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SummaryEven though beneficial plant-microbe interactions are commonly observed in nature, direct evidence for the evolution of bacterial mutualism in the rhizosphere remains elusive. Here we use experimental evolution to causally show that initially plant-antagonistic Pseudomonas protegens bacterium evolves into mutualists in the rhizosphere of Arabidopsis thaliana within six plant growth cycles (6 months). This evolutionary transition was accompanied with increased mutualist fitness via two mechanisms: i) improved competitiveness for root exudates and ii) enhanced capacity for activating the root-specific transcription factor gene MYB72, which triggers the production of plant-secreted scopoletin antimicrobial for which the mutualists evolved relatively higher tolerance to. Genetically, mutualism was predominantly associated with different mutations in the GacS/GacA two-component regulator system, which conferred high fitness benefits only in the presence of plants. Together, our results show that bacteria can rapidly evolve along the parasitism-mutualism continuum in the plant rhizosphere at an agriculturally relevant evolutionary timescale.

In nature and agriculture, plants interact with an astonishing number of microbes, collectively referred to as the "plant microbiome." Roots are a microbial hotspot where beneficial plant-microbe interactions are established that support plant growth and provide protection against pathogens and insects. Recently, we discovered that in response to foliar pathogen attack, plant roots can recruit specific protective microbes into the rhizosphere. Root exudates play an essential role in the interaction between plant roots and rhizosphere microbiota. In order to study the chemical communication between plant roots and the rhizosphere microbiome, it is essential to study the metabolite profile of root exudates. Here, we describe a detailed protocol for the collection of sterile root exudates that are secreted by Arabidopsis thaliana roots in response to inoculation of the leaves with the biotrophic pathogen Hyaloperonospora arabidopsidis.

Plant roots are associated with a diverse microbial community that consists of antagonistic, commensal and mutualistic organisms. While beneficial plant-microbe interactions are important for plant performance, direct evidence for the evolution of rhizosphere mutualism remains elusive. Here we experimentally show that initially plant-antagonistic Pseudomonas protegens bacterium can rapidly evolve into a mutualist in the rhizosphere of Arabidopsis thaliana only within six plant growth cycles (6 months). Using an in vivo experimental evolution system, we observed that P. protegens rapidly diversified into distinct phenotypes, with some having clearly positive effects on the plant performance. Crucially, this evolution of plant facilitation was also accompanied with increased bacterial fitness indicative of a mutualistic interaction. At the genetic level, P. protegens mutualism was associated with mutations in the GacS/GacA two-component global regulator system, which is known to affect several aspects of bacterial physiology including carbon metabolism, host-microbe signalling, and downregulation of phytotoxic secondary metabolites. At the phenotypic level, the evolution of mutualism could be explained via two mechanisms: improved competition for root exudates and enhanced capacity for activating the root-specific transcription factor gene MYB72, which is essential for the production and excretion of the antimicrobial scopoletin for which the mutualists evolved enhanced tolerance. Together, these results show that plant-bacteria mutualism can rapidly evolve at an agriculturally relevant evolutionary timescale in the rhizosphere.

The plant root microbiome consists of commensal, pathogenic and plant-beneficial microbes. Most members of the root microbiome possess microbe-associated molecular patterns (MAMPs) similar to those of plant pathogens. Their recognition by the cell surface-localized pattern recognition receptors can lead to the initiation of host immune signaling and suppression of plant growth due to growth-defense tradeoffs. We found that 42% of the tested root microbiota, including the plant growth-promoting rhizobacteria Pseudomonas capeferrum WCS358 (WCS358) and Pseudomonas simiae WCS417, are able to quench local Arabidopsis thaliana root immune responses that were triggered by the synthetic MAMP flg22, suggesting that this is an important function of the root microbiome. In a screen for WCS358 mutants that lost their capacity to suppress flg22-induced CYP71A12pro:GUS MAMP-reporter gene expression, we identified the bacterial genes pqqF and cyoB in WCS358. CyoB and pqqF are required for the production of gluconic acid and its derivative 2-keto gluconic acid. Both WCS358 mutants are impaired in the production of these organic acids and consequently lowered their extracellular pH to a lesser extent than wild-type WCS358. Acidification of the plant growth medium similarly suppressed flg22-induced CYP71A12pro:GUS and MYB51pro:GUS expression as well as the flg22-mediated oxidative burst, suggesting a role for rhizobacterial gluconic acid-mediated modulation of the extracellular pH in the suppression of local root immunity. Rhizosphere population densities of the mutants were significantly reduced compared to wild-type. Collectively, these findings show that suppression of local root immune responses is an important function of the root microbiome, as it facilitates colonization by beneficial root microbiota.

Significance Plant roots nurture a large diversity of soil microbes via exudation of chemical compounds into the rhizosphere. In turn, beneficial root microbiota promote plant growth and immunity. The root-specific transcription factor MYB72 has emerged as a central regulator in this process. Here, we show that MYB72 regulates the excretion of the coumarin scopoletin, an iron-mobilizing phenolic compound with selective antimicrobial activity that shapes the root-associated microbial community. Selected soil-borne fungal pathogens appeared to be highly sensitive to the antimicrobial activity of scopoletin, while two MYB72-inducing beneficial rhizobacteria were tolerant. Our results suggest that probiotic root-associated microbes that activate the iron-deficiency response during colonization stimulate MYB72-dependent excretion of scopoletin, thereby potentially improving their niche establishment and enhancing plant growth and protection., Plant roots nurture a tremendous diversity of microbes via exudation of photosynthetically fixed carbon sources. In turn, probiotic members of the root microbiome promote plant growth and protect the host plant against pathogens and pests. In the Arabidopsis thaliana–Pseudomonas simiae WCS417 model system the root-specific transcription factor MYB72 and the MYB72-controlled β-glucosidase BGLU42 emerged as important regulators of beneficial rhizobacteria-induced systemic resistance (ISR) and iron-uptake responses. MYB72 regulates the biosynthesis of iron-mobilizing fluorescent phenolic compounds, after which BGLU42 activity is required for their excretion into the rhizosphere. Metabolite fingerprinting revealed the antimicrobial coumarin scopoletin as a dominant metabolite that is produced in the roots and excreted into the rhizosphere in a MYB72- and BGLU42-dependent manner. Shotgun-metagenome sequencing of root-associated microbiota of Col-0, myb72, and the scopoletin biosynthesis mutant f6′h1 showed that scopoletin selectively impacts the assembly of the microbial community in the rhizosphere. We show that scopoletin selectively inhibits the soil-borne fungal pathogens Fusarium oxysporum and Verticillium dahliae, while the growth-promoting and ISR-inducing rhizobacteria P. simiae WCS417 and Pseudomonas capeferrum WCS358 are highly tolerant of the antimicrobial effect of scopoletin. Collectively, our results demonstrate a role for coumarins in microbiome assembly and point to a scenario in which plants and probiotic rhizobacteria join forces to trigger MYB72/BGLU42-dependent scopolin production and scopoletin excretion, resulting in improved niche establishment for the microbial partner and growth and immunity benefits for the host plant.

Disease suppressive soils typically develop after a disease outbreak due to the subsequent assembly of protective microbiota in the rhizosphere. The role of the plant immune system in the assemblage of a protective rhizosphere microbiome is largely unknown. In this study, we demonstrate that Arabidopsis thaliana specifically promotes three bacterial species in the rhizosphere upon foliar defense activation by the downy mildew pathogen Hyaloperonospora arabidopsidis. The promoted bacteria were isolated and found to interact synergistically in biofilm formation in vitro. Although separately these bacteria did not affect the plant significantly, together they induced systemic resistance against downy mildew and promoted growth of the plant. Moreover, we show that the soil-mediated legacy of a primary population of downy mildew infected plants confers enhanced protection against this pathogen in a second population of plants growing in the same soil. Together our results indicate that plants can adjust their root microbiome upon pathogen infection and specifically recruit a group of disease resistance-inducing and growth-promoting beneficial microbes, therewith potentially maximizing the chance of survival of their offspring that will grow in the same soil.

The rhizosphere is the infection court where soil-borne pathogens establish a parasitic relationship with the plant. To infect root tissue, pathogens have to compete with members of the rhizosphere microbiome for available nutrients and microsites. In disease-suppressive soils, pathogens are strongly restricted in growth by the activities of specific rhizosphere microorganisms. Here, we sequenced metagenomic DNA and RNA of the rhizosphere microbiome of sugar beet seedlings grown in a soil suppressive to the fungal pathogen Rhizoctonia solani. rRNA-based analyses showed that Oxalobacteraceae, Burkholderiaceae, Sphingobacteriaceae and Sphingomonadaceae were significantly more abundant in the rhizosphere upon fungal invasion. Metatranscriptomics revealed that stress-related genes (ppGpp metabolism and oxidative stress) were upregulated in these bacterial families. We postulate that the invading pathogenic fungus induces, directly or via the plant, stress responses in the rhizobacterial community that lead to shifts in microbiome composition and to activation of antagonistic traits that restrict pathogen infection.

Systemically induced resistance is a promising strategy to control plant diseases, as it affects numerous pathogens. However, since induced resistance reduces one or both growth and activity of plant pathogens, the indigenous microflora may also be affected by an enhanced defensive state of the plant. The aim of this study was to elucidate how much the bacterial rhizosphere microflora of Arabidopsis is affected by induced systemic resistance (ISR) or systemic acquired resistance (SAR). Therefore, the bacterial microflora of wild-type plants and plants affected in their defense signaling was compared. Additionally, ISR was induced by application of methyl jasmonate and SAR by treatment with salicylic acid or benzothiadiazole. As a comparative model, we also used wild type and ethylene-insensitive tobacco. Some of the Arabidopsis genotypes affected in defense signaling showed altered numbers of culturable bacteria in their rhizospheres; however, effects were dependent on soil type. Effects of plant genotype on rhizosphere bacterial community structure could not be related to plant defense because chemical activation of ISR or SAR had no significant effects on density and structure of the rhizosphere bacterial community. These findings support the notion that control of plant diseases by elicitation of systemic resistance will not significantly affect the resident soil bacterial microflora.

Summary Pseudomonas putida KT2440 is an efficient colonizer of the rhizosphere of plants of agronomical and basic interest. We have demonstrated that KT2440 can protect the model plant Arabidopsis thaliana against infection by the phytopathogen Pseudomonas syrin- gae pv. tomato DC3000. P. putida extracellular haem- peroxidase (PP2561) was found to be important for competitive colonization and essential for the induc- tion of plant systemic resistance. Root exudates of plants elicited by KT2440 exhibited distinct patterns of metabolites compared with those of non-elicited plants. The levels of some of these compounds were dramatically reduced in axenic plants or plants colo- nized by a mutant defective in PP2561, which has increased sensitiveness to oxidative stress with respect to the wild type. Thus high-level oxidative stress resistance is a bacterial driving force in the rhizosphere for efficient colonization and to induce systemic resistance. These results provide important new insight into the complex events that occur in order for plants to attain resistance against foliar pathogens.

The rhizosphere is a complex and dynamic environment in which microbes introduced to control root pathogens must establish and maintain populations of sufficient size and activity to antagonize pathogens directly or by manipulating the host plant’s own defenses. Genetic and physiological studies of rhizobacteria with the capacity to control root pathogens have given considerable insight into the microbial side of these interactions, but much remains to be learned about the physical conditions and the chemical and biological activities that take place at the root-microbe interface. This chapter focuses on advances in our understanding of the constraints to the successful introduction of microbial agents for the control of soil-borne root pathogens and the mechanisms involved in pathogen suppression. Chapters elsewhere in this volume address related topics including plant growth promotion , stress control, the activation of the plant’s own defense mechanisms by introduced microbes, and powerful new biotechnological advances available to gain insight into rhizosphere processes.

To investigate the impact of genetically modified, antibiotic-producing rhizobacteria on the indigenous microbial community, Pseudomonas putida WCS358r and two transgenic derivatives were introduced as a seed coating into the rhizosphere of wheat in two consecutive years (1999 and 2000) in the same field plots. The two genetically modified microorganisms (GMMs), WCS358r::phz and WCS358r::phl, constitutively produced phenazine-1-carboxylic acid (PCA) and 2,4-diacetylphloroglucinol (DAPG), respectively. The level of introduced bacteria in all treatments decreased from 10 7 CFU per g of roots soon after sowing to less than 10 2 CFU per g after harvest 132 days after sowing. The phz and phl genes remained stable in the chromosome of WCS358r. The amount of PCA produced in the wheat rhizosphere by WCS358r::phz was about 40 ng/g of roots after the first application in 1999. The DAPG-producing GMMs caused a transient shift in the indigenous bacterial and fungal microflora in 1999, as determined by amplified ribosomal DNA restriction analysis. However, after the second application of the GMMs in 2000, no shifts in the bacterial or fungal microflora were detected. To evaluate the importance of the effects induced by the GMMs, these effects were compared with those induced by crop rotation by planting wheat in 1999 followed by potatoes in 2000. No effect of rotation on the microbial community structure was detected. In 2000 all bacteria had a positive effect on plant growth, supposedly due to suppression of deleterious microorganisms. Our research suggests that the natural variability of microbial communities can surpass the effects of GMMs.

Pseudomonas putida WCS358r, genetically modified to have improved activity against soil-borne pathogens, was released into the rhizosphere of wheat. Two genetically modified derivatives carried the phz or the phl biosynthetic gene loci and constitutively produced either the antifungal compound phenazine-1-carboxylic acid (PCA) or the antifungal and antibacterial compound 2,4-diacetylphloroglucinol (DAPG). In 1997 and 1998, effects of single introductions of PCA producing derivatives on the indigenous microflora were studied. A transient shift in the composition of the total fungal microflora, determined by amplified ribosomal DNA restiction analysis (ARDRA), was detected. Starting in 1999, effects of repeated introduction of genetically modified microorganisms (GMMs) were studied. Wheat seeds coated with the PCA producer, the DAPG producer, a mixture of the PCA and DAPG producers, or WCS358r, were sown and the densities, composition and activities of the rhizosphere microbial populations were measured. All introduced strains decreased from 10(7) CFU per gram of rhizosphere sample to below the detection limit after harvest of the wheat plants. The phz genes were stably maintained in the PCA producers, and PCA was detected in rhizosphere extracts of plants treated with this strain or with the mixture of the PCA and DAPG producers. The phl genes were also stably maintained in the DAPG producing derivative of WCS358r. Effects of the genetically modified bacteria on the rhizosphere fungi and bacteria were analyzed by using amplified ribosomal DNA restriction analysis. Introduction of the genetically modified bacterial strains caused a transient change in the composition of the rhizosphere microflora. However, introduction of the GMMs did not affect the several soil microbial activities that were investigated in this study.

There is increasing interest in commercial application of genetically modified microorganisms (GMMs) with improved biocontrol properties toward soil-borne plant pathogens. Despite long-term experience with the introduction of nonmodified microorganisms, concern about the ecological impact of large-scale release of GMMs remains. To date, risk assessment studies under field conditions have focused mainly on microorganisms genetically modified with markers, such as antibiotic resistance, lacZY, or xylE, and attention has been given to the potential impact of GMMs on the indigenous soil microflora (8, 29). Biologically mediated processes are central to the ecological functioning of the soil system. Microorganisms play a crucial role in nutrient cycling, and they contribute to suppression of soil-borne plant pathogens (7, 28). The introduction of large numbers of GMMs into the soil could alter microbial populations and disturb microbially driven soil processes. Effects of introduced GMMs on soil ecosystems have been studied mainly in microcosm experiments. Transient perturbations have been observed in indigenous bacterial (26), fungal (30), and protozoal (3) populations, in carbon turnover (38), and in soil enzyme activities (24). However, these microcosms lack the full biotic and abiotic components of a field environment. Most studies of nontarget effects of GMMs examine only the culturable microflora. This limitation reduces the value of the results, because only a small proportion (0.5 to 2%) of the fungal and bacterial microflora can be cultured using currently available media (37). Techniques such as amplified ribosomal DNA (rDNA) restriction analysis (ARDRA), temperature gradient gel electrophoresis (TGGE), or denaturing gradient gel electrophoresis (DGGE) (23) can be used to more accurately monitor microbial communities without cultivation. Shifts in microbial communities, at the 16S rDNA level, have been studied mainly in soil polluted with heavy metals or with pesticides (12, 13, 31). Reports on the effects of functional genetically modified bacteria on microbial communities, using molecular methods, are scarce. Robleto et al. (29) demonstrated a reduction in the diversity of trifolitoxin-sensitive bacteria after inoculation of field-grown Phaseolus vulgaris with Rhizobium strains differing in their trifolitoxin production. As far as we are aware, no reports have been published on the effects of genetically modified biocontrol bacteria on the composition of the fungal microflora at the 18S rDNA level. We modified Pseudomonas putida WCS358r to produce the antifungal agent phenazine-1-carboxylic acid (PCA) (35), resulting in improved biocontrol activity toward fungal pathogens, such as Gaeumannomyces graminis var. tritici (D. Glandorf et al., unpublished data). Our objective in this study was to determine if genetically improved biocontrol bacteria could exert effects on nontarget members of the fungal rhizosphere microflora by using both cultivation-dependent and cultivation-independent methods.

Fusarium wilt diseases, caused by the fungus Fusarium oxysporum, lead to significant yield losses of crops. One strategy to control fusarium wilt is the use of antagonistic, root-colonizing Pseudomonas spp. It has been demonstrated that different strains of these bacteria suppress disease by different mechanisms. Therefore, application of a mixture of these biocontrol strains, and thus of several suppressive mechanisms, may represent a viable control strategy. A prerequisite for biocontrol by combinations of biocontrol agents can be the compatibility of the co-inoculated micro-organisms. Hence, compatibility between several Pseudomonasspp. strains, that have the ability to suppress fusarium wilt of radish, was tested in vitro on KB agar plates. Growth of P. fluorescens strain RS111 was strongly inhibited by Pseudomonas spp. strains RE8, RS13, RS56 and RS158, whereas a mutant of strain RS111 (RS111-a) was insensitive to inhibition by these strains. Strains RS111 and RS111-a only slightly inhibited some other strains. Suppression of fusarium wilt of radish in a potting soil bioassay by the incompatible combination of RE8 and RS111 was comparable to the effects of the single strains. However, disease suppression by the compatible combination of RE8 and RS111-a was significantly better as compared to the single strains. In contrast, the incompatible combination of RS56 with RS111 resulted in enhanced disease suppression as compared to the single strains. Increased disease suppression by combinations of RS13 or RS158 with RS111 or RS111-a was not observed. This indicates that specific interactions between biocontrol strains influence disease suppression by combinations of these strains.

▪ Abstract Nonpathogenic rhizobacteria can induce a systemic resistance in plants that is phenotypically similar to pathogen-induced systemic acquired resistance (SAR). Rhizobacteria-mediated induced systemic resistance (ISR) has been demonstrated against fungi, bacteria, and viruses in Arabidopsis, bean, carnation, cucumber, radish, tobacco, and tomato under conditions in which the inducing bacteria and the challenging pathogen remained spatially separated. Bacterial strains differ in their ability to induce resistance in different plant species, and plants show variation in the expression of ISR upon induction by specific bacterial strains. Bacterial determinants of ISR include lipopolysaccharides, siderophores, and salicylic acid (SA). Whereas some of the rhizobacteria induce resistance through the SA-dependent SAR pathway, others do not and require jasmonic acid and ethylene perception by the plant for ISR to develop. No consistent host plant alterations are associated with the induced state, but upon challenge inoculation, resistance responses are accelerated and enhanced. ISR is effective under field conditions and offers a natural mechanism for biological control of plant disease.

In this study, the potential of different Pseudomonas strains to utilize heterologous siderophores was compared with their competitiveness in the rhizosphere of radish. This issue was investigated in interactions between Pseudomonas putida WCS358 and Pseudomonas fluoresceins WCS374 and in interactions between strain WCS358 and eight indigenous Pseudomonas strains capable of utilizing pseudobactin 358. During four successive plant growth cycles of radish, strain WCS358 significantly reduced rhizosphere population densities of the wild-type strain WCS374 by up to 30 times, whereas derivative strain WCS374(pMR), harboring the siderophore receptor PupA for ferric pseudobactin 358, maintained its population density. Studies involving interactions between strain WCS358 and eight different indigenous Pseudomonas strains demonstrated that despite the ability of these indigenous isolates to utilize pseudobactin 358, their rhizosphere population densities were significantly reduced by strain WCS358 by up to 20 times. Moreover, rhizosphere colonization by WCS358 was not affected by any of these indigenous strains, even though siderophore-mediated growth inhibition of WCS358 by a majority of these strains was demonstrated in a plate bioassay. In conclusion, it can be stated that siderophore-mediated competition for iron is a major determinant in interactions between WCS358 and WCS374 in the rhizosphere. Moreover, our findings support the common assumption that cloning of siderophore receptor genes from one Pseudomonas strain into another can confer a competitive advantage in interactions in the rhizosphere. Interactions between WCS358 and the selected indigenous rhizosphere isolates, however, indicate that other traits also contribute to the rhizosphere competence of fluorescent Pseudomonas spp.Key words: siderophore, siderophore receptors, root colonization, fluorescent Pseudomonas.

Crop specificity of rhizosphere-inhabiting fluorescent Pseudomonas spp was studied for potato, grass and wheat grown in a silty loam soil. For each crop about 50 dominant Pseudomonas isolates were obtained from the rhizosphere. Isolates were distinguished by comparing their lipopolysaccharide (LPS) and cell envelope protein (CEP) patterns. About 30 distinct patterns were detected for each crop. The majority of LPS and CEP patterns observed for each crop was not detected on the other crops, suggesting a crop specificity for certain Pseudomonas populations. From each crop distinct Pseudomonas isolates were used to study crop-specific root colonization by growing potato, grass and wheat plants in soil bacterized with a mixture of these isolates of the three crops. Crop specificity in root colonization could not be demonstrated. The involvement of root agglutinins in the composition of rhizosphere Pseudomonas populations was examined. Reactions of distinct Pseudomonas isolates from each crop with crude or partially-purified agglutinin preparations of potato, grass or wheat did not correlate. We concluded that the composition of rhizosphere Pseudomonas populations differed between crops. However, crop-specific root colonization or crop-specific agglutination could not be demonstrated with the experimental approach used.

The stability of rifampicin resistance in plant growth-promoting Pseudomonas putida strain WCS358 was studied in potato rhizosphere in the field. Three out of seven rifampicin-resistant mutants of strain WCS358 were selected in this study. Their specific growth rate, competitive growth in liquid medium and colonization of potato roots in non-sterile soil, was comparable to that of their parental strain. These rifampicin-resistant mutants were used to treat potato seed tubers, which were thereafter sown in the field. To test the stability of the rifampicin resistance in the field, about 1200 fluorescent Pseudomonas isolates obtained from underground plant parts at 82, 95, 109 and 130 days after seeding, were tested for rifampicin resistance and for agglutination with an antiserum specific for strain WCS358. Ail fluorescent Pseudomonas isolates that showed a positive agglutination reaction with the antiserum, were also rifampicin-resistant. Twelve agglutination-positive isolates, selected at random, were all identified as strain WCS358 from patterns of lipopolysaccharides after sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Thus, rifampicin resistance seems to be a stable marker in the mutants of strain WCS358 tested, also under field conditions. It is concluded that rifampicin resistance can be used as a reliable marker for ecological studies on rhizosphere pseudomonads.

Iron is an essential micronutrient for plants and associated microorganisms. Iron nutrition of these organisms relies on the soil supply. However, bioavailability of iron in cultivated soils is low. Plants and microorganisms have thus evolved active strategies of iron uptake based on acidification, chelation, and/or reduction processes. Iron acquisition by these organisms leads to complex interactions ranging from mutualism to competition. In the rhizosphere, plants support abundant and active microbial communities through the release of rhizodeposits. Iron uptake by these microorganisms and by the host plant decrease even more the concentration of iron in solution. Therefore, there is an intense competition for iron among rhizosphere microorganisms, favoring those with the most efficient iron uptake strategy. This is the case for fluorescent Pseudomonas bacteria that synthesize siderophores, called pyoverdines or pseudobactines, which have a high affinity for iron and suppress fungal phytopathogens and deleterious microorganisms. Pyoverdines also elicit plant defense reactions and contribute to plant iron acquisition. Taken together, these mutual effects promote plant growth and health. However, competition for iron may also occur between plants and microbes during pathogenesis. Siderophores contribute to the iron uptake of the host plant and to the virulence of pathogens; conversely, host plants activate mechanisms aimed at depriving pathogens of nutritional iron. The iron-withholding mechanisms of the host plant rely on controlling its iron homeostasis. In this chapter, we describe the strategies of iron uptake of plants and microorganisms, the resulting complex interactions between them, and the challenges represented by their monitoring in agroecology.

A long-term field experiment (1999-2002) was conducted to monitor effects on the indigenous microflora of Pseudomonas putida WCS358r and two transgenic derivatives constitutively producing phenazine-1-carboxylic acid (PCA) or 2,4-diacetylphloroglucinol (DAPG). The strains were introduced as seed coating on wheat into the same field plots each year. Rhizosphere populations of ascomycetes were analysed using denaturing gradient gel electrophoresis (DGGE). To evaluate the significance of changes caused by the genetically modified microorganisms (GMMs), they were compared with effects caused by a crop rotation from wheat to potato. In the first year, only the combination of both GMMs caused a significant shift in the ascomycete community. After the repeated introductions this effect was no longer evident. However, cropping potato significantly affected the ascomycete community. This effect persisted into the next year when wheat was grown. Clone libraries were constructed from samples taken in 1999 and 2000, and sequence analysis indicated ascomycetes of common genera to be present. Most species occurred in low frequencies, distributed almost evenly in all treatments. However, in 1999 Microdochium occurred in relatively high frequencies, whereas in the following year no Microdochium species were detected. On the other hand, Fusarium-like organisms were low in 1999, and increased in 2000. Both the DGGE and the sequence analysis revealed that repeated introduction of P. putida WCS358r had no major effects on the ascomycete community in the wheat rhizosphere, but demonstrated a persistent difference between the rhizospheres of potato and wheat.

To assess effects of plant crop species on rhizosphere ascomycete communities in the field, we compared a wheat monoculture and an alternating crop rotation of wheat and potato. Rhizosphere soil samples were taken at different time points during the growing season in four consecutive years (1999-2002). An ascomycete-specific primer pair (ITS5-ITS4A) was used to amplify internal transcribed spacer (ITS) sequences from total DNA extracts from rhizosphere soil. Amplified DNA was analyzed by denaturing gradient gel electrophoresis (DGGE). Individual bands from DGGE gels were sequenced and compared with known sequences from public databases. DGGE gels representing the ascomycete communities of the continuous wheat and the rotation site were compared and related to ascomycetes identified from the field. The effect of crop rotation exceeded that of the spatial heterogeneity in the field, which was evident after the first year. Significant differences between the ascomycete communities from the rhizospheres of wheat in monoculture and one year after a potato crop were found, indicating a long-term effect of potato. Sequencing of bands excised from the DGGE gels revealed the presence of ascomycetes that are common in agricultural soils.

Seventy bacterial isolates from the rhizosphere of tomato were screened for antagonistic activity against the tomato foot and root rot-causing fungal pathogen Fusarium oxysporum f. sp. radicis-lycopersici. One isolate, strain PCL1391, appeared to be an efficient colonizer of tomato roots and an excellent biocontrol strain in an F. oxysporum/tomato test system. Strain PCL1391 was identified as Pseudomonas chlororaphis and further characterization showed that it produces a broad spectrum of antifungal factors (AFFs), including a hydrophobic compound, hydrogen cyanide, chitinase(s), and protease(s). Through mass spectrometry and nuclear magnetic resonance, the hydrophobic compound was identified as phenazine-1-carboxamide (PCN). We have studied the production and action of this AFF both in vitro and in vivo. Using a PCL1391 transposon mutant, with a lux reporter gene inserted in the phenazine biosynthetic operon (phz), we showed that this phenazine biosynthetic mutant was substantially decreased in both in vitro antifungal activity and biocontrol activity. Moreover, with the same mutant it was shown that the phz biosynthetic operon is expressed in the tomato rhizosphere. Comparison of the biocontrol activity of the PCN-producing strain PCL1391 with those of phenazine-1-carboxylic acid (PCA)-producing strains P. fluorescens 2-79 and P. aureofaciens 30-84 showed that the PCN-producing strain is able to suppress disease in the tomato/F. oxysporum system, whereas the PCA-producing strains are not. Comparison of in vitro antifungal activity of PCN and PCA showed that the antifungal activity of PCN was at least 10 times higher at neutral pH, suggesting that this may contribute to the superior biocontrol performance of strain PCL1391 in the tomato/F. oxysporum system.

Under low iron conditions, Pseudomonas putida WCS358 produces a specific siderophore, pseudobactin 358, and a specific receptor for this siderophore, pupA. Addition of pseudobactin to an agar medium, such that all Fe 3+ in the medium is bound to this siderophore, facilitated selective reisolation of wild-type P. putida WCS358 from soil and potato rhizosphere.

Crop specificity of fluorescent Pseudomonas spp. was studied on potato, wheat, grass, tomato and carnation. Pseudomonas isolates derived from the rhizospheres of these crops were distinquished by comparing their lipopolysaccharide (LPS) and cell envelope protein (CEP) patterns, using sodium dodecyl sulfate polyacrylamide gel electroforesis (SDS-PAGE). The majority of LPS and CEP patterns detected for each crop, were not detected on the other crops. These results suggest a crop specificity for certain Pseudomonas populations. Agglutinins derived from root washings of potato, wheat, grass, tomato and carnation were used to study the involvement of agglutinins in the composition of rhizosphere Pseudomonas populations. Isolates from wheat and grass reacted more frequently with wheat agglutinins compared to isolates from other crops. For the other agglutinins no differential results were obtained with isolates from different crops. The composition of the rhizosphere Pseudomonas populations appeares to differ between the crops studied. For wheat, agglutinins may be involved in this phenomenon.

Rhizobacteria live around roots but also inside the cortical root tissues by utilizing organic substances released from root cells into the intercellular spaces and the root environment. The effects of metabolites of these rhizosphere-inhabiting bacteria on root physiology and plant development have hardly been studied. However, recent studies indicate that, depending on environmental factors and plant species, certain strains of rhizosphere Pseudomonas spp. and some of their metabolites such as HCN may inhibit or enhance plant establishment or inhibit development of plant disease. Cultural practices such as cropping frequency, no tillage, and soilless cultivation, as well as edaphic factors seem to determine these rhizosphere interactions.

The dose-response relationships in the suppression of Fusarium wilt of radish by Pseudomonas putida strain WCS358 and P. fluorescens strain WCS374 were investigated. The sole mechanism involved in the suppression of Fusarium wilt of radish by strain WCS358 is siderophore-mediated competition for iron(III), whereas strain WCS374 suppresses this disease by induction of systemic resistance. The efficacy of siderophore-mediated disease suppression and induced resistance was highly dependent on the level of disease incidence. Both mechanisms of biological control were effective over a wide range of disease incidences. The absolute disease reduction reached a maximum of approximately 30% at an average level of disease incidence of 50% for both mechanisms. The rhizosphere population density of strain WCS358 and of strain WCS374 was an important determinant of their efficacy to suppress Fusarium wilt of radish. Regression analysis demonstrated significant nonlinear asymptotic relationships between the rhizosphere population densities of strain WCS358 or strain WCS374 and the level of suppression of Fusarium wilt of radish. At moderate to relatively high levels of disease incidence for both mechanisms, a threshold population density of the bacterial strain of approximately 10 5 CFU per gram of root is required for a significant suppression of Fusarium wilt of radish. When rhizosphere population densities of both strains dropped below this threshold level, a relatively small decline in the population density had a major effect on their efficacy to suppress Fusarium wilt of radish. Increasing the rhizosphere population density of both strains to levels higher than the threshold level did not result in a significant improvement of disease suppression.

Pseudomonas putida strain WCS358, P. fluorescens strain WCS374, and siderophore‐negative mutants of these strains obtained by Tn5 transposon mutagenesis, were used in experiments to assess the role of siderophores in potato growth stimulation by WCS358. The siderophore‐negative mutants used were still able to use the siderophores produced by their respective wild‐types. WCS358 was able to use the siderophore produced by WCS374, but WCS374 could not use the siderophore produced by WCS358. Population densities of siderophore‐negative mutants of WCS358 on potato roots were high in the presence of the wild‐type strains of WCS358 or WCS374, whereas population densities of siderophore‐negative mutants of WCS374 were high only in the presence of wild‐type strain WCS374. It is concluded from these experiments that siderophores of WCS358 and WCS374 are produced in the potato rhizosphere. Root development of potato stem cuttings and potato tuber yields in soils frequently cropped to potato were increased b...

Evidence is increasing that the saprophytic microflora of the rhizosphere includes both deleterious and beneficial elements that have the potential to influence plant growth and crop yields significantly (13, 16, 25, 27, 57, 72, 77, 78). The deleterious microorganisms affect plant growth negatively, but they do not necessarily parasitize the plant tissue. Their deleterious activities include alterations of the supply of water, ions, and plant growth substances by changing root functions and/or by limiting root growth (1,57, 67, 77). The mechanisms by which beneficial nonsymbiotic microorganisms are considered to affect plant growth positively include promotion of the availability and uptake of mineral nutrients (6, 7, 27), provision of plant-growth substances (27, 56, 57), and, most of all, suppression of deleterious rhizosphere microorganisms (13, 27, 28, 72, 77). Beneficial rhizobacteria that promote plant growth, supposedly by competing for iron with deleterious rhizosphere microorganisms, have recently become known as plant growth-promoting rhizobacteria (PGPR) (13, 41, 67, 77). This paper reviews recent knowledge on deleterious and beneficial rhizosphere bacteria, their interactions, and their effect on yield as related to cropping practices.

Organic compounds released by roots stimulate the activity of a variety of micro-organisms in the root environment, the rhizosphere (Rovira and Davey 1974). Of these micro-organisms, soil-borne plant pathogens and nitrogen fixing micro-organisms have obtained most attention. Relatively little is known about the majority of the rhizosphere micro-organisms, the saprophytes and their influence on root functioning, plant development and crop yield.

Treatment of seed potatoes with certain root-colonizing Pseudomonas putida and fluorescens strains has resulted in protection of the potato tuber yield against the effects of narrow rotation cropping (1:3) and against the effects of certain microbial pathogens. This protective activity of the bacteria is thought to be caused by the production and excretion of large quantities of siderophores with high affinity for binding of iron(III) and uptake of the siderophore-iron(III) complex. The subsequent decrease in iron(III) around the root-surface prevents or delays the growth of other (pathogenic) micro-organisms.

Microbial factors, responsible for potato tuber yield reductions in high frequency potato cropping at the experimental farm ‘De Schreef’ in a Dutch polder, were investigated. Besides Streptomyces spp. and Verticillium dahliae, another bacterial factor seemed to be involved. The mechanisms by which this bacterial factor limits potato root growth and tuber yield and the mechanisms of its counteraction by the plant growthpromoting Pseudomonas putida strain WCS358 were analyzed. Based on experimental data, we hypothesize that part of the potato rhizosphere Pseudomonas populations produces more cyanide in short rotations of potato (at ‘De Schreef’) than in long rotations of potato; the cyanide permeates the roots and negatively influences the root energy metabolism.

Although potato is an economically profitable crop, its cultivation in the Netherlands is limited to once every three years on the same soil, since diseases (e.g., caused by nematodes or pathogenic microorganisms) may occur in soil more frequently cropped to potato. Even in this rotation schedule, crops are reduced when compared to potato crops from soils cropped only once every six years. Presently, this crop reduction is often due to an increase in the population of harmful microorganisms (Schippers et al 1985, 1986). This crop reduction can be abolished by applying antagonistic Pseudomonas spp. to the seed-potatoes (Geels and Schippers 1983; Bakker et al, submitted for publication).

The stimulation of plant growth by fluorescent pseudomonads has recently been reviewed (Schippers et al., 1985; Schippers et al., in press). Field- and pot experiments in the Netherlands strongly suggest that potato growth stimulation by seed tuber bacterization with pseudomonads selected for efficient siderophore mediated Fe3+-uptake, is most noticable in soils where frequent potato cropping causes a significant reduction in potato yield (Geels and Schippers, 1983b; Geels and Schippers, 1983c; Schippers et al., 1985). Such yield reductions were more severe with increasing potato cropping frequency in long term rotational experiments (Hoekstra, 1981; Lamers, 1981; Schippers et al., 1985). They are largely caused by as yet unknown harmful rhizosphere microorganisms (HMO) rather than by well-known soil-borne pathogens (Scholte, et al.,1985; Schippers et al., 1985). The yield reductions were reproduced in pot experiments which could be almost eliminated if potato tubers were treated with selected pseudomonads before planting (Geels and Schippers, 1983a; Geels and Schippers, 1983b). In pot experiments significant stimulation of potato growth by bacterization of tubers was only observed in soil from fields frequently cropped with potato and not in soil from fields with no recent history of potato cropping. Similarly, in field experiments yield increases by tuber bacterization were only obtained in soils frequently cropped with potato. In some experiments yield increases were detected only during the first three months following planting and differences between treated tubers and controls were not significant at harvest-time. However, the rifampicin resistant Pseudomonas isolates used could be reisolated from roots of treated plants until harvest although their numbers gradually declined during the season (Geels et al., submitted for publication). Failure to increase yield in the field is one of the major problems. This is possibly due to inadequate colonization by PGSP of root parts that are responsible for tuber development later in the season. Failure of HMO to develop their harmful activity could be another factor, because yield reductions in short rotations were not apparent to the same extent every season and in every field plot.