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In July 2020, plants with crinkled, chlorotic, occasionally necrotic leaves, typical for Soybean Mosaic Virus (SMV), were observed in eight soybean fields (Glycine max L.) in Flevoland, The Netherlands (Supp. Fig. 1). Disease incidence varied from 5-50% and the plants affected often occurred in small or extensive patches. Leaves from several symptomatic plants were sampled from each of two fields planted with soybean variety Green Shell or Summer Shell. Total RNA was extracted from one plant leaf sample per field using InviTrap Spin Plant RNA Mini Kit (Invitek, Germany). One-tube RT-PCRs employing potyvirus generic primers P9502 and CPUP (Van der Vlugt et al, 1999) and SMV-specific primers SMV-dT (5'-TTTTTTTTTTTTTTTAGGACAAC-3') and SMV-Nib-Fw (5'-CAAGGATGARTTTAAGGAG-3') combined with Sanger sequencing confirmed the presence of SMV in all leaf samples. To exclude the presence of other agents in the samples, total RNA from each cultivar was used in standard Illumina library preparation with ribosomal RNA depletion followed by sequencing on an Illumina NovaSeq6000 (paired-end, 150 bp) which yielded 66,579,158 reads (Summer Shell) and 223,953,206 reads (Green Shell). After quality trimming in CLC Genomics Workbench 20.0.4 (CLC-GWB; Qiagen, Hilden), four million reads were randomly sampled for de novo assembly. Contigs over 500 nucleotides (nts) in length with a minimum of 500 reads were annotated by BLASTn against NCBI GenBank. This identified one contig of 9,883 nts (6,233,397 reads) in Summer Shell and one contig of 9,727 nts (3,139,927 reads) in Green Shell with clear homology to SMV (E-value = 0.0). No other viruses were identified in the datasets. Reference assemblies against the SMV reference sequence (NC_002634) mapped 24,090,763 reads (36.2%) for Summer Shell and 175,459,637 reads (78.3%) for Green Shell. Extracted consensus sequences for SMV in both soybean cultivars were 9,584 nts long (excluding the poly-A tail). Sequence data from the de novo and reference assemblies were combined into consensus sequences which showed over 98% overall nt sequence identity to NC_002634 and 99.6% to each other. Both consensus sequences were deposited in GenBank under accession numbers MW822167 (SMV-Summer Shell) and MW822168 (SMV-Green Shell). In addition, the presence of SMV in the field samples was confirmed with an inoculation assay. Leaf samples from both fields were ground in phosphate buffer (0.1M, pH 7.2) and inoculated on cotyledons and first expanded leaves of soybean plants (unknown cv.) 12 days post-germination. Plants showed veinal chlorosis in systemic leaves from 12 days post-inoculation, which developed into veinal necrosis. SMV infections were confirmed by RT-PCR in systemic, chlorotic leaf samples of all symptomatic plants using the SMV-specific primers described above. To our knowledge, this is the first report of SMV in The Netherlands. As soybean is a relatively new but expanding crop in this country, information about emerging diseases is highly relevant. SMV can be transmitted via seeds and aphids, where seeds can serve as primary source of virus inoculum (Cui et al., 2011; Hartman et al., 2016; Hajimorad et al., 2018). Weeds and non-commercial plants can also serve as origin of SMV, particularly in subsequent growing seasons, although this virus infects a limited host range of six plant families (Cui et al., 2011; Hill & Whitham, 2014). Special monitoring would be advised for the recurrence and possible damage by SMV in Dutch soybean fields.

Pseudomonas simiae WCS417 is a root-colonizing bacterium with well-established plant-beneficial effects. Upon colonization of Arabidopsis roots, WCS417 evades local root immune responses while triggering an induced systemic resistance (ISR) in the leaves. The early onset of ISR in roots shows similarities with the iron deficiency response, as both responses are associated with the production and secretion of coumarins. Coumarins can mobilize iron from the soil environment and have a selective antimicrobial activity that impacts microbiome assembly in the rhizosphere. Being highly coumarin-tolerant, WCS417 induces the secretion of these phenolic compounds, likely to improve its own niche establishment, while providing growth and immunity benefits for the host in return. To investigate the possible signaling function of coumarins in the mutualistic Arabidopsis-WCS417 interaction, we analyzed the transcriptome of WCS417 growing in root exudates of coumarin-producing Arabidopsis Col-0 and the coumarin-biosynthesis mutant f6′h1. We found that coumarins in F6′H1-dependent root exudates significantly affected the expression of 439 bacterial genes (8% of the bacterial genome). Of those, genes with functions related to transport and metabolism of carbohydrates, amino acids, and nucleotides were induced, whereas genes with functions related to cell motility, the bacterial mobilome, and energy production and conversion were repressed. Strikingly, most genes related to flagellar biosynthesis were down-regulated by F6′H1-dependent root exudates and we found that application of selected coumarins reduces bacterial motility. These findings suggest that coumarins’ function in the rhizosphere as semiochemicals in the communication between the roots and WCS417. Collectively, our results provide important novel leads for future functional analysis of molecular processes in the establishment of plant-mutualist interactions.

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.

The rhizosphere microbiome of plants is essential for plant growth and health. Recent studies have shown that upon infection of leaves with a foliar pathogen, the composition of the root microbiome is altered and enriched with bacteria that in turn can systemically protect the plant against the foliar pathogen. This protective effect is extended to successive populations of plants that are grown on soil that was first conditioned by pathogen-infected plants, a phenomenon that was coined "the soil-borne legacy." Here we provide a detailed protocol for soil-borne legacy experiments with the model plant Arabidopsis thaliana after infection with the obligate biotrophic pathogen Hyaloperonospora arabidopsidis. This protocol can easily be extended to infection with other pathogens or even infestation with herbivorous insects and can function as a blueprint for soil-borne legacy experiments with crop species.

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.

Trichoderma species and fluorescent Pseudomonas spp. have been reported to induce systemic resistance in plants. In this study the effectiveness of a combination of these biological control agents on the efficacy of induced resis- tance was investigated in cucumber and the model plant Arabidopsis thaliana. Trichoderma harzianum Tr6, and Pseudomonas sp. Ps14, both isolated from the rhizosphere of cucumber, were tested as a single application and in combination for their abilities to elicit induced resistance in cucumber against Fusarium oxysporum f. sp. radicis cucumerinum and in A. thaliana against Botrytis cinerea. The combination of Tr6 and Ps14 induced a significantly higher level of resistance in cucumber, which was associated with the primed expression of a set of defense-related genes upon challenge with Fusarium. In Arabidopsis both Ps14 and Tr6 triggered ISR against B. cinerea but their combination did not show enhanced effects. In the induced systemic resistance-defective Arabidopsis mutant myb72, none of the treatments protected against B. cinerea, whereas in the SA-impaired mutant sid2 all treatments were effective. Taken together, these results indicate that in Arabidopsis Ps14 and Tr6 activate the same signaling pathway and thus have no enhanced effect in combination. The enhanced protection in cucumber by the combi- nation is most likely due to activation of different signaling pathways by the two biocontrol agents.

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.

The plant growth-promoting rhizobacterium Pseudomonas fluorescens WCS374r produces several iron-regulated metabolites, including the fluorescent siderophore pseudobactin (Psb374), salicylic acid (SA), and pseudomonine (Psm), a siderophore that contains a SA moiety. After purification of Psb374 from culture supernatant of WCS374r, its structure was determined following isoelectrofocusing and tandem mass spectrometry, and found to be identical to the fluorescent siderophore produced by P. fluorescens ATCC 13525. To study the role of SA and Psm production in colonization of Arabidopsis thaliana roots and in induced systemic resistance (ISR) against Pseudomonas syringae pv. tomato (Pst) by strain WCS374r, mutants disrupted in the production of these metabolites were obtained by homologous recombination. These mutants were further subjected to transposon Tn5 mutagenesis to generate mutants also deficient in Psb374 production. The mutants behaved similar to the wild type in both their Arabidopsis rhizosphere-colonizing capacity and their ability to elicit ISR against Pst. We conclude that Psb374, SA, and Psm production by P. fluorescens WCS374r are not required for eliciting ISR in Arabidopsis.

Pseudomonas fluorescens strains that produce the polyketide antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) are among the most effective rhizobacteria that suppress root and crown rots, wilts, and damping-off diseases of a variety of crops, and they play a key role in the natural suppressiveness of some soils to certain soilborne pathogens. Root colonization by 2,4-DAPG-producing P. fluorescens strains Pf-5 (genotype A), Q2-87 (genotype B), Q8r1-96 (genotype D), and HT5-1 (genotype N) produced induced systemic resistance (ISR) in Arabidopsis thaliana accession Col-0 against bacterial speck caused by P. syringae pv. tomato. The ISR-eliciting activity of the four bacterial genotypes was similar, and all genotypes were equivalent in activity to the well-characterized strain P. fluorescens WCS417r. The 2,4-DAPG biosynthetic locus consists of the genes phlHGF and phlACBDE. phlD or phlBC mutants of Q2-87 (2,4-DAPG minus) were significantly reduced in ISR activity, and genetic complementation of the mutants restored ISR activity back to wild-type levels. A phlF regulatory mutant (overproducer of 2,4-DAPG) had ISR activity equivalent to the wild-type Q2-87. Introduction of DAPG into soil at concentrations of 10 to 250 μM 4 days before challenge inoculation induced resistance equivalent to or better than the bacteria. Strain Q2-87 induced resistance on transgenic NahG plants but not on npr1-1, jar1, and etr1 Arabidopsis mutants. These results indicate that the antibiotic 2,4-DAPG is a major determinant of ISR in 2,4-DAPG-producing P. fluorescens, that the genotype of the strain does not affect its ISR activity, and that the activity induced by these bacteria operates through the ethylene- and jasmonic acid-dependent signal transduction pathway.

Summary Dry bubble disease is a major problem in the commercial cultivation of the white button mushroom Agaricus bisporus and is caused by the ascomycete Lecanicillium fungicola. In the casing layer, germination of L. fungicola spores is inhibited by the microflora, a phenomenon known as fungistasis. Fungistasis is annulled when the casing is colonized by A. bisporus hyphae. We demonstrated that addition of A. bisporus-associated sugars, similarly annulled the casing fungistasis. However, casing fungistasis does not seem to be based on competition for resources as L. fungicola spores germinate regardless of nutrient availability. Pseudomonas bacteria are a dominant group of bacteria in the casing and have previously been implied to be essential for the development of fungistasis in soils. Antibiotics produced by model strain Pseudomonas fluorescens CHA0 inhibited L. fungicola spore germination. Addition of glucose desensitized spores of L. fungicola, which resulted in germination in the presence of antibiotics. We conclude that antibiotics produced by the microflora most likely cause fungistasis.

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.

Kwak, Y.-S., Bakker, P. A. H. M., Glandorf, D. C. M., Rice, J. T., Paulitz, T. C., and Weller, D. M. 2010. Isolation, characterization, and sensitivity to 2,4-diacetylphloroglucinol of isolates of Phialophora spp. from Washington wheat fields. Phytopathology 100:404-414. Dark pigmented fungi of the Gaeumannomyces–Phialophora complex were isolated from the roots of wheat grown in fields in eastern Washington State. These fungi were identified as Phialophora spp. on the basis of morphological and genetic characteristics. The isolates produced lobed hyphopodia on wheat coleoptiles, phialides, and hyaline phialospores. Sequence comparison of internal transcribed spacer regions indicated that the Phialophora isolates were clearly separated from other Gaeumannomyces spp. Primers AV1 and AV3 amplified 1.3-kb portions of an avenacinase-like gene in the Phialophora isolates. Phylogenetic trees of the avenacinase-like gene in the Phialophora spp. also clearly separated them from other Gaeumannomyces spp. The Phialophora isolates were moderately virulent on wheat and barley and produced confined black lesions on the roots of wild oat and two oat cultivars. Among isolates tested for their sensitivity to 2,4-diacetylphloroglucinol (2,4-DAPG), the 90% effective dose values were 11.9 to 48.2 µg ml–1. A representative Phialophora isolate reduced the severity of take-all on wheat caused by two different isolates of Gaeumannomyces graminis var. tritici. To our knowledge, this study provides the first report of an avenacinase-like gene in Phialophora spp. and demonstrated that the fungus is significantly less sensitive to 2,4-DAPG than G. graminis var. tritici.

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.

International audience; Peatlands are natural sources of atmospheric methane (CH4), an important greenhouse gas. It is established that peatland methane dynamics are controlled by both biotic and abiotic conditions, yet the interactive effect of these drivers is less studied and consequently poorly understood. Climate change affects the distribution of vascular plant functional types (PFTs) in peatlands. By removing specific PFTs, we assessed their effects on peat organic matter chemistry, microbial community composition and on potential methane production (PMP) and oxidation (PMO) in two microhabitats (lawns and hummocks). Whilst PFT removal only marginally altered the peat organic matter chemistry, we observed considerable changes in microbial community structure. This resulted in altered PMP and PMO. PMP was slightly lower when graminoids were removed, whilst PMO was highest in the absence of both vascular PFTs (graminoids and ericoids), but only in the hummocks. Path analyses demonstrate that different plant-soil interactions drive PMP and PMO in peatlands and that changes in biotic and abiotic factors can have auto-amplifying effects on current CH4 dynamics.Synthesis. Changing environmental conditions will, both directly and indirectly, affect peatland processes, causing unforeseen changes in CH4 dynamics. The resilience of peatland CH4 dynamics to environmental change therefore depends on the interaction between plant community composition and microbial communities.

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.

Ran, L. X., van Loon, L. C., and Bakker, P. A. H. M. 2005. No role for bacterially produced salicylic acid in rhizobacterial induction of systemic resistance in Arabidopsis. Phytopathology 95:1349-1355. The role of bacterially produced salicylic acid (SA) in the induction of systemic resistance in plants by rhizobacteria is far from clear. The strong SA producer Pseudomonas fluorescens WCS374r induces resistance in radish but not in Arabidopsis thaliana, whereas application of SA leads to induction of resistance in both plant species. In this study, we compared P. fluorescens WCS374r with three other SA-producing fluorescent Pseudomonas strains, P. fluorescens WCS417r and CHA0r, and P. aeruginosa 7NSK2 for their abilities to produce SA under different growth conditions and to induce systemic resistance in A. thaliana against bacterial speck, caused by P. syringae pv. tomato. All strains produced SA in vitro, varying from 5 fg cell –1 for WCS417r to >25 fg cell –1 for WCS374r. Addition of 200 µM FeCl3 to standard succinate medium abolished SA production in all strains. Whereas the incubation temperature did not affect SA production by WCS417r and 7NSK2, strains WCS374r and CHA0r produced more SA when grown at 33 instead of 28°C. WCS417r, CHA0r, and 7NSK2 induced systemic resistance apparently associated with their ability to produce SA, but WCS374r did not. Conversely, a mutant of 7NSK2 unable to produce SA still triggered induced systemic resistance (ISR). The possible involvement of SA in the induction of resistance was evaluated using SA-nonaccumulating transgenic NahG plants. Strains WCS417r, CHA0r, and 7NSK2 induced resistance in NahG Arabidopsis. Also, WCS374r, when grown at 33 or 36°C, triggered ISR in these plants, but not in ethylene-insensitive ein2 or in non-plant pathogenesis-related protein-expressing npr1 mutant plants, irrespective of the growth temperature of the bacteria. These results demonstrate that, whereas WCS374r can be manipulated to trigger ISR in Arabidopsis, SA is not the primary determinant for the induction of systemic resistance against bacterial speck disease by this bacterium. Also, for the other SAproducing strains used in this study, bacterial determinants other than SA must be responsible for inducing resistance.

The ability of selected strains of fluorescent Pseudomonas spp. to cause induced systemic resistance (ISR) in Eucalyptus urophylla against bacterial wilt caused by Ralstonia solanacearum was investigated. Four of the five strains used can produce salicylic acid (SA) in vitro and, therefore, chemical SA, that is known to induce resistance in many plant species, was used as a reference treatment. Whereas a soil drench with SA did induce systemic resistance in E. urophylla, infiltration of SA into leaves did not. None of the fluorescent Pseudomonas spp. strains caused ISR against bacterial wilt when applied to the soil, but two strains, P. putida WCS358r and P. fluorescens WCS374r triggered ISR when infiltrated into two lower leaves 3–7 days before challenge inoculation. A mutant of strain WCS358r defective in the biosynthesis of the fluorescent siderophore pseudobactin, did not cause ISR, while the purified siderophore of WCS358r did, suggesting that pseudobactin358 is the ISR determinant of WCS358. A siderophore-minus mutant of WCS374r induced the same level of disease resistance as its parental strain, but the purified siderophore induced resistance as well, indicating that both the siderophore and another, unknown, inducing determinant(s) of WCS374r can trigger ISR in Eucalyptus. A possible role of WCS374r-produced SA remains uncertain. Transformation of a siderophore-minus mutant of WCS358 with the SA biosynthetic gene cluster from WCS374 did not enable this transformant to cause ISR in E. urophylla.

Transgenic tobacco plants (Tetr) expressing the mutant etr1-1 gene from Arabidopsis thaliana are insensitive to ethylene and develop symptoms of wilting and stem rot when grown in nonautoclaved soil. Several isolates of Fusarium, Thielaviopsis, and Pythium were recovered from stems of diseased Tetr plants. Inoculation with each of these isolates of 6-week-old plants growing in autoclaved soil caused disease in Tetr plants but not in nontransformed plants. Also, when 2-week-old seedlings were used, nontransformed tobacco appeared nonsusceptible to the Fusarium isolates, whereas Tetr seedlings did develop disease. Tetr seedlings were not susceptible to several nonhost Fusarium isolates. In contrast to results with Fusarium isolates, inoculation of 2-week-old seedlings with a Thielaviopsis isolate resulted in equal symptom development of nontransformed and Tetr tobacco. In order to explore the potential range of pathogens to which Tetr tobacco plants display enhanced susceptibility, the pathogenicity of several root and leaf pathogens was tested. Tetr plants were more susceptible to the necrotrophic fungi Botrytis cinerea and Cercospora nicotianae and the bacterium Erwinia carotovora, but only marginally more to the bacterium Ralstonia solanacearum. In contrast, the biotrophic fungus Oidium neolycopersici, the oomycete Peronospora tabacina, and Tobacco mosaic virus caused similar or less severe symptoms on Tetr plants than on nontransformed plants. Total peroxidase activity of Tetr plants was lower than that of nontransformed plants, suggesting a role for peroxidases in resistance against necrotrophic microorganisms. A comparable range of pathogens was examined on Arabidopsis and its ethylene-insensitive mutants etr1-1 and ein2-1. With the exception of one Fusarium isolate, ethylene insensitivity increased susceptibility of Arabidopsis plants to a similar spectrum of necrotizing pathogens as in tobacco. Thus, both ethylene-insensitive tobacco and Arabidopsis plants appear to be impaired in their resistance to necrotrophic pathogens.

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.

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 syringae pv. tomato DC3000. P. putida extracellular haem-peroxidase (PP2561) was found to be important for competitive colonization and essential for the induction 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 colonized 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.

Dry bubble disease is a major problem in the commercial cultivation of the white button mushroom Agaricus bisporus and is caused by the ascomycete Lecanicillium fungicola. In the casing layer, germination of L. fungicola spores is inhibited by the microflora, a phenomenon known as fungistasis. Fungistasis is annulled when the casing is colonized by A. bisporus hyphae. We demonstrated that addition of A. bisporus-associated sugars, similarly annulled the casing fungistasis. However, casing fungistasis does not seem to be based on competition for resources as L. fungicola spores germinate regardless of nutrient availability. Pseudomonas bacteria are a dominant group of bacteria in the casing and have previously been implied to be essential for the development of fungistasis in soils. Antibiotics produced by model strain Pseudomonas fluorescens CHA0 inhibited L. fungicola spore germination. Addition of glucose desensitized spores of L. fungicola, which resulted in germination in the presence of antibiotics. We conclude that antibiotics produced by the microflora most likely cause fungistasis.

Jasmonates, together with other plant hormones, are important orchestrators of the plant immune system. The different hormone-controlled signaling pathways cross-communicate in an antagonistic or a synergistic manner, providing the plant with a powerful capacity to finely regulate its immune response. Jasmonic acid (JA) signaling is required for plant resistance to harmful organisms, such as necrotrophic pathogens and herbivorous insects. Furthermore, JA signaling is essential in interactions of plants with beneficial microbes that induce systemic resistance to pathogens and insects. The role of JA signaling components in plant immunity can be studied by performing bioassays with different interacting organisms. Determination of the level of resistance and the induction of defense responses in plants with altered JA components, through mutation or ectopic expression, will unveil novel mechanisms of JA signaling. We provide detailed protocols of bioassays with the model plant Arabidopsis thaliana challenged with the pathogens Botrytis cinerea and Pseudomonas syringae, the insect herbivore Pieris rapae, and the beneficial microbe Pseudomonas fluorescens. In addition, we describe pharmacological assays to study the modulation of JA-regulated responses by exogenous application of combinations of hormones, because a simultaneous rise in hormone levels occurs during interaction of plants with other organisms.

Jasmonates, together with other plant hormones, are important orchestrators of the plant immune system. The different hormone-controlled signaling pathways cross-communicate in an antagonistic or a synergistic manner, providing the plant with a powerful capacity to finely regulate its immune response. Jasmonic acid (JA) signaling is required for plant resistance to harmful organisms, such as necrotrophic pathogens and herbivorous insects. Furthermore, JA signaling is essential in interactions of plants with beneficial microbes that induce systemic resistance to pathogens and insects. The role of JA signaling components in plant immunity can be studied by performing bioassays with different interacting organisms. Determination of the level of resistance and the induction of defense responses in plants with altered JA components, through mutation or ectopic expression, will unveil novel mechanisms of JA signaling. We provide detailed protocols of bioassays with the model plant Arabidopsis thaliana challenged with the pathogens Botrytis cinerea and Pseudomonas syringae, the insect herbivore Pieris rapae, and the beneficial microbe Pseudomonas fluorescens. In addition, we describe pharmacological assays to study the modulation of JA-regulated responses by exogenous application of combinations of hormones, because a simultaneous rise in hormone levels occurs during interaction of plants with other organisms.

Dry bubble disease caused by Lecanicillium fungicola is a persistent problem in the cultivation of the white button mushroom (Agaricus bisporus). Because control is hampered by chemicals becoming less effective, new ways to control dry bubble disease are urgently required. 1- Octen-3-ol is a volatile that is produced by A. bisporus and many other fungi. In A. bisporus, it has been implicated in self-inhibition of fruiting body formation while it was shown to inhibit spore germination in ascomycetes. Here, we show that 1-octen-3-ol inhibits germination of L. fungicola and that enhanced levels of 1-octen-3-ol can effectively control the malady. In addition, application of 1- octen-3-ol stimulates growth of bacterial populations in the casing and of Pseudomonas spp. specifically. Pseudomonas spp. and other bacteria have been demonstrated to play part in both the onset of mushroom formation in A. bisporus, as well as the inhibition of L. fungicola spore germination. A potential role of 1-octen-3-ol in the ecology of L. fungicola is discussed.

In an earlier study, treatment of radish seed with the bacteriumPseudomonas fluorescens WCS374 suppressed fusarium wilt of radish (Fusarium oxysporum f. sp.raphani) in a commercial greenhouse [Leemanet al., 1991b, 1995a]. In this greenhouse, the areas with fusarium wilt were localized or expanded very slowly, possibly due to disease suppressiveness of the soil. To study this phenomenon, fungi were isolated from radish roots collected from the greenhouse soil. Roots grown from seed treated with WCS374 were more abundantly colonized by fungi than were roots from nonbacterized plants. Among these were several species known for their antagonistic potential. Three of these fungi,Acremonium rutilum, Fusarium oxysporum andVerticillium lecanii, were evaluated further and found to suppress fusarium wilt of radish in a pot bioassay. In an induced resistance bioassay on rockwool,F. oxysporum andV. lecanii suppressed the disease by the apparent induction of systemic disease resistance. In pot bioassays with thePseudomonas spp. strains, the pseudobactin-minus mutant 358PSB− did not suppress fusarium wilt, whereas its wild type strain (WCS358) suppressed disease presumably by siderophore-mediated competition for iron. The wild type strains of WCS374 and WCS417, as well as their pseudobactin-minus mutants 374PSB− and 417PSB− suppressed fusarium wilt. The latter is best explained by the fact that these strains are able to induce systemic resistance in radish, which operates as an additional mode of action. Co-inoculation in pot bioassays, ofA. rutilum, F. oxysporum orV. lecanii with thePseudomonas spp. WCS358, WCS374 or WCS417, or their pseudobactin-minus mutants, significantly suppressed disease (except forA. rutilum/417PSB− and all combinations with 358PSB−), compared with the control treatment, if the microorganisms were applied in inoculum densities which were ineffective in suppressing disease as separate inocula. If one or both of the microorganism(s) of each combination were applied as separate inocula in a density which suppressed disease, no additional suppression of disease was observed by the combination. The advantage of the co-inoculation is that combined populations significantly suppressed disease even when their individual population density was too low to do so. This may provide more consistent biological control. The co-inoculation effect obtained in the pot bioassays suggests that co-operation ofP. fluorescens WCS374 and indigenous antagonists could have been involved in the suppression of fusarium wilt of radish in the commercial greenhouse trials.

Summary-.Dispersal of wild-type and genetically-modified strains of plant growth-promoting fluorescent Pseudomonas spp to aerial parts of radish plants was investigated using different inoculation strategies. P. putida strain WCS358rr colonized the surface of cotyledons of radish plants when seeds were coated with this strain or when non-coated seeds were sown in soil treated with WCS358rr. The cotyledons were not colonized by WCS358rr when suspensions of this strain were inoculated onto roots of radish seedlings that had already grown for 4 d in soil. Strain WCS358rr not only colonized the surface, but also the interior of cotyledons. For all types of inoculation strategies tested, strain WCS358rr colonized the root surface but did not penetrate the interior of the root. These results indicate that WCS358rr was not transported up, or downwards through the vascular system. When coated on seed, dispersal to cotyledons of radish was also demonstrated for P.fluorescens WCS374 and its genetically-modified derivative WCS374pMR. Plasmid pMR was maintained in WCS374pMR throughout the experiment. No differences were observed between modified strain WCS374pMR and wild-type strain WCS374 with regard to dispersal to and colonization of cotyledons and roots of radish. Strains WCS358rr, WCS374 and WCS374pMR survived on cotyledons for a period of 21-31 d. Dispersal of the applied bacterial strains to the first true leaves of radish could not be demonstrated. The results indicate that cotyledons of radish become contaminated with strains of Pseudomonas spp during germination of treated seeds or during seedling emergence from treated soil. The implications of dispersal of wild-type and genetically-modified pseudomonads to aerial parts of the plant are discussed.

Pseudomonas fluorescens-mediated induction of systemic resistance in radish against fusarium wilt (Fusarium oxysporum f. sp.raphani) was studied in a newly developed bioassay using a rockwool system. In this bioassay the pathogen and bacterium were confirmed to be confined to spatially separate locations on the plant root, throughout the experiment. Pathogen inoculum obtained by mixing peat with microconidia and subsequent incubation for four days at 22 °C, yielded a better percentage of diseased plants than a microconidial suspension drench, an injection of a microconidial suspension into the hypocotyl, or a talcum inoculum.Pseudomonas fluorescens strain WCS374 applied in talcum or peat, but not as a suspension drench, induced systemic resistance. A minimal initial bacterial inoculum density of ≥105 CFU WCS374 root−1 was required to significantly reduce the percentage diseased plants. At least one day was necessary between bacterization of strain WCS374 in talcum on the root tips and inoculation of the pathogen in peat on the root base, for an optimal induction of systemic resistance. Strain WCS374 induced systemic resistance in six radish cultivars differing in their susceptibility toF. oxysporum f. sp.raphani. Significant suppression of disease by bacterial treatments was generally observed when disease incidence in the control treatment, depending on pathogen inoculum density, ranged between approximately 40 to 80%. Strains WCS374 and WCS417 ofPseudomonas fluorescens induced systemic resistance against fusarium wilt, whereasP. putida WCS358 did not. This suggests that the induction of systemic resistance byPseudomonas spp. is dependent on strain-specific traits.

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.

The plant growth-promoting rhizobacterium Pseudomonas fluorescens WCS374r produces several iron-regulated metabolites, including the fluorescent siderophore pseudobactin (Psb374), salicylic acid (SA), and pseudomonine (Psm), a siderophore that contains a SA moiety. After purification of Psb374 from culture supernatant of WCS374r, its structure was determined following isoelectrofocusing and tandem mass spectrometry, and found to be identical to the fluorescent siderophore produced by P. fluorescens ATCC 13525. To study the role of SA and Psm production in colonization of Arabidopsis thaliana roots and in induced systemic resistance (ISR) against Pseudomonas syringae pv. tomato (Pst) by strain WCS374r, mutants disrupted in the production of these metabolites were obtained by homologous recombination. These mutants were further subjected to transposon Tn5 mutagenesis to generate mutants also deficient in Psb374 production. The mutants behaved similar to the wild type in both their Arabidopsis rhizosphere-colonizing capacity and their ability to elicit ISR against Pst. We conclude that Psb374, SA, and Psm production by P. fluorescens WCS374r are not required for eliciting ISR in Arabidopsis.

The mechanisms of suppression of fusarium wilt of carnation by two fluorescentPseudomonas strains were studied.