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ELSEVIER FEMS Microbiology Ecology 17 (199.5) 221-228 MiniReview Colonization of the rhizosphere of crop plants by plant-beneficial pseudomonads Letty A. de Weger *, Arjan J. van der Bij, Linda C. Dekkers, Marco Simons, Care1 A. Wijffelman, Ben J.J. Lugtenberg Leiden University, Institute of Molecular Plant Sciences, Clusius Laboratory, Wassenaarseweg 64, 2333 AL Leiden, the Netherlands Received 21 December 1994; revised 28 April 1995; accepted 10 May 1995 Abstract Efficient colonization of the plant root is thought to be crucial for the plant-beneficial effect of particular Pseudomonas strains. Since root colonization is often the limiting step for successful plant growth stimulation, this process needs improvement. It is therefore important to acquire more information as regards (i) the conditions in the rhizosphere, and (ii) the bacterial traits that are involved in colonization. This review discusses some recent studies that focus on these two issues. Keywords: Pseudomonas; Rhizosphere colonization; Biocontrol 1. Introduction Nowadays agriculture requires the extensive use of chemical pesticides to protect crops against pests and diseases. Several of these chemicals pollute our ground water and drinking water and therefore some governments have decided to reduce these chemical inputs substantially. This urges the need for altemative crop protectants. One of these alternatives is the use of biological control agents, among which are microorganisms that can protect the plant against diseases. Many different microbial genera (fungi and bacteria) have been described as potential biocontrol agents against soil-related diseases [ 11, but especially the group of fluorescent Pseudomonas spp. is receiving much attention. Spectacular results have been ’ Corresponding author. Tel: (31)-71-275056; Fax: (31)71275088; E-mail: [email protected] obtained after treatment of the seeds with particular strains, but often their performance is not consistent. Therefore a better understanding of the crucial steps involved in this plant-beneficial bacterium interaction is required for a successful commercial application of these microbes. The mechanisms by which Pseudomonas spp. exert their beneficial effect on plants can be very diverse [1,2]. Under conditions of Fe3+ limitation, some Pseudomonas spp. produce siderophores that scavenge the traces of Fe3+ in the soil and thereby deprive other microbes, including deleterious and pathogenic ones, of this essential element. Other Pseudomonas spp. secrete antibiotics inhibitory to pathogens, or enzymes that degrade fungal cell walls. Considering these different modes of action it is crucial that the beneficial bacterium delivers the active compound at the place where it is required. When protection of the whole root is required, an efficient colonization of the root system is consid- 016%6496/95/$09.50 0 1995 Federation of European Microbiological Societies. AR rights reserved SSDI 0168-6496(95)00031-3 222 L.A. de Weger et al. /FEMS Microbiology ered to be the best delivery system of a successful biocontrol activity. This notion is supported by two findings: (i) unsuccessful field trials are often accompanied by inadequate colonization of the roots [3], and (ii) the number of lesions in wheat roots caused by the pathogenic fungus Gaeumannomyces graminis var. tritici decreased with an increasing number of plant protecting bacteria of Pseudomonas fluorescens 2-79 on the roots [4]. To improve this limiting step in the biocontrol performance of these bacteria, a better knowledge of the mechanism of root colonization is important. This review focuses on research that intends to increase our knowledge of root colonization. Firstly, we will focus on the growth conditions in the rhizosphere, since this is the site where the bacteria have to be active in producing their biological control compound. Secondly, bacterial traits and genes that are involved in the colonization process will be discussed. 2. Growth conditions in the rhizosphere The rhizosphere is an ecological niche which is mainly a black box. Hardly anything is known about the availability of nutrients (organic or inorganic), the dangers which must be survived (e.g. predation, harmful compounds) or the interactions with other rhizosphere organisms. The deficiency in our knowledge on the growth conditions in the rhizosphere is illustrated by the fact that only 1 to 10% of the microorganisms present in the rhizosphere can be cultured on laboratory media [5]. The classical methods (e.g. chemical methods) have helped us to determine the amount of various nutrients like nitrate, phosphate, or iron. However, they did not demonstrate the proportion of these nutrients that are really available for microbes growing in the rhizosphere. Novel molecular techniques, which can be used for measurements in situ should help us to increase our knowledge on this ecological niche. The in situ determinations on the availability of nutrients have been studied in different ways. Using a microelectrode the oxygen gradient in the rhizosphere of barley was measured [6]. The oxygen concentration in bulk soil was much higher than in the rhizosphere soil, due to the high microbial respi- Ecology I7 (1995) 221-228 ration in the rhizosphere and the oxygen uptake by the root. Furthermore the measurements showed that the rhizosphere can easily develop very low oxygen concentrations and even complete anoxia. Employment of the denitrifying activity of bacteria appeared to be very helpful in measuring two different forms of nitrogen, nitrate and nitrite, in the rhizosphere. Besides nitrate fertilizer which is often added to agricultural soils, nitrification activity is the only other source of nitrate. Nitrite is usually present in small amounts in natural soils, probably because it is an intermediate in both nitrification and denitrification. A very sensitive bioassay was developed, employing denitrifying Pseudomonas aeruginosa cells [7]. A mutant strain was used that was unable to reduce nitrate, but still able to convert nitrite into N,O, which can be detected using gas chromatography. The remaining nitrate in the samples can subsequently be determined after addition of wild-type P. aeruginosa cells capable of reducing nitrate to N,O. This bioassay was used to measure the nitrate and nitrite pools in 10 mg soil samples taken along a microgradient from various parts of the rhizosphere of field-grown barley plants. The relative amounts of nitrate and nitrite in the rhizosphere were higher than in bulk soil, indicating a net accumulation of these two compounds from nitrification activity at the root surface. Wetting of the soil reduced the amount of nitrate in the rhizosphere, probably due to a decrease in the O2 concentration in the rhizosphere, which in turn may favour denitrifying activities [7]. For measuring the bioavailability of phosphate and iron in the soil and in the rhizosphere so-called reporter bacteria were constructed, which can report whether growth conditions in situ are limiting for a certain compound [8,9]. Phosphate reporter bacteria were constructed which respond to phosphate-limiting conditions by the production of P-galactosidase [8]. The level of this enzyme can easily be quantified using the Miller test employing ONPG (Onitrophenyl-/3-galactopyranoside) as the substrate. When phosphate reporter cells were growing under gnotobiotic conditions in the rhizosphere of tomato, potato or radish plants or in the bulk soil, they sensed phosphate limitation as judged from the significant increase in /3-galactosidase activity [s]. Iron-reporter bacteria were constructed using another reporter protein, InaZ, which catalyses ice formation LA. de Weger et al. / FEMS Microbiology at - 2 to - 10°C [9]. When these cells experience iron-limitation they start to produce the InaZ-protein, which can be quantified by a droplet freezing assay. Using this reporter system it was shown that Pseudomonas cells growing in the rhizosphere of bean experience a moderate but not very severe iron limitation. Comparing the two reporter systems discussed here, it is evident that the InaZ-based system is more sensitive than pgalactosidase. Therefore, the former system will be better suited for situations where low transcriptional activity of the promoter of interest is to be expected or when only low numbers of bacteria can be reisolated from the microniche. However, when enough cells can be recovered from the environment and when transcriptional activity is high, /3-galactosidase is a very fast and easy reporter system for whose detection no special equipment is required. Plant roots can lose a large portion (up to 31%) of their assimilates, mainly in the form of amino acids, organic acids and sugars [lo]. However, using HPLC analysis, only small amounts of amino acids including histidine, leucine and isoleucine could be detected in sterile tomato root exudate (M. Simons et al., unpublished results). Auxotrophic mutants impaired in the synthesis of these amino acids were used to study whether tomato roots are able to supply these auxotrophs with a sufficient amount of the relevant amino acid to support colonization. In contrast to the parent strain the mutants appeared to be unable to colonize the root tips of tomato. Addition of the relevant amino acid to the sand in which the plants were growing restored the colonization ability of the mutants (M. Simons et al., unpublished results). These results show that these mutants are able to colonize tomato roots provided that the required amino acid is present, which apparently cannot be supplied by the tomato roots in amounts sufficient to support colonization of these auxotrophs. Predation is one of the hazards that bacteria in the rhizosphere can encounter. This may either be predation by protozoa or by bacteriophages. The presence of a bacteriophage in the soil specific for a given strain significantly reduces the numbers of cells of this strain present in the rhizosphere [ll]. Attachment to soil particles may protect the cells from predation by protozoa [12]. Using Zux marked bacte- Ecology 17 (1995) 221-228 223 ria it was nicely demonstrated that cells present in the small pores of soil particles were better protected from protozoan predation than the cells in the larger pores (J. Presser, personal communication). Recently a new class of signalling molecules have been discovered in a wide range of Gram-negative bacteria, which enable the bacterial cells to monitor the growth of its population [13]. When the bacterial population reaches high densities, these signalling molecules with a common structure (N-acyl homoserine lactones = acyl-HSLs) mediate the induction of new sets of genes responsible for e.g. bioluminescence in Vibrio fischeri [14], antibiotic and exoenzyme production in Erwinia carotovora [ 151, and Ti-plasmid conjugation in Agrobacterium tumefaciens [16]. Also the phenazine production in the biocontrol bacterium Pseudomonas aureofaciens strain 30-84 appeared to be quorum-regulated [17]. This stresses again the relevance of an efficient colonization of the plant root which must lead to the formation of microcolonies in which the cell density is high enough to induce the production of this pathogen-inhibiting antibiotic. Interestingly, certain bacteria can respond to the signalling molecules of other bacterial species. Using an Agrobacterium tumefaciens indicator strain with a 1acZ gene introduced into the acyl-HSL responsive tra operon [16], a series of 25 different Pseudomonas root isolates and a Rhizobium leguminosarum strain have been tested for their ability to induce the A. tumefaciens tra operon. One biocontrol Pseudomonas putida strain, strain WCS358, and the R. leguminosarum strain appeared to produce signalling molecules which were recognized by the A. tumefaciens induction machinery (de Weger, unpublished results). This inter-species signalling challenges our traditional view of microbial life in the rhizosphere. Rather than an independent entity, bacterial cells may communicate with many of its neighbouring cells either of its own kind or of other species [13]. 3. Bacterial traits involved in colonization We hypothesised that structures located at the outer surface of the cell could be important for the interaction with the root, and therefore for colonization. By comparing the colonization abilities of mu- 224 L.A. de Weger et al. / FEMS Microbiology tants deficient in particular structures with that of the parent strain, the role of these structures in colonization could be determined. Flagellaless mutants of P. fluorescens strain WCS374 appeared to be impaired in the colonization of the deeper root parts of potato plants [18]. In contrast Howie et al. [19] did not observe any difference between nonmotile mutants and the parent strain in the colonization of wheat roots. These results may be due to differences in experimental design (e.g. plant species, Pseudomonas strain, soil type). De Mot et al. [20] showed that mutants of P. fluorescens strain OE28.3 which are deficient in the major outer membrane protein OprF are impaired in their adhesion to wheat roots. Pili, filamentous, proteinaceous extrusions, have been found to be involved in the adhesion of many plant pathogenic bacteria to plant surfaces, e.g. Erwinia spp., Pseudomonas syringae [21]. Vesper showed that also in the plant growth-promoting P. fluorescens strain 2-79 the presence of pili was correlated with attachment to corn roots [22]. In neither of these studies the question whether attachment is involved in colonization is addressed and this remains a matter of debate. Some studies do find a correlation between the abilities to attach to and to colonize the root [23], while in other studies such a correlation was not found [24]. Polysaccharides at the bacterial cell surface are involved in the Rhizobium-legume interaction [25]. Two polysaccharides at the surface of plant-beneficial Pseudomonas strains have been studied for their role in root colonization. Mutants of P. jluorescens WCS374 and P. putida WCS358 that lacked the 0-antigenic side chain of their lipopolysaccharide were impaired in the colonization of the deeper potato root parts [24]. This reduced colonization ability appeared neither to be due to reduced binding to the plant root, nor to poor survival of these mutants. Apparently the 0-antigenic side chain is relevant for the distribution of the cells along the root system. In contrast to the O-antigen, a polysaccharide located at the cell surface of P. putida strain WCS358, which shares characteristics with the K-antigens present in Escherichia cob, appeared not to be relevant for colonization (de Weger et al., submitted for publication). The major factors responsible for the stimulation Ecology 17 (1995) 221-228 of microorganisms in the rhizosphere are the excretion of organic compounds and the sloughing off of root hairs and epidermal cells 1261. The ability to utilize these various nutrients is assumed to play an important role in colonization. Cells which can utilize many major rhizosphere nutrients efficiently may therefore have an advantage over cells that are fastidious. On the other hand a complete dependence on specific rhizosphere nutrients seemed to be a disadvantage since it appeared that amino acid auxotrophs of P. fluorescens WCS365, in contrast to the wildtype, were unable to colonize the tomato rhizosphere unless the required amino acid was added to the medium (see also above, M. Simons et al., unpublished). So, to be competitive in the rhizosphere the cells have to be able to synthesise their own amino acids. Another aspect of life in the rhizosphere is that the cells will have to be able to compete with the indigenous microorganisms for the available resources in the rhizosphere. Production of compounds that reduce the growth of competing colleagues are likely to be favourable for effective colonization of the rhizosphere. Siderophores play a role in the antagonistic activity of certain Pseudomonas strains (see above), but they can also be relevant for the establishment of the cells in the rhizosphere. P. putida strain WCS358 is a unique strain in that it cannot only utilize its own iron-siderophore but also the iron-siderophores of a diversity of other strains, for example that of strain P. fluorescens strain WCS374 [27]. When a siderophore-negative mutant of strain WCS358 is mixed through soil, its cell numbers in the rhizosphere are increased by the presence of wild-type (siderophore-producing) cells of WCS358 as well as those of WCS374. In contrast, the rhizosphere population of a siderophore-negative mutant of WCS374, which cannot utilize the siderophore of WCS358 [27] is increased by the presence of its own wild-type cells but reduced by the presence of cells of strain WCS358 [28]. This result shows that the production of siderophores and the ability to utilize the iron-siderophore complexes of other microorganisms is an important trait for the establishment of cells in the rhizosphere. Many Pseudomonas spp. produce antibiotics which can play a role in the competition with other rhizosphere microorganisms. When a mutant of P. fluorescens LA. de Weger et al. / FEMS Microbiology strain 2-79, deficient in the synthesis of the antibiotic, is studied for its wheat root colonization ability, the mutant was present at population levels comparable to those of the parent strain [29]. However, when the mutant and parent strain were compared after five successive 20-day plant-harvest cycles of wheat in the same soil, the mutant strain declined more rapidly in population size than the wild-type strain [30]. This indicates that the presence of antibiotic production can be a relevant factor for growth and survival in the rhizosphere. Another approach which may lead to the discovery of new unexpected traits involved in colonization is the direct isolation of mutants defective in colonization. The impaired functions in these mutants will teach us more about novel traits involved in colonization. Using P. fluorescens strain WCS365 with excellent root colonizing properties, we have screened approximately 900 mutants on individual potato, tomato or wheat plants in a gnotobiotic colonization assay (Fig. 1) for their colonization ability in competition with the parental strain. We isolated approximately 20 mutants which show an impaired colonization phenotype on either of these plants. Among them were auxotrophic mutants, mutants impaired in motility, mutants lacking the 0-antigenic side chain of LPS and mutants with a reduced growth rate. Three mutants were found that did not show any known defects. These mutants are the most interesting since they can lead to new colonization traits after cloning and screening of the corresponding wild-type genes. 4. Future prospects The current efforts to understand the mechanisms of plant growth promotion by Pseudomonas spp. will teach us which steps in this process are inefficient. Future research will have to focus on the regulation of crucial genes, like genes for antibiotic synthesis or genes involved in colonization. Since environmental factors in the rhizosphere may influence the expression of these genes and thus influence the biocontrol activities of the strains, we will have to increase our knowledge on the growth conditions in the rhizosphere. Once we know these conditions we may be able to construct more efficient biocon- Ecology 17 (I 995) 221-228 wildtype : mutant = 1 1 Fig. 1. Schematic representation of the colonization assay used to screen colonization-negative mutants. Sterile plants are inoculated with a 1:l mixture of the wild-type and the In&marked mutant. After one week of growth the ratio of wild-type and mutant cells on the root tips is determined on plates containing X-gal (5. bromo-4-chloro-3-indolyl-P-r>galactoside). trol bacteria for instance by manipulating antibiotic production with respect to timing and site. Furthermore, a better understanding of the rhizosphere conditions may allow us to mimic the rhizosphere conditions in the laboratory such that non-culturable microorganisms, which may comprise 90% to 99% of the bacterial population present in the rhizosphere, can be cultured. This would allow us to study this anonymous group of bacteria. Several bacterial traits have been identified as being relevant for efficient root colonization, i.e. presence of flagella, presence of the 0-antigenic side chain, and the ability to synthesise amino acids. Other traits reported to be relevant for the ability to compete with other rhizosphere microorganisms are 226 L.A. de Weger et al. / FEMS Microbiology e.g. production of antibiotics or siderophores and the ability to utilize siderophores of other rhizosphere bacteria. The present knowledge can be used by companies involved in the development of inoculants to improve strains by genetic engineering or for screening of isolates on these particular traits. Efficient colonization of plant roots can also be employed in other areas of research e.g. for bioremediation of polluted soil by employing bacterial colonizers of a plant with an extensive root system. Furthermore, the relevance of colonization and microcolony formation is stressed by the newly discovered quorum-sensing signal molecules (acyl-HSL’s). These molecules mediate production of antibiotics and secretion of exoenzymes only when cell numbers are high. Therefore the establishment of high populations on the root is not only relevant for the delivery of the biocontrol compound but even already for its synthesis. Disclosure of the complex network of interactions which comprises life in the rhizosphere can only be reached by a multidisciplinary approach. 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