a test system we used tomato root and foot rot caused by the fungus Fusarium oxysporum f. sp. radicis lycopersici. Strain PCL1391 of P. chlororaphis controls this disease through the production of the antifungal metabolite phenazine-1-carboxamide (PCN) (19). Different colonization-defective mutants of this strain were constructed that were nonmotile, auxotrophic for phenylalanine, or contained a tetracyclin cassette in the sss gene. The mutants appeared not to be altered in the production of the extracellular metabolites PCN, HCN, chitinase, and protease and were still antagonistic against the fungus in a plate test. All three mutants were impaired in colonization in the gnotobiotic system as well as in potting soil. In contrast to the parental strain, none of the mutants showed biocontrol activity. Therefore, these results showed for the first time that root colonization plays a crucial role in biocontrol, presumably 25 Jun 2001 10:48 AR AR138-18.tex AR138-18.SGM ARv2(2001/05/10) P1: GJB RHIZOSPHERE COLONIZATION BY PSEUDOMONAS 477 by functioning as the delivery system for cells producing the antifungal metabolite PCN (19). One biocontrol mechanism is not based on direct attack of the pathogen by the biocontrol microbe but on induction of systemic resistance of the plant by the microbe (reviewed in 141). Biocontrol by P. fluorescens WCS365 has been described to act by induced systemic resistance (ISR) (50), in which the mechanism is different from that of systemically acquired resistance (SAR) in that salicylic acid and pathogenesis-related (PR) proteins do not play a role in ISR. Mutations in various colonization genes of WCS365 did not consistently decrease the biocontrol activity of the strain (27), in contrast to observations for similar mutations in P. chlororaphis PCL1391. The results suggest that colonization plays a less important role, if any, in ISR-induced biocontrol. We propose that a temporary or local presence of ISR-inducing cells is sufficient to cause long-lasting resistance of the whole root system (27). QUORUM SENSING AND MICROCOLONIES Quorum sensing-dependent processes such as conjugation (99) and PCN production (16) require a minimal critical bacterial cell density. The mechanism of quorum sensing is based on the intracellular requirement of membrane-permeable N-acyl homoserine lactones (N-AHLs) as autoinducers and transcription factors (47). Pseudomonas biocontrol bacteria form microcolonies on plant roots (4, 21, 27, 47). We hypothesize that microcolonies are perfect sites for quorum sensing to take place because the bacterial cell density is high and the mucigel layer covering the bacteria could delay diffusion of N-AHLs. Also the observation by Van Elsas et al (140), that conjugation between pseudomonads is significantly and unexpectedly stimulated in the rhizosphere, has been attributed to quorum-sensing stimulated conjugation in microcolonies (21). COLONIZATION AND BIOFILM FORMATION In most natural, clinical, and industrial settings, bacteria are predominantly present in biofilms and not as planktonic cells such as those grown under laboratory conditions (23, 100). O’Toole & Kolter (92) have isolated mutants of strain WCS365 of P. fluorescens impaired in the initiation of biofilm formation, i.e., in the attachment to the abiotic polyvinylchloride (PVC) surface of microtiter dishes. They observed that protein synthesis is required for the initiation of biofilm formation and that the ClpP protein, a component of the cytoplasmic Clp protease, participates in biofilm formation. Biofilm formation is strongly influenced by environmental signals: The presence of Fe3+ and casamino-acids increased biofilm formation whereas high osmolarity decreased it (92). Among the surface attachment defective (sad ) mutants were many mutants in genes of unknown function (92). 25 Jun 2001 10:48 478 AR AR138-18.tex LUGTENBERG ¥ AR138-18.SGM DEKKERS ¥ ARv2(2001/05/10) P1: GJB BLOEMBERG Since microcolony formation on plant root surfaces can be considered as a form of biofilm formation, it was interesting to see that strains impaired in flagellar synthesis were found among sad mutants (92). OTHER COLONIZATION MUTANTS The Tn5lacZ competitive colonization mutant PCL1207 (29) spontaneously forms white colonies on the root after a single inoculation. The colonization defect is apparently caused by the fact that the transposon is unstable in this mutant, resulting in an altered ratio of blue and white colonies. The mutation of the competitive colonization mutant PCL1204 (29) is located in moxR, which encodes a regulatory gene of methanol dehydrogenase (L. Dekkers, S. de Weert, C. Mulders & B. Lugtenberg, unpublished). The mutations in PCL1208 and PCL1217 (29) are in genes with no significant homology to known genes. Espinoza-Urgel et al (40) have isolated KT2440 mutants of P. putida impaired in adhesion to seeds. Three of the eight sequences obtained show no obvious similarities with known genes, and therefore a novel function has been attributed to them. One gene showed limited similarities to surface proteins whereas the other four correspond to putative surface and membrane proteins: a calciumbinding protein, a hemolysin, a peptide transporter, and a potential multidrug efflux pump (8). PROMOTORS INDUCED BY EXUDATE AND IN THE RHIZOSPHERE Corn root exudates strongly induce put genes of P. putida that are involved in proline uptake and utilization (13). Van Overbeek & Van Elsas (142) described a Tn5lacZ R2f mutant of P. fluorescens in which the reporter gene was induced by wheat exudate and by proline. The induction is specific since root exudates of maize and grass were less effective, whereas clover root exudate was inactive. Rainey (104) identified promoters that are active in the rhizosphere but not in laboratory growth medium. COMPARISON OF GENES AND TRAITS INVOLVED IN COLONIZATION OF ROOTS AND OF ANIMAL TISSUES Several of the genes and traits important for root colonization have also been implicated in colonization of animal tissues by pathogenic bacteria. Flagella and motility were shown to be virulence factors in burn wound sepsis by P. aeruginosa (11). Motility plays a role in colonization of the host light organ by Vibrio fischeri 25 Jun 2001 10:48 AR AR138-18.tex AR138-18.SGM ARv2(2001/05/10) P1: GJB RHIZOSPHERE COLONIZATION BY PSEUDOMONAS 479 (53). LPS plays a role in colonization of the large intestine by S. typhimurium in mouse (89). The htrB gene, which is impaired in the competitive rhizosphere colonization mutant PCL1216 of P. fluorescens (29), encoding lauroyl transferase involved in the biosynthesis of the lipid A part of LPS (22), plays a role in colonizing the organs of the lymphatic system by S. typhimurium in mouse (63). LPS of the human pathogens Vibrio cholera, Klebsiella pneumoniae (95), Salmonella typhimurium, and Shiga toxin-producing E. coli (81) is essential to enable the pathogen to colonize animal tissues. Found among mutants of Vibrio cholerae impaired in colonization of the mouse intestine were auxotrophic mutants as well as mutants in the biosynthesis of biotin and LPS (15). Colonization mutants of a strain of E. coli that lost the capacity to colonize the GI tract of infant rats included mutants affected in LPS biosynthesis, tip adhesin on type 1 pili, and methionine biosynthesis (82). Interestingly, strain PAO-1 of the human pathogen P. aeruginosa appears to be a biocontrol strain since it is able to protect wheat and cucumber from the fungal pathogens Gaeumannomyces graminis and Pythium ultimum, respectively (see 132). Furthermore, the two established prerequisites for biocontrol of strain PCL1391, colonization (19) and PCN biosynthesis (20), are the same as the traits found to be important for pathogenesis by strain PA14 of the human opportunistic pathogen P. aeruginosa (130). CAN COLONIZATION BE IMPROVED? It seems reasonable to assume that colonization plays an important role in agroindustrial applications of microbial inoculants such as biocontrol (19, 27), biofertilization (124), phytostimulation, and phytoremediation (69). Three ways to improve colonization have been identified. First, introduction of multiple copies of an ssscontaining fragment from strain WCS365 into the poor colonizer P. fluorescens WCS307 and into the good colonizer P. fluorescens F113 increased the competitive tomato root tip colonization ability of the latter strains 16- to 40-fold, and 8- to 16-fold, respectively. These results show that improvement of the colonization ability of wild-type Pseudomonas strains by genetic engineering is a realistic goal (27). Note that introduction of the same fragment into WCS365 tended to decrease competitive root colonization, albeit not significantly. We interpret the results by assuming that multiple copies of sss result in an equilibrium between the various phases. According to this interpretation, the majority of the wild-type WCS365 cells would be in a colonization-competent state and multiple sss copies would lead to a decrease in colonization. In contrast, most cells of strains F113 and WCS307 would be in a colonization-incompetent state and multiple copies of sss would lead to a higher percentage of colonization-competent cells (27). Second, we have isolated enhanced colonizers by enrichment. To our knowledge, this classical microbiological approach had never been used to isolate plant-beneficial bacteria. In this particular case, the goal was to combine the abilities to colonize plant roots and to degrade the xenobiotic naphtalene. A suspension of bacteria 25 Jun 2001 10:48 480 AR AR138-18.tex LUGTENBERG ¥ AR138-18.SGM DEKKERS ¥ ARv2(2001/05/10) P1: GJB BLOEMBERG from the rhizosphere of a plant grown in naphtalene-contaminated soil was used to inoculate seedlings. After growth, bacteria that had reached the root tip were isolated, grown as a mixture on naphtalene as the sole carbon source, and harvested. The procedure was repeated twice. This approach resulted in the isolation of a bacterium, P. putida PCL1444, which is a 10-fold and 100-fold better root tip colonizer than strain WCS365 in the absence and presence, respectively, of naphtalene. We predict that this approach will also be applicable to isolating more effective biopesticides, biofertilizers, and phytostimulators owing to enhanced colonization properties (69). A third way to enhance colonization was found to be Tn5 mutagenesis of P. fluorescens WCS365 and subsequent enrichment for enhanced root tip colonizers, as described previously. This resulted in a variety of different mutants that are currently being characterized. Preliminary results indicate that one mutation is located in a homologue of the ferrisiderophore receptor gene (L. Dekkers, S. de Weert & B. Lugtenberg, unpublished). Consistent with this observation is the finding that the ability to utilize ferric pseudobactin M114 does not improve the ecological fitness of Pseudomonas sp. B24 in the rhizosphere (88). Webster et al (146) reported that endophytic colonization of wheat roots by Azorhizobium caulinodans can be improved by the presence of the flavonoid naringenin. Consistent with this result is the finding by Batinic et al (3) that biocontrol of Pythium ultimum Trow on cucumber by strain W24 of P. fluorescens can be significantly increased by various flavonoids. We did not find a significant effect of the presence of 10-µM naringenin in the gnotobiotic system on root tip colonization by P. fluorescens WCS365 (S. de Weert & B. Lugtenberg, unpublished). COLONIZATION AND FUNCTIONAL GENOMICS The complete nucleotide sequence of P. aeruginosa PAO-1 has been determined (123). Interestingly, this strain is an excellent rhizosphere colonizer of wheat (132). Once the DNA corresponding with open reading frames has been spotted on microarrays, many interesting questions can be answered: 1. Which genes are expressed during seed coating, on the seed, and in the rhizosphere? 2. Which global and specific regulatory genes next to phzI, phzR, gacS (17), gacA (106), and lexA (18) are involved in the synthesis of the antifungal metabolite PCN? 3. Through which genes and processes do environmental factors such as glycerol, oxygen limitation, and Fe3+ (54) exert their regulatory action on PCN expression? 4. What are the target genes of the regulatory genes colR/colS (25), moxR, and sss (28) involved in rhizosphere colonization? 25 Jun 2001 10:48 AR AR138-18.tex AR138-18.SGM ARv2(2001/05/10) P1: GJB RHIZOSPHERE COLONIZATION BY PSEUDOMONAS 481 5. Which novel genes are regulated by quorum sensing (17)? 6. Which genes are influenced by the presence of fungi? FUTURE PERSPECTIVES Two developments should dramatically influence our knowledge of rhizosphere microbiology. First, visualization technology, using CLSM in combination with various forms of GFP, will allow detailed studies on interactions between various organisms as well as temporal-spatial aspects of gene expression. Second, functional genomics (including proteomics and metabolomics) will allow us to identify all genes expressed in the rhizosphere. The use of promoters that are specifically expressed in the rhizosphere will allow the engineering of microorganisms for beneficial purposes with minimal loss of energy. Finally, the interactions between bacteria and fungi in the rhizosphere (41, 73) is an exciting new field of research. ACKNOWLEDGMENTS The research described here was supported by numerous grants from the Netherlands Organization for Scientific Research, either directly for Crop Protection and in cooperation with the Russian Federation or through the Foundations for Earth and Life Sciences, Chemical Sciences and Technological Sciences. Moreover, grants were obtained from the European Community through the programs BIOTECH (BIO2-CT93.0053, BIO2-CT93.0196, BIO4-CT96.0027, BIO4-CT96. 0181, and BIO4-CT98.0254), and Marie Curie fellowships (ERBFMBI-CT982930 and HPMF-CT-1999-00377). We thank the following colleagues for sending us relevant preprints and reprints: Dieter Haas, Thierry Heulin, Ann Matthijsse, Ole Nybrö, Jos Raaymakers, Paul Rainey, Juan Ramos, Jan Sørensen, Linda Tomashow, Jan Tommassen, and David Weller. We also thank Ludmila Frankova, Janneke van Gaal, Martijn E. Lugtenberg, and Martha Rodrigues Gomes for typing and literature searches. Visit the Annual Reviews home page at www.AnnualReviews.org LITERATURE CITED 1. Achouak W, Sutra L, Heulin T, Meyer J-M, Fromin N, et al. 2000. Pseudomonas brassicacearum sp. nov. and Pseudomonas thivervalensis sp. nov., two root-associated bacteria isolated form Arabidopsis thaliana and Brassica napus. Int. J. Syst. Bacteriol. 50:9–18 2. Andersen JB, Sternberg C, Poulsen LK, Bjorn SP, Givskov M, Molin S. 1998. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64:2240–46 3. Batinic T, Redecker D, Schulz U, 25 Jun 2001 10:48 482 4. 5. 6. 7. 8. 9. 10. AR AR138-18.tex LUGTENBERG ¥ AR138-18.SGM DEKKERS ¥ ARv2(2001/05/10) P1: GJB BLOEMBERG Vinuesa P, Werner D. 2000. Rhizobacteria as biocontrol agents. In Book of Abstracts, Eur. COST830 Workshop Selection Strategies for Plant-Beneficial Microorganisms, ed. N Champenoux, J Garbaye Bloemberg GV, O’Toole GA, Lugtenberg BJJ, Kolter R. 1997. Green fluorescent protein as a marker for Pseudomonas spp. Appl. Environ. Microbiol. 63:4543–51 Bloemberg GV, Wijfjes AHM, Lamers GEM, Stuurman N, Lugtenberg BJJ. 2000. Simultaneous imaging of Pseudomonas fluorescens WCS365 populations expressing three different autofluorescent proteins in the rhizosphere: new perspectives for studying microbial communities. Mol. Plant-Microbe Interact. 13:1170–76 Brand J, Lugtenberg BJJ, Glandorf DCM, Bakker PAHM, Schippers B, de Weger LA. 1991. Isolation and characterization of a superior potato root-colonizing Pseudomonas strain. In Plant GrowthPromoting Rhizobacteria—Progress and Prospects, ed. C Keel, B Knoller, G Défago, pp. 350–54. Interlaken, Switz: Int. Org. Biol. Integr. Control Noxious Anim. Plants Bull CT, Weller DM, Thomashow LS. 1991. Relationship between root colonization and suppression of Gaeumannomyces graminis var. triciti by Pseudomonas strain 2-79. Phytopathology 81: 954–59 Camacho Carvajal MM. 2001. Molecular characterization of the roles of type 4 pili, NDH-I and PyrR in rhizosphere colonization of Pseudomonas fluorescens WCS365. PhD thesis, Univ. Leiden, The Netherlands Campbell R, Greaves MP. 1990. Anatomy and community structure of the rhizosphere. In The Rhizosphere, ed. JM Lynch, pp. 11–34. Chichester, UK: Wiley & Sons Campbell R, Rovira AD. 1973. The study of the rhizosphere by scanning electron microscopy. Soil Biol. Biochem. 5:747–52 11. Camprubi S, Merino S, Guillot JF, Tomas JM. 1993. The role of the Oantigen lipopolysaccharide on the colonization in vivo of the germfree chicken gut by Klebsiella pneumoniae. Microb. Pathog. 14:433–40 12. Chabeaud P, de Groot A, Bitter W, Tommassen J, Heulin T, Achouak W. 2001. Phase-variable expression of an operon encoding extracellular alkaline protease, serine protease homologue and lipase in Pseudomonas brassicacearum. J. Bacteriol. 183:2117–20 13. Chabot R, Antoun H, Kloepper JW, Beauchamp CJ. 1996. Root colonization of maize and lettuce by bioluminescent Rhizobium leguminosarum biovar phaseoli. Appl. Environ. Microbiol. 62:2767–72 14. Cheng WX, Coleman DC, Carroll CR, Hoffman CA. 1993. In situ measurement of root respiration and soluble Cconcentrations in the rhizosphere. Soil Biol. Biochem. 25:1189–96 15. Chiang SL, Mekalanos JJ. 1998. Use of signature-tagged transposon mutagenesis to identify Vibrio cholerae genes critical for colonization. Mol. Microbiol. 27:797– 805 16. Chin-A-Woeng TFC, Van den Broek D, De Voer G, Van der Drift KMGM, Thomas-Oates JE, et al. 2001. Phenazine1-carboxamide production in the biocontrol strain Pseudomonas chlororaphis PCL1391 is regulated by multiple factors secreted in the growth medium. Mol. Plant-Microbe Interact. In press 17. Chin-A-Woeng TFC, Van den Broek D, Bloemberg GV, Lugtenberg BJJ. 2001. Phenazine-1-carboxamide production in the biocontrol strain Pseudomonas chlororaphis PCL1391: positive and negative regulation by GacS and LexA. Submitted 18. Chin-A-Woeng TFC. 2000. Molecular basis of biocontrol of tomato foot and root rot by Pseudomonas chlororaphis strain PCL1391. PhD thesis, Univ. Leiden, The Netherlands 19. Chin-A-Woeng TFC, Bloemberg GV, 25 Jun 2001 10:48 AR AR138-18.tex AR138-18.SGM ARv2(2001/05/10) P1: GJB RHIZOSPHERE COLONIZATION BY PSEUDOMONAS 20. 21. 22. 23. 24. 25. 26. Mulders IHM, Dekkers LC, Lugtenberg BJJ. 2000. Root colonization is essential for biocontrol of tomato foot and root rot by the phenazine-1-carboxamide-producing bacterium Pseudomonas chlororaphis PCL1391. Mol. PlantMicrobe Interact. 13:1340–45 Chin-A-Woeng TFC, Bloemberg GV, van der Bij AJ, van der Drift KMGM, Schripsema J, et al. 1998. Biocontrol by phenazine-1-carboxamide-producing Pseudomonas chlororaphis PCL1391 of tomato root rot caused by Fusarium oxysporum f.sp. radicis-lycopersici. Mol. Plant-Microbe Interact. 11:1069–77 Chin-A-Woeng TFC, de Priester W, van der Bij AJ, Lugtenberg BJJ. 1997. Description of the colonization of a gnotobiotic tomato rhizosphere by Pseudomonas fluorescens biocontrol strain WCS365 using scanning electron microscopy. Mol. Plant-Microbe Interact. 10:79– 86 Clementz T, Bednarski JJ, Raetz CRH. 1996. Function of the htrB high temperature requirement gene of Escherichia coli in the acylation of lipidA. J. Biol. Chem. 271:12095–102 Costerton JW, Lewandowski Z, Caldwell DE, Korber DR, Lappin-Scott HM. 1995. Microbial biofilms. Annu. Rev. Microbiol. 49:711–45 Darzins A, Russell MA, 1997. Molecular genetic analysis of type-4 pilus biogenesis and twitching motility using Pseudomonas aeruginosa as a model system-a review. Gene 192:109–15 Dekkers LC, Bloemendaal CJP, de Weger LA, Wijffelman CA, Spaink HP, Lugtenberg BJJ. 1998. A two-component system plays an important role in the root-colonizing ability of Pseudomonas fluorescens strain WCS365. Mol. PlantMicrobe Interact. 11:45–56 Dekkers LC, Lugtenberg BJJ. 2001. Two component colR/colS system of Pseudomonas fluorescens WCS365 plays a role in rhizosphere competence through 27. 28. 29. 30. 31. 32. 483 maintaining the integrity of the outer membrane. Submitted Dekkers LC, Mulders IHM, Phoelich CC, Chin-A-Woeng TFC, Wijfjes AHM, Lugtenberg BJJ. 2000. The sss colonization gene of the tomato-Fusarium oxysporum f. sp. radicis-lycopersici biocontrol strain Pseudomonas fluorescens WCS365 can improve root colonization of other wild type Pseudomonas bacteria. Mol. Plant-Microbe Interact. 13:1177– 83 Dekkers LC, Phoelich C.C., van der Fits L, Lugtenberg BJJ. 1998. A site specific recombinase is required for competitive root colonization by Psudomonas fluorescens WCS365. Proc. Natl. Acad. Sci. USA 95:7051–56 Dekkers LC, van der Bij AJ, Mulders IHM, Phoelich CC, Wentwoord RAR, et al. 1998. Role of the O-antigen of lipopolysaccharide, and possible roles of growth rate and NADH:ubiquinone oxidoreductase (nuo) in competitive tomato root-tip colonization by Pseudomonas fluorescens WCS365. Mol. Plant-Microbe Interact. 11:763–71 de Mot R, Vanderleyden J. 1991. Purification of a root-adhesive outer membrane protein of root-colonising Pseudomonas fluorescens. FEMS Microbiol. Lett 81:323–28 de Weger LA, Bakker PAHM, Schippers B, van Loosdrecht MCM, Lugtenberg BJJ. 1989. Pseudomonas spp. with mutational changes in the O-antigenic side chain of their lipopolysaccharide are affected in their ability to colonize potato roots. In Signal Molecules in Plants and Plant-Microbe Interactions, ed. BJJ Lugtenberg, pp. 197–202. Berlin: Springer Verlag de Weger LA, Bloemberg GV, van Wezel T, van Raamsdonk M, Glandorf DCM, et al. 1996. A novel cell surface polysaccharide in Pseudomonas putida WCS358, which shares characteristics with Escherichia coli K antigens, is not involved in 25 Jun 2001 10:48 484 33. 34. 35. 36. 37. 38. 39. 40. 41. AR AR138-18.tex LUGTENBERG ¥ AR138-18.SGM DEKKERS ¥ ARv2(2001/05/10) P1: GJB BLOEMBERG root colonization. J. Bacteriol. 178:1955– 61 de Weger LA, Dunbar P, Mahafee W, Lugtenberg BJJ, Sayler GS. 1991. Use of bioluminescence markers for detection of Pseudomonas bacteria in the rhizosphere. Appl. Environ. Microbiol. 57:3641– 44 de Weger LA, Jann B, Jann K, Lugtenberg B. 1987. Lipolysaccharides of Pseudomonas spp. that stimulate plant growth: composition and use for strain identification. J. Bacteriol. 169:1441–46 de Weger LA, Kuiper I, van der Bij AJ, Lugtenberg BJJ. 1997. Use of a lux-based procedure to rapidly visualize root colonisation by Pseudomonas fluorescens in the wheat rhizosphere. Antonie van Leeuwenhoek J. Microbiol. Serol. 72:365–72 de Weger LA, van der Vlugt CIM, Wijfjes AHM, Bakker PAHM, Schippers B, Lugtenberg BJJ. 1987. Flagella of a plant growth stimulating Pseudomonas fluorescens strain are required for colonization of potato roots. J. Bacteriol. 169:2769–73 Dorr J, Hurek T, Reinhold HB. 1998. Type IV pili are involved in plant-microbe and fungus-microbe interactions. Mol. Microbiol. 30:7–17 Drake D, Montie TC. 1988. Flagella, motility and invasive virulence of Pseudomonas aeruginosa. J. Gen. Microbiol. 134:43–52 Dybvig K. 1993. DNA rearrangements and phenotypic switching in prokaryotes. Mol. Microbiol. 10:465–71 Espinosa-Urgel M, Salido A, Ramos JL. 2000. Genetic analysis of functions involved in adhesion of Pseudomonas putida to seeds. J. Bacteriol. 182:2363– 69 Fedi S, Tola E, Moënne-Loccoz Y, Dowling DN, Smith LM, O’Gara F. 1997. Evidence for signalling between the phytopathogenic fungus Pythium ultimum and Pseudomonas fluorescens F113: P. ultimum represses the expression of genes 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. in P. fluorescens F113, resulting in altered ecological fitness. Appl. Environ. Microbiol. 63:4261–66 Foster RC. 1981. The ultrastructure and histochemistry of the rhizosphere. New Phytol. 89:263–73 Foster RC. 1982. The fine structure of epidermal cell mucilages of roots. New Phytol. 91:727–40 Foster RC, Rovira AD. 1976. Ultrastructure of wheat rhizosphere. New Phytol. 76:343–52 Foster RC, Rovira AD, Cock TW. 1983. Ultrastructure of the Root-Soil Interface. 1983. St Paul: Am. Phytopathol. Soc. Fukui R, Poinar EI, Bauer PH, Schroth MN, Hendson M, et al. 1994. Spatial colonization patterns and interaction of bacteria on inoculated sugar beet seed. Phytopathology 84:1338–45 Fuqua WC, Winans SC, Greenberg EP. 1994. Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators. J. Bacteriol. 176:269–75 Gamliel A, Katan J. 1992. Influence of seed and root exudates on fluorescent pseudomonads and fungi in solarized soil. Phytopathology 82:320–27 Geels FP, Schippers B. 1983. Reduction of yield depressions in high frequency potato cropping soil after seed tuber treatments with antagonistic fluorescent Pseudomonas spp. Phytopathol. Z. 108:207– 14 Gerrits JPL, Weisbeek PJ. 1996. Induction of systemic acquired resistance by saprophytic Pseudomonas spp. in the model plant Arabidopsis thaliana. In NWO-LNV Priority Progr. Crop Prot. Prog. Rep., pp. 13–14. Lunteren, The Netherlands: NWO, The Hague Glandorf DCM. 1992. Root colonization by fluorescent pseudomonads. PhD thesis. Univ. Utrecht, The Netherlands Goldman RC, Leive L. 1980. Heterogeneity in antigenic side chain length in lipopolysaccharide from Escherichia coli 25 Jun 2001 10:48 AR AR138-18.tex AR138-18.SGM ARv2(2001/05/10) P1: GJB RHIZOSPHERE COLONIZATION BY PSEUDOMONAS 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 0111 and Salmonella typhimurium LT2. Eur. J. Biochem. 107:145–53 Graf J, Dunlap PV, Ruby EG. 1994. Effect of transposon-induced motility mutations on colonization of the host light organ by Vibrio fischeri. J. Bacteriol. 176:6986–91 Haas D, Blumer C, Keel C. 2000. Biocontrol ability of fluorescent pseudomonads genetically dissected: importance of positive feedback regulation. Curr. Opin. Biotechnol. 11:290–97 Hahn HP. 1997. The type-4 pilus is the major virulence-associated adhesin of Pseudomonas aerogunisa—a review. Gene 192:99–108 Heeb S, Itoh Y, Nishijyo T, Schnider U, Keel C, et al. 2000. Small, stable shuttle vectors based on the minimal pVS1 replicon for use in gram-negative, plantassociated bacteria. Mol. Plant-Microbe Interact. 13:232–37 Henderson IR, Navarro-Garcia F, Nataro JP. 1998. The great escape: structure and function of the autotransporter proteins. Trends Microbiol. 6:370–78 Hiltner L. 1904. Über neuere Erfahrungen und Probleme auf dem Gebiete der Bodenbakteriologie unter bessonderer Berücksichtigung der Gründung und Brache. Arb. Dtsch. Landwirtsch. Ges. Berl. 98:59–78 Höfte M, Seong KY, Jurkevitch E, Verstraete H. 1991. Pyoverdin production by the plant growth beneficial Pseudomonas strain 7NSK2: ecological significance in soil. Plant Soil 130:249–57 Howie WJ, Cook RJ, Weller DM. 1987. Effects of soil matric potential and cell motility on wheat root colonization by fluorescent pseudomonads suppressive to take-all. Phytopathology 77:286–92 Deleted in proof Jenny H, Grossenbacher K. 1963. Rootsoil boundary zones as seen in the electron microscope. Soil Sci. Soc. Am. Proc. 27:273–77 Jones BD, Nichols WA, Gibson BW, 64. 65. 66. 67. 68. 69. 70. 71. 72. 485 Sunshine MG, Apicella MA. 1997. Study of the role of the htrB gene in Salmonella typhimurium virulence. Infect. Immun. 65:4778–83 Klein DA, Frederick BA, Biondini M, Trlica MJ. 1988. Rhizosphere microorganism effects on soluble amino acids, sugars and organic acids in the root zone of Agropyron cristatum, A. Smithii and Bouteloua gracilis. Plant Soil 110:19– 25 Kloepper JW, Beauchamp CJ. 1992. A review of issues related to measuring colonization of plant roots by bacteria. Can. J. Microbiol. 38:1219–32 Kloepper JW, Lifshitz R, Zablotowicz RM. 1989. Free-living bacterial inocula for enhancing crop productivity. Trends Biotechnol. 7:39–44 Kravchenko LV, Makarova NM. 1993. Kinetics of cereal root surface colonization after introduction of associative bacteria. Microbiology 62:324–27 Kuiper I, Bloemberg GV, Noreen S, Thomas-Oates JE, Lugtenberg BJJ. 2001. Increased uptake of putrescine inhibits competitive root colonization by Pseudomonas fluorescens strain WCS365. Mol. Plant Microbe Interact. In press Kuiper I, Bloemberg GV, Lugtenberg BJJ. 2001. Selection of a plant-bacterium pair of which the bacterium prevents xenobiotic phytotoxicity and of which the plant injects the xenobiotic-degrading bacterium into the soil. Submitted Lagopodi AL, Ram AFJ, Lamers GEM, Punt PJ, van den Hondel CAMJJ, et al. 2001. Confocal laser scanning microscopy analyses of tomato root colonization and infection by Fusarium oxysporum f.sp. radicis-lycopersici using the green fluorescent protein as a marker. Submitted Lam ST, Ellis DM, Ligon JM. 1990. Genetic approaches for studying rhizosphere colonization. Plant Soil 129:11–18 Lee N, Sunshine MG, Engstrom JJ, Gibsons BW, Apicella MA. 1995. Mutation 25 Jun 2001 10:48 486 73. 74. 75. 76. 77. 78. 79. 80. AR AR138-18.tex LUGTENBERG ¥ AR138-18.SGM DEKKERS ¥ ARv2(2001/05/10) P1: GJB BLOEMBERG of the htrB locus of Haemophilus influenzae nontypable strain 2019 is associated with modifications of lipidA and phosphorylation of the lipo-oligosaccharide. J. Biol. Chem. 270:27151–59 Lee S-W, Cooksey DA. 2000. Genes expressed in Pseudomonas putida during colonization of a plant-pathogenic fungus. Appl. Environ. Microbiol. 66:2764– 72 Loper JE, Suslow TV, Schroth MN. 1984. Lognormal distribution of bacterial populations in the rhizosphere. Phytopathology 74:1454–60 Lugtenberg BJJ. 2000. Use of bacteria for plant growth promotion and plant protection. In Book of Abstracts, New Approaches and Techniques in Breeding Sustainable Fodder Crops and Amenity Grasses, ed. NA Provorov, IA Tikhonovich, F Veronesi. St. Petersburg, Russia: Russ. Inst. Agric. Microbiol. Lugtenberg BJJ, van Alphen L. 1983. Molecular architecture and functioning of the outer membrane of Escherichia coli and other gram-negative bacteria. Biochim. Biophys. Acta 737:51–115 Lugtenberg BJJ, Dekkers LC, Bansraj M, Bloemberg GV, Camacho M, et al. 1999. Pseudomonas genes and traits involved in tomato root colonization. In Biology of Plant-Microbe Interactions, ed. PJGM de Wit, T Bisseling, WJ Stiekema, pp. 433–40. St. Paul: Am. Phytopathol. Soc. Lugtenberg BJJ, Kravchenko LV, Simons M. 1999. Tomato seed and root exudate sugars: composition, utilization by Pseudomonas biocontrol strains and role in rhizosphere colonization. Environ. Microbiol. 1:439–46 Lugtenberg BJJ, Phoelich C, Simons M, Kravchenko LV, Tikhonovich I, Wijfjes AHM. 2001. Utilization of exudate organic acids is the nutritional basis of tomato root colonization by Pseudomonas fluorescens WCS365. Submitted Lynch JM. 1990. Introduction: some con- 81. 82. 83. 84. 85. 86. 87. 88. 89. sequences of microbial rhizosphere competence for plant and soil. In The Rhizosphere, ed. JM Lynch, pp. 1–10. Chichester, UK: Wiley & Sons Mahajan MS, Tan MW, Rahme LG, Ausubel FM. 1999. Molecular mechanisms of bacterial virulence elucidated using a Pseudomonas aeruginosa Caenorhabditis elegans pathogenesis model. Cell 96:47–56 Martindale J, Stroud D, Moxon ER, Tang CM. 2000. Genetic analysis of Escherichia coli K1 gastrointestinal colonization. Mol. Microbiol. 37:1293–305 Matthysse AG, McMahan S. 1998. Root colonization by Agrobacterium tumefaciens is reduced in cel, attB, attD, and attR mutants. Appl. Environ. Microbiol. 64:2341–45 Matthysse AG, White S, Lightfoot R. 1995. Genes required for cellulose synthesis in Agrobacterium tumefaciens. J. Bacteriol. 177:1069–75 Matthysse AG, Yarnall HA, Young N. 1996. Requirement for genes with homology to ABC transport systems for attachment and virulence of Agrobacterium tumefaciens. J. Bacteriol. 178:5302– 8 Mazzola M, Cook RJ, Thomashow LS, Weller DM, Pierson LS. 1992. Contribution of phenazine antibiotic biosynthesis to the ecological competence of fluorescent Pseudomonads in soil habitats. Appl. Environ. Microbiol. 58:2616–24 Meharg AA, Killham K. 1995. Loss of exudates from the roots of perennial ryegrass inoculated with a range of microorganisms. Plant Soil 170:345–49 Moënne-Loccoz Y van, McHugh B, Stephens PM, McConnel FI, Glennon JD, et al. 1996. Rhizosphere competence of fluorescent Pseudomonas sp. B24 genetically modified to utilise additional ferric siderophores. FEMS Microbiol. Ecol. 19:215–25 Nevola JJ, Stocker BAD, Laux DC, Cohen PS. 1995. Colonization of the 25 Jun 2001 10:48 AR AR138-18.tex AR138-18.SGM ARv2(2001/05/10) P1: GJB RHIZOSPHERE COLONIZATION BY PSEUDOMONAS 90. 91. 92. 93. 94. 95. 96. 97. 98. mouse intestine by an avirulent Salmonella typhimurium strain and its lipopolysaccharide-defective mutants. Infect. Immun. 50:152–59 Newman EI, Bowen HJ. 1974. Patterns of distribution of bacteria on root surfaces. Soil Biol. Biochem. 6:205–9 Ojanen-Reuhs T, Kalkkinen N, Westerlund-Wikstrom B, van Doorn J, Haahtela K, et al. 1997. Characterization of the fimA gene encoding bundleforming fimbriae of the plant pathogen Xanthomonas campestris pv. vesicatoria. J. Bacteriol. 179:1280–90 O’Toole GA, Kolter R. 1998. Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis. Mol. Microbiol. 28:449–61 Palumbo JD, Kado CI, Phillips DA. 1998. An isoflavonoid-inducible efflux pump in Agrobacterium tumefaciens is involved in competitive colonization of roots. J. Bacteriol. 180:3107–13 Palva ET, Mäkelä PH. 1980. Lipopolysaccharide heterogeneity in Salmonella typhimurium analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Eur. J. Biochem. 107:137–43 Paton AW, Voss E, Manning PA, Paton JC. 1998. Antibodies to lipopolysaccharide block adherence of Shiga toxinproducing Escherichia coli to human intestinal epithelial (Henle 407) cells. Microb. Pathog. 24:57–63 Phillips DA, Streit W. 1995. Legume signals to rhizobial symbionts: a new approach for defining rhizosphere colonization. In Plant-Microbe Interactions, ed. G Stacey, NT Keen, pp. 236–71. New York: Chapman & Hall Pier GB. 1985. Pulmonary disease associated with Pseudomonas aeruginosa in cystic fibrosis: current status of the host-bacterium interaction. J. Infect. Dis. 151:575–80 Pierson EA, Wood DW, Cannon JA, Blachere FM, Pierson III LS. 1998. 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 487 Interpopulation signaling via N-acylhomoserine lactones among bacteria in the wheat rhizosphere. Mol. PlantMicrobe Interact. 11:1078–84 Piper KR, Beck von Bodman S, Farrand SK. 1993. Conjugation factor of Agrobacterium tumefaciens regulates Ti plasmid transfer by autoinduction. Nature 362:448–50 Potera C. 1996. Research news: biofilms invade microbiology. Science 273:1795– 97 Preston GM, Bertrand N, Rainey PB. 2001. Type III (Hrp) secretion in plant growth-promoting Pseudomonas fluorescens SBW25. Submitted Prikryl Z, Vancura V. 1980. Root exudates of plants VI. Wheat root exudation as dependent on growth, concentration gradient of exudates and the presence of bacteria. Plant Soil 57:69–83 Raaijmakers JM, van der Sluis I, Koster M, Bakker PAHM, Weisbeek PJ, Schippers B. 1995. Utilization of heterologous siderophores and rhizosphere competence of fluorescent Pseudomonas spp. Can J. Microbiol. 41:126–35 Rainey PB. 1999. Adaption of Pseudomonas fluorescens to the plant rhizosphere. Environ. Microbiol. 1:243–57 Rainey PB, Bailey MJ. 1996. Physical and genetic map of the Pseudomonas fluorescens SBW25 chromosome. Mol. Microbiol. 19:521–33 Ramos C, Licht TR, Sternberg C, Krogfelt KA, Molin S. 2001. Monitoring bacterial growth activity in biofilms from laboratory flow-chambers, plant rhizosphere and animal intestine. Methods Enzymol. In press Ramos C, Mølbak L, Molin S. 2000. Bacterial activity in the rhizosphere analyzed at the single-cell level by monitoring ribosome contents and synthesis rates. Appl. Environ. Microbiol. 66:801–9 Roberts DP, Short NMJ, Maloney AP, Nelson EB, Schaff DA. 1994. Role of colonization in biocontrol: studies with 25 Jun 2001 10:48 488 109. 110. 111. 112. 113. 114. 115. 116. 117. 118. AR AR138-18.tex LUGTENBERG ¥ AR138-18.SGM DEKKERS ¥ ARv2(2001/05/10) P1: GJB BLOEMBERG Enterobacter cloacae. Plant Sci. 101:83– 89 Roine E, Raineri DM, Romantschuk M, Wilson M. Nunn DN. 1998. Characterization of type IV pilus genes in Pseudomonas syringae pv. tomato DC3000. Mol. Plant-Microbe Interact. 11:1048–56 Rovira AD. 1956. A study of the development of the root surface microflora during the initial stages of plant growth. J. Appl. Bacteriol. 19:72–79 Rovira AD, Campbell R. 1975. A scanning electron microscope study of interactions between micro-organisms and Gaeumannomyces graminis (Syn. Ophiobolus graminis) on wheat roots. Microb. Ecol. 3:177–85 Rovira AD, Harris JR. 1961. Plant root excretions in relation tot the rhizosphere effect. Plant Soil 14:199–214 Sadowski P. 1986. Site specific recombinases: changing partners and doing the twist. J. Bacteriol. 165:341–37 Sahashi N, Tsuji H, Shishiyama J. 1989. Barley plants grown under germ-free conditions have increased susceptibility to two powdery mildew fungi. Physiol. Mol. Plant Pathol. 34:163–70 Scher FM, Kloepper JW, Singleton C, Zaleska I, Laliberte M. 1988. Colonization of soybean roots by Pseudomonas and serratia species: relationship to bacterial motility, chemotaxis and generation time. Phytopathology 78:1055–59 Schippers B, Bakker AW, Bakker PAHM. 1987. Interactions of deleterious and beneficial rhizosphere microorganisms and the effect on cropping practices. Annu. Rev. Phytopathol. 25:339–58 Shanahan P, O’Sullivan DJ, Simpson P, Glennon JD, O’Gara F. 1992. Isolation of 2,4-diacetylphloroglucinol from a fluorescent Pseudomonad and investigation of physiological parameters influencing its production. Appl. Environ. Microbiol. 58:353–58 Shaw JJ, Dane F, Geiger D, Kloepper JW. 1992. Use of bioluminescence for detec- 119. 120. 121. 122. 123. 124. 125. 126. 127. 128. tion of genetically-engineered microbes released into the environment. Appl. Environ. Microbiol. 58:267–73 Simons M, Permentier HP, de Weger LA, Wijffelman CA, Lugtenberg BJJ. 1997. Amino acid synthesis is necessary for tomato root colonization by Pseudomonas fluorescens strain WCS365. Mol.PlantMicrobe Int. 10:102–6 Simons M, van der Bij AJ, Brand J, de Weger LA, Wijffelman CA., Lugtenberg BJJ. 1996. Gnotobiotic system for studying rhizosphere colonization by plant growth-promoting Pseudomonas bacteria. Mol. Plant-Microbe Interact. 9:600–7 Smit G, Kijne JW, Lugtenberg BJJ. 1986. Correlation between extracellular fibrils and attachment of Rhizobium leguminosarum to pea root hair tips. J. Bacteriol. 168:821–27 Smit G, Kijne JW, Lugtenberg BJJ. 1987. Both cellulose fibrils and a Ca2+dependent adhesin are involved in the attachment of Rhizobium leguminosarum to pea root hair tips. J. Bacteriol. 169:4294– 301 Stover CK, Pham XQ, Erwin AL, Mizoguchi SD, Warrener P, et al. 2000. Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogen. Nature 406:959–64 Streeter JG. 1994. Failure of inoculant rhizobia to overcome the dominance of indigenous strains for nodule formation. Can. J. Microbiol. 40:513–22 Streit WR, Joseph CM, Phillips DA 1996 Biotin and other water-soluble vitamins are key growth factors for alfalfa root colonization by Rhizobium meliloti 1021. Mol. Plant-Microbe Int. 9:330–38 Streit WR, Phillips DA 1997. A biotinregulated locus, bioS, in a possible survival operon of Rhizobium meliloti. Mol. Plant-Microbe Interact. 10:933–37 Strom MS, Lory S. 1993. Structurefunction and biogenesis of the type IV pili. Annu. Rev. Microbiol. 47:565–96 Stuurman N, Pacios Bras C, Schlaman 25 Jun 2001 10:48 AR AR138-18.tex AR138-18.SGM ARv2(2001/05/10) P1: GJB RHIZOSPHERE COLONIZATION BY PSEUDOMONAS 129. 130. 131. 132. 133. 134. 135. 136. HRM, Wijfjes AHM, Bloemberg GV, Spaink HP. 2000. The use of GFP color variants expressed on stable broad-host range vectors to visualize rhizobia interacting with plants. Mol. Plant-Microbe Interact. 13:1163–69 Swart S, Logman TJJ, Lugtenberg BJJ, Kijne JW. 1994. Purification and partial charactization of a glycoprotein from pea (Pisum sativum) with receptor activity for rhicadhesin, an attachment protein of Rhizobiacea. Plant Mol. Biol. 24:171–83 Tan M-W, Rahme LG, Sternberg JA, Tompkins RG, Ausubel FM. 1999. Pseudomonas aeruginosa killing of Caenorhabditis elegans used to identify P. aeruginosa virulence factors. Proc. Natl. Acad. Sci. USA 96:2408–13 Thomashow LS, Weller DM. 1996. Current concepts in the use of introduced bacteria for biological disease control: mechanisms and antifungal metabolites. Plant-Microbe Interact. 6:187–235 Troxler J, Azelvandre P, Zala M, Défago G, Haas D. 1997. Conjugative transfer of chromosomal genes between fluorescent pseudomonads in the rhizosphere of wheat. Appl. Environ. Microbiol. 63:213– 19 Unge A, Tombolini R, Molbak L, Jansson J. 1999. Simultaneous monitoring of cell number and metabolic activity of specific bacterial populations with a dual gfpluxAB marker system. Appl. Environ. Microbiol. 65:813–21 Vancura V. 1964. Root exudates of plants. I. Analysis of root exudates of barley and wheat in their initial phases of growth. Plant Soil 21:231–48 Vancura V. 1967. Root exudates of plants. III. Effect of temperature and ‘cold shock’ on the exudation of various compounds from seeds and seedlings of maize and cucumber. Plant Soil 27:319–28 Vancura V, Hanzlikova A. 1972. Root exudates of plant IV. difference in chemical composition of seed and seedlings exudates. Plant Soil 36:271–82 489 137. Vancura V, Hovadik A. 1965. Root exudates of plants II. Composition of root exudates of some vegetables. Plant Soil 22:21–32 138. Vancura V, Stanek M. 1975. Root exudates of plants V. Kinetics of exudates from bean roots as related to the presence of reserve compounds in cotyledons. Plant Soil 43:547–59 139. Van Egeraat AWSM. 1972. Pea root exudates and their effect upon root-nodule bacteria. PhD thesis. Agric. Univ., Wageningen, The Netherlands 140. Van Elsas JD, Trevors JT, Starodub ME. 1998. Bacterial conjugation between pseudomonads in the rhizosphere of wheat. FEMS Microbiol. Lett. 53:299–306 141. Van Loon LC, Bakker PAHM, Pieterse CMJ. 1998. Systemic resistance induced by rhizosphere bacteria. Annu. Rev. Phytopathol. 36:453–83 142. Van Overbeek LS, van Elsas JD. 1995. Root exudate induced promoter activity in Pseudomonas fluorescens mutants in the wheat rhizosphere. Appl. Environ. Microbiol. 61:890–98 143. Vesper SJ. 1987. Production of pili (fimbriae) by Pseudomonas fluorescens and correlation with attachment to corn roots. Appl. Environ. Microbiol. 53:1397–405 144. Vı́lchez S, Molina L, Ramos C, Ramos JL. 2000. Proline catabolism by Pseudomonas putida: cloning, characterization, and expression of the put genes in the presence of root exudates. J. Bacteriol. 182:91–99 145. Volpin H, Burdman S, Castro-Sowinski S, Kapulnik Y, Okon Y. 1996. Inoculation with Azospirillum increased exudation of rhizobial nod-gene inducers by alfalfa roots. Mol. Plant-Microbe Interact. 9:388–94 146. Webster G, Jain V, Davey MR, Gough C, Vasse J, et al. 1998. The flavonoid naringenin stimulates the intercellular colonization of wheat roots by Azorhizobium caulinodans. Plant Cell Environ. 21:373– 83 25 Jun 2001 10:48 490 AR AR138-18.tex LUGTENBERG ¥ AR138-18.SGM DEKKERS ¥ ARv2(2001/05/10) P1: GJB BLOEMBERG 147. Weidner U, Geier S, Ptock A, Friedrich T, Leif H, Weiss H. 1993. The gene locus of the proton-translocating NADH: ubiquinone oxidoreductase in Escherichia coli. J. Mol. Biol. 233:109–22 148. Whitchurch CB, Hobbs M, Livingston SP, Krishnapillai V, Mattick JS. 1991. Characterisation of a Pseudomonas aeruginosa twitching motility gene and evidence for a specialised protein export system widespread in eubacteria. Gene 101:33–44 149. Young IG, Jaworowski A, Poulis MI. 1978. Amplification of the respiratory NADH dehydrogenase of Escherichia coli by gene cloning. Gene 4:25–36 150. Young IG, Rogers BL, Campbell HD, Jaworowski A, Shaw DC. 1981. Nucleotide sequence coding for the respiratory NADH dehydrogenase of Escherichia coli. FEBS Lett. 116:165–70 151. Zaat SAJ, Van Brussel AAN, Tak T, Pees E, Lugtenberg BJJ. 1987. Flavonoids induce Rhizobium leguminosarum to produce nodABC gene-related factors that cause thick short roots and root hair responses on common vetch. J. Bacteriol. 169:3388–91 11 Jul 2001 13:55 AR AR138-18-COLOR.tex AR138-18-COLOR.SGM ARv2(2001/05/10) P1: FUI Figure 1 Confocal laser scanning microscopical analysis of a tomato root surface colonized by Pseudomonas fluorescens WCS365 expressing ds-red and Fusarium oxysporum f. sp. radicis lycopersici expressing g f p (image taken by A. Lagopodi).