Root-colonizing Pseudomonas Research Articles

Discovery of an Antibiotic-Related Small Protein of Biocontrol Strain Pseudomonas sp. Os17 by a Genome-Mining Strategy.

Many root-colonizing Pseudomonas spp. exhibiting biocontrol activities produce a wide range of secondary metabolites that exert antibiotic effects against other microbes, nematodes, and insects in the rhizosphere. The expression of these secondary metabolites depends on the Gac/Rsm signal transduction pathway. Based on the findings of a previous genomic study on newly isolated biocontrol pseudomonad strains, we herein investigated the novel gene cluster OS3, which consists of four genes (Os1348–Os1351) that are located upstream of putative efflux transporter genes (Os1352–Os1355). Os1348 was predicted to encode an 85-aa small precursor protein, the expression of which was under the control of GacA, and an X-ray structural analysis suggested that the Os1348 protein formed a dimer. The mutational loss of the Os1348 gene decreased the antibiotic activity of Pseudomonas sp. Os17 without changing its growth rate. The Os1349–1351 genes were predicted to be involved in post-translational modifications. Intracellular levels of the Os1348 protein in the deficient mutant of each gene differed from that in wild-type cells. These results suggest that Os1348 is involved in antibiotic activity and that the structure or expression of this protein is under the control of downstream gene products.

Persistence of root-colonizing Pseudomonas protegens in herbivorous insects throughout different developmental stages and dispersal to new host plants.

The discovery of insecticidal activity in root-colonizing pseudomonads, best-known for their plant-beneficial effects, raised fundamental questions about the ecological relevance of insects as alternative hosts for these bacteria. Since soil bacteria are limited in their inherent abilities of dispersal, insects as vectors might be welcome vehicles to overcome large distances. Here, we report on the transmission of the root-colonizing, plant-beneficial and insecticidal bacterium Pseudomonas protegens CHA0 from root to root by the cabbage root fly, Delia radicum. Following ingestion by root-feeding D. radicum larvae, CHA0 persisted inside the insect until the pupal and adult stages. The emerging flies were then able to transmit CHA0 to a new plant host initiating bacterial colonization of the roots. CHA0 did not reduce root damages caused by D. radicum and had only small effects on Delia development suggesting a rather commensal than pathogenic relationship. Interestingly, when the bacterium was fed to two highly susceptible lepidopteran species, most of the insects died, but CHA0 could persist throughout different life stages in surviving individuals. In summary, this study investigated for the first time the interaction of P. protegens CHA0 and related strains with an insect present in their rhizosphere habitat. Our results suggest that plant-colonizing pseudomonads have different strategies for interaction with insects. They either cause lethal infections and use insects as food source or they live inside insect hosts without causing obvious damages and might use insects as vectors for dispersal, which implies a greater ecological versatility of these bacteria than previously thought.

Plant-mediated restriction of Salmonella enterica on tomato and spinach leaves colonized with Pseudomonas plant growth-promoting rhizobacteria

Reducing Salmonella enterica association with plants during crop production could reduce risks of fresh produce-borne salmonellosis. Plant growth-promoting rhizobacteria (PGPR) colonizing plant roots are capable of promoting plant growth and boosting resistance to disease, but the effects of PGPR on human pathogen-plant associations are not known. Two root-colonizing Pseudomonas strains S2 and S4 were investigated in spinach, lettuce and tomato for their plant growth-promoting properties and their influence on leaf populations of S. enterica serovar Newport. Plant roots were inoculated with Pseudomonas in the seedling stage. At four (tomato) and six (spinach and lettuce) weeks post-germination, plant growth promotion was assessed by shoot dry weight (SDW) and leaf chlorophyll content measurements. Leaf populations of S. Newport were measured after 24h of leaf inoculation with this pathogen by direct plate counts on Tryptic Soy Agar. Root inoculation of spinach cv. ‘Tyee’, with Pseudomonas strain S2 or S4 resulted in a 69% and 63% increase in SDW compared to non-inoculated controls (p<0.005 and p<0.01, respectively). Similarly, Romaine lettuce cv. ‘Parris Island Cos’ responded positively to S2 and S4 inoculation (53% and 48% SDW increase, respectively; p<0.05), and an increase in leaf chlorophyll content (p<0.001), compared to controls. Tomato cv. ‘Nyagous’ yielded significantly greater SDW (74%, p<0.01 and 54%, p<0.05 for S2 and S4, respectively), and also higher leaf chlorophyll content (19% and 29%, p<0.001, respectively) relative to controls. Leaf chlorophyll content only increased in S4-inoculated tomato cv. ‘Moneymaker’ plants (27%, p<0.001), although both S2 and S4 promoted plant growth by over 40% compared to controls (p<0.01 and p<0.05, respectively). No significant growth promotion was detected in tomato cv. ‘BHN602’, but S2-inoculated plants had elevated leaf chlorophyll content (13%, p<0.01). Root inoculation with Pseudomonas S4 restricted S. Newport populations inoculated on leaves of spinach (p<0.001) and all three tomato cultivars (p<0.05), compared to controls, 24h post Salmonella inoculation. Impairment of S. Newport leaf populations was also observed on spinach when plant roots were inoculated with S2 (p<0.01). With an initial leaf inoculum of approximately 6.0logCFU of S. Newport/plant, the significantly greater reduction of S. Newport populations on Pseudomonas-treated plants than those on non-inoculated control plants after 24h was modest with differences of one log or less. By contrast, the survival of S. Newport on the leaves of Romaine lettuce was not influenced by Pseudomonas root colonization. These findings provide evidence that root inoculation of certain specialty crops with beneficial Pseudomonas strains exhibiting PGPR properties may not only promote plant growth, but also reduce the fitness of epiphytic S. enterica in the phyllosphere. Plant-mediated effects induced by PGPR may be an effective strategy to minimize contamination of crops with S. enterica during cultivation.

Microbial cooperation in the rhizosphere improves liquorice growth under salt stress

ABSTRACTLiquorice (Glycyrrhiza uralensis Fisch.) is one of the most widely used plants in food production, and it can also be used as an herbal medicine or for reclamation of salt-affected soils. Under salt stress, inhibition of plant growth, nutrient acquisition and symbiotic interactions between the medicinal legume liquorice and rhizobia have been observed. We recently evaluated the interactions between rhizobia and root-colonizing Pseudomonas in liquorice grown in potting soil and observed increased plant biomass, nodule numbers and nitrogen content after combined inoculation compared to plants inoculated with Mesorhizobium alone. Several beneficial effects of microbes on plants have been reported; studies examining the interactions between symbiotic bacteria and root-colonizing Pseudomonas strains under natural saline soil conditions are important, especially in areas where a hindrance of nutrients and niches in the rhizosphere are high. Here, we summarize our recent observations regarding the combined application of rhizobia and Pseudomonas on the growth and nutrient uptake of liquorice as well as the salt stress tolerance mechanisms of liquorice by a mutualistic interaction with microbes. Our observations indicate that microbes living in the rhizosphere of liquorice can form a mutualistic association and coordinate their involvement in plant adaptations to stress tolerance. These results support the development of combined inoculants for improving plant growth and the symbiotic performance of legumes under hostile conditions.

Alleviation of salt stress of symbiotic Galega officinalis L. (goat's rue) by co-inoculation of Rhizobium with root-colonizing Pseudomonas

Salt stress is an increasing problem in agricultural soils in many parts of the world, and salt tolerant cropping systems are in great demand. We investigated the effect of co-inoculation of Galega officinalis with Rhizobium galegae and two plant growth promoting Pseudomonas species on plant growth, nodulation, and N content under salt stress. The effect of inoculation with R. galegae sv. officinalis HAMBI 1141 alone and in combination with the root-colonizing Pseudomonas extremorientalis TSAU20 or Pseudomonas trivialis 3Re27 on the growth of G. officinalis exposed to salt stress (50 and 75 mM NaCl) was studied under gnotobiotic and greenhouse conditions. The growth of goat’s rue was reduced at 50 and 75 mM NaCl both in the gnotobiotic sand system and in low-fertilized potting soil in the greenhouse. Co-inoculation of unstressed and salt-stressed goat’s rue with R. galegae HAMBI 1141 and either P. extremorientalis TSAU20 or P. trivialis 3Re27 significantly improved root and shoot growth and increased nodulation of plant roots in both growth systems compared with plants inoculated with R. galegae alone. The nitrogen content of co-inoculated plant roots was significantly increased at 75 mM NaCl in potting soil. Co-inoculation of G. officinalis with either of the two plant growth promoting (PGPR) Pseudomonas strains also improved root tip-colonization by R. galegae cells. The co-inoculation of goat’s rue with Rhizobium and PGPR Pseudomonas strains alleviated salt stress of plants grown in NaCl-amended gnotobiotic sand systems and in potting soil in the greenhouse.

Promise for plant pest control: root-associated pseudomonads with insecticidal activities

Insects are an important and probably the most challenging pest to control in agriculture, in particular when they feed on belowground parts of plants. The application of synthetic pesticides is problematic owing to side effects on the environment, concerns for public health and the rapid development of resistance. Entomopathogenic bacteria, notably Bacillus thuringiensis and Photorhabdus/Xenorhabdus species, are promising alternatives to chemical insecticides, for they are able to efficiently kill insects and are considered to be environmentally sound and harmless to mammals. However, they have the handicap of showing limited environmental persistence or of depending on a nematode vector for insect infection. Intriguingly, certain strains of plant root-colonizing Pseudomonas bacteria display insect pathogenicity and thus could be formulated to extend the present range of bioinsecticides for protection of plants against root-feeding insects. These entomopathogenic pseudomonads belong to a group of plant-beneficial rhizobacteria that have the remarkable ability to suppress soil-borne plant pathogens, promote plant growth, and induce systemic plant defenses. Here we review for the first time the current knowledge about the occurrence and the molecular basis of insecticidal activity in pseudomonads with an emphasis on plant-beneficial and prominent pathogenic species. We discuss how this fascinating Pseudomonas trait may be exploited for novel root-based approaches to insect control in an integrated pest management framework.

Metabolic and transcriptomic changes induced in Arabidopsis by the rhizobacterium Pseudomonas fluorescens SS101.

Systemic resistance induced in plants by nonpathogenic rhizobacteria is typically effective against multiple pathogens. Here, we show that root-colonizing Pseudomonas fluorescens strain SS101 (Pf.SS101) enhanced resistance in Arabidopsis (Arabidopsis thaliana) against several bacterial pathogens, including Pseudomonas syringae pv tomato (Pst) and the insect pest Spodoptera exigua. Transcriptomic analysis and bioassays with specific Arabidopsis mutants revealed that, unlike many other rhizobacteria, the Pf.SS101-induced resistance response to Pst is dependent on salicylic acid signaling and not on jasmonic acid and ethylene signaling. Genome-wide transcriptomic and untargeted metabolomic analyses showed that in roots and leaves of Arabidopsis plants treated with Pf.SS101, approximately 1,910 genes and 50 metabolites were differentially regulated relative to untreated plants. Integration of both sets of "omics" data pointed to a prominent role of camalexin and glucosinolates in the Pf.SS101-induced resistance response. Subsequent bioassays with seven Arabidopsis mutants (myb51, cyp79B2cyp79B3, cyp81F2, pen2, cyp71A12, cyp71A13, and myb28myb29) disrupted in the biosynthesis pathways for these plant secondary metabolites showed that camalexin and glucosinolates are indeed required for the induction of Pst resistance by Pf.SS101. Also for the insect S. exigua, the indolic glucosinolates appeared to play a role in the Pf.SS101-induced resistance response. This study provides, to our knowledge for the first time, insight into the substantial biochemical and temporal transcriptional changes in Arabidopsis associated with the salicylic acid-dependent resistance response induced by specific rhizobacteria.

Induction of systemic resistance in banana (Musa spp.) against Banana bunchy top virus (BBTV) by combining chitin with root-colonizing Pseudomonas fluorescens strain CHA0

Pseudomonas fluorescens strains CHA0 and Pf1 were investigated for their biocontrol efficacy against Banana bunchy top virus (BBTV) in banana (Musa spp.) alone and in combination with chitin under glasshouse and field conditions. Bioformulation of P. fluorescens strain CHA0 with chitin was effective in reducing the banana bunchy top disease (BBTD) incidence in banana under glasshouse and field conditions. In addition to disease control, the bioformulation increased the economic yield significantly compared to the untreated control. Increased accumulation of oxidative enzymes, peroxidase (PO), polyphenol oxidase (PPO), phenylalanine ammonia lyase (PAL), pathogenesis-related (PR) proteins, chitinase, β-1,3-glucanase and phenolics were observed in CHA0 bioformulation amended with chitin-treated plants challenged with BBTV under glasshouse conditions. Indirect ELISA indicated the reduction in viral antigen concentration in P. fluorescens strain CHA0 with chitin-treated banana plants corresponding to reduced disease ratings. The present study revealed that induction of defence enzymes by P. fluorescens with chitin amendment reduced the BBTD incidence and increased bunch yield in banana.

Plant responses to plant growth-promoting rhizobacteria

Non-pathogenic soilborne microorganisms can promote plant growth, as well as suppress diseases. Plant growth promotion is taken to result from improved nutrient acquisition or hormonal stimulation. Disease suppression can occur through microbial antagonism or induction of resistance in the plant. Several rhizobacterial strains have been shown to act as plant growth-promoting bacteria through both stimulation of growth and induced systemic resistance (ISR), but it is not clear in how far both mechanisms are connected. Induced resistance is manifested as a reduction of the number of diseased plants or in disease severity upon subsequent infection by a pathogen. Such reduced disease susceptibility can be local or systemic, result from developmental or environmental factors and depend on multiple mechanisms. The spectrum of diseases to which PGPR-elicited ISR confers enhanced resistance overlaps partly with that of pathogen-induced systemic acquired resistance (SAR). Both ISR and SAR represent a state of enhanced basal resistance of the plant that depends on the signalling compounds jasmonic acid and salicylic acid, respectively, and pathogens are differentially sensitive to the resistances activated by each of these signalling pathways. Root-colonizing Pseudomonas bacteria have been shown to alter plant gene expression in roots and leaves to different extents, indicative of recognition of one or more bacterial determinants by specific plant receptors. Conversely, plants can alter root exudation and secrete compounds that interfere with quorum sensing (QS) regulation in the bacteria. Such two-way signalling resembles the interaction of root-nodulating Rhizobia with legumes and between mycorrhizal fungi and roots of the majority of plant species. Although ISR-eliciting rhizobacteria can induce typical early defence-related responses in cell suspensions, in plants they do not necessarily activate defence-related gene expression. Instead, they appear to act through priming of effective resistance mechanisms, as reflected by earlier and stronger defence reactions once infection occurs.

Use of green fluorescent protein-based reporters to monitor balanced production of antifungal compounds in the biocontrol agentPseudomonas fluorescensCHA0

To develop reporter constructs based on stable and unstable variants of the green fluorescent protein (GFP) for monitoring balanced production of antifungal compounds that are crucial for the capacity of the root-colonizing Pseudomonas fluorescens strain CHA0 to control plant diseases caused by soil-borne pathogenic fungi. Pseudomonas fluorescens CHA0 produces the three antifungal metabolites 2,4-diacetylphloroglucinol (DAPG), pyoluteorin (PLT) and pyrrolnitrin (PRN). The gfp[mut3] and gfp[AAV] reporter genes were fused to the promoter regions of the DAPG, PLT and PRN biosynthetic genes. The reporter fusions were then used to follow the kinetics of expression of the three antifungal metabolites in a microplate assay. DAPG and PLT were found to display an inverse relationship in which each metabolite activates its own biosynthesis while repressing the synthesis of the other metabolite. PRN appears not to be involved in this balance. However, the microbial and plant phenolic metabolite salicylate was found to interfere with the expression of both DAPG and PLT. The results obtained provide evidence that P. fluorescens CHA0 may keep the antifungal compounds DAPG and PLT at a fine-tuned balance that can be affected by certain microbial and plant phenolics. To our knowledge, the present study is the first to use stable and unstable GFP variants to study antibiotic gene expression in a biocontrol pseudomonad. The developed reporter fusions will be a highly valuable tool to study in situ expression of this bacterial biocontrol trait on plant roots, i.e. at the site of pathogen suppression.

The Arabidopsis ISR1 Locus is Required for Rhizobacteria‐Mediated Induced Systemic Resistance Against Different Pathogens

Abstract: In Arabidopsis thaliana, non‐pathogenic, root‐colonizing Pseudomonas fluorescens WCS417r bacteria trigger an induced systemic resistance (ISR) that is phenotypically similar to pathogen‐induced systemic acquired resistance (SAR). In contrast to SAR, WCS417r‐mediated ISR is controlled by a salicylic acid (SA)‐independent signalling pathway that requires an intact response to the plant hormones jasmonic acid (JA) and ethylene (ET). Arabidopsis accessions RLD1 and Ws‐0 fail to express ISR against Pseudomonas syringae pv. tomato and show enhanced disease susceptibility to this pathogen. Genetic analysis of progeny from crosses between WCS417r‐responsive and non‐responsive accessions demonstrated that ISR inducibility and basal resistance against P. syringae pv. tomato are controlled by a single dominant locus (ISR1) on chromosome III (Ton et al., 1999[294]). Here, we investigated the role of the ISR1 locus in ISR, SAR and basal resistance against three additional pathogens: Xanthomonas campestris pv. armoraciae, Peronospora parasitica and turnip crinkle virus (TCV), using accessions Col‐0 (ISR1), RLD1 (isr1) and Ws‐0 (isr1) as host plants.

Combining fluorescent Pseudomonas spp. strains to enhance suppression of fusarium wilt of radish

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 Pseudomonas spp. 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.

A novel signaling pathway controlling induced systemic resistance in Arabidopsis.

Plants have the ability to acquire an enhanced level of resistance to pathogen attack after being exposed to specific biotic stimuli. In Arabidopsis, nonpathogenic, root-colonizing Pseudomonas fluorescens bacteria trigger an induced systemic resistance (ISR) response against infection by the bacterial leaf pathogen P. syringae pv tomato. In contrast to classic, pathogen-induced systemic acquired resistance (SAR), this rhizobacteria-mediated ISR response is independent of salicylic acid accumulation and pathogenesis-related gene activation. Using the jasmonate response mutant jar1, the ethylene response mutant etr1, and the SAR regulatory mutant npr1, we demonstrate that signal transduction leading to P. fluorescens WCS417r-mediated ISR requires responsiveness to jasmonate and ethylene and is dependent on NPR1. Similar to P. fluorescens WCS417r, methyl jasmonate and the ethylene precursor 1-aminocyclopropane-1-carboxylate were effective in inducing resistance against P. s. tomato in salicylic acid-nonaccumulating NahG plants. Moreover, methyl jasmonate-induced protection was blocked in jar1, etr1, and npr1 plants, whereas 1-aminocyclopropane-1-carboxylate-induced protection was affected in etr1 and npr1 plants but not in jar1 plants. Hence, we postulate that rhizobacteria-mediated ISR follows a novel signaling pathway in which components from the jasmonate and ethylene response are engaged successively to trigger a defense reaction that, like SAR, is regulated by NPR1. We provide evidence that the processes downstream of NPR1 in the ISR pathway are divergent from those in the SAR pathway, indicating that NPR1 differentially regulates defense responses, depending on the signals that are elicited during induction of resistance.

Variable specificity of Tn7::lacZY insertion into the chromosome of root‐colonizing Pseudomonas putida strains

Molecular EcologyVolume 6, Issue 1 p. 85-87 Variable specificity of Tn7::lacZY insertion into the chromosome of root-colonizing Pseudomonas putida strains T. E. Staley, T. E. Staley USDA-ARS-NAA, Beaver, WV 25813-0400, USASearch for more papers by this authorE. G. Lawrence, E. G. Lawrence USDA-ARS-NAA, Beaver, WV 25813-0400, USASearch for more papers by this authorD. J. Drahos, D. J. Drahos Sybron Chemicals, Inc., 111 Kesler Mill Road, PO Box 808, Salem, VA 24153, USASearch for more papers by this author T. E. Staley, T. E. Staley USDA-ARS-NAA, Beaver, WV 25813-0400, USASearch for more papers by this authorE. G. Lawrence, E. G. Lawrence USDA-ARS-NAA, Beaver, WV 25813-0400, USASearch for more papers by this authorD. J. Drahos, D. J. Drahos Sybron Chemicals, Inc., 111 Kesler Mill Road, PO Box 808, Salem, VA 24153, USASearch for more papers by this author First published: 31 October 2003 https://doi.org/10.1046/j.1365-294X.1997.00157.xCitations: 8 T. E. Staley, Tel.:+1-304-256-2821: Fax: +1-304-256-2921; [email protected] Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article.Citing Literature Volume6, Issue1January 1997Pages 85-87 RelatedInformation

Amino Acid Synthesis Is Necessary for Tomato Root Colonization by Pseudomonas fluorescens Strain WCS365

In this work the bio-availability of amino acids for the root-colonizing Pseudomonas fluorescens strain WCS365 in the tomato rhizosphere was studied. The amino acid composition of axenically collected tomato root exudate was determined. The results show that aspartic acid, glutamic acid, isoleucine, leucine, and lysine are the major amino acid components. The concentrations of individual amino acids in the rhizosphere of gnotobiotically grown tomato plants were estimated and considered to be too low to support growth of rhizosphere micro-organisms to numbers usually found in the tomato rhizosphere. To test this experimentally, mutants of P. fluorescens WCS365 auxotrophic for the amino acids leucine, arginine, histidine, isoleucine plus valine, and tryptophan were isolated after mutagenesis with Tn5lacZ. Root tip colonization of these mutants was measured after inoculation of germinated tomato seeds and subsequent growth in a gnotobiotic quartz sand system (M. Simons, A. J. van der Bij, I. Brand, L. A. de Weger, C. A. Wijffelman, and B. J. J. Lugtenberg. 1996. Gnotobiotic system for studying rhizo-sphere colonization by plant growth-promoting Pseudomonas bacteria. Mol. Plant-Microbe Interact. 9:600–607). In contrast to the wild-type strain, none of the five amino acid auxotrophs tested was able to colonize the tomato root tip, neither alone nor after co-inoculation with the wild-type strain. However, addition of the appropriate amino acid to the system restored colonization by the auxotrophic mutants, usually to wild-type levels. Analysis of the root base showed that cells of auxotrophic mutants were still present there. The results show that, although amino acids are present in root exudate, the bio-availability of the tested amino acids is too low to support root tip colonization by auxotrophic mutants of P. fluorescens strain WCS365. The genes that are required for amino acid synthesis are therefore necessary for root colonization. Moreover, these compounds apparently play no major role as nutrients in the tomato rhizosphere.

Rhizosphere effects on biodegradation of 2,5-dichlorobenzoate by a bioluminescent strain of root-colonizing Pseudomonas fluorescens

A recombinant strain of bioluminescent Pseudomonas fluorescens 2–79 RLD containing a catabolic pathway for degradation of 2,5-dichlorobenzoate (2,5-DCB) was monitored in soil microcosms to examine the influence of plants on its growth and activity in a contaminated soil. Recombinant P. fluorescens 2–79 RLD was generated by mating a versatile chlorobenzoate utilizer, P. putida P 111, containing plasmid pPB111, with a bioluminescent strain of P. fluorescens that had been transformed previously with a Tn7- luxCDABE marker. Plasmid pPB111 contains genes encoding for a chlorobenzoate-1,2-dioxygenase that converts ortho-chlorobenzoates to their corresponding catechols. DNA hybridization experiments and cell-free extract assays with parental and recombinant P. fluorescens 2–79 RLD suggested that the reaction product of the plasmid pPB111 encoded chlorobenzoate dioxygenase was degraded by an endogenous catechol dioxygenase in P. fluorescens 2–79. After introduction of P. fluorescens 2–79 RLD into soil containing 10 mg kg −1 2,5-DCB, normally recalcitrant 2,5-DCB was degraded rapidly over a period of 2 to 4 days in soil with plants. In contrast, 2,5-DCB disappearance in nonplanted soil was significantly slower, requiring 7 days in one experiment, and more than 2 weeks in a second experiment. Population numbers of the degrader were similar in planted and nonplanted soil for the first 7 days, but declined in nonplanted soils by day 14. Physiological status, measured using an assay based on lag-phase duration, was similar in planted and nonplanted soils at day 2, but rapidly declined in nonplanted soil by day 7. At day 14, plasmid stability in P. fluorescens 2–79 RLD was significantly greater in rhizosphere soil; only 10% of P. fluorescens 2–79 RLD cells in rhizosphere soil had lost the ability to degrade 2,5-DCB, versus 94% of the cells in nonplanted soil. The plasmid also was transferred to indigenous bacteria in both planted and nonplanted soils, as determined by the appearance of novel degraders. The results demonstrate that the presence of plants promoted rapid degradation of DCB and provided a niche that enhanced maintenance of plasmid pPB111 in the degrader bacterium.

Rhizosphere effects on biodegradation of 2,5-dichlorobenzoate by a bioluminescent strain of root-colonizing Pseudomonas fluorescens

A recombinant strain of bioluminescent Pseudomonas fluorescens 2–79 RLD containing a catabolic pathway for degradation of 2,5-dichlorobenzoate (2,5-DCB) was monitored in soil microcosms to examine the influence of plants on its growth and activity in a contaminated soil. Recombinant P. fluorescens 2–79 RLD was generated by mating a versatile chlorobenzoate utilizer, P. putida P 111, containing plasmid pPB111, with a bioluminescent strain of P. fluorescens that had been transformed previously with a Tn7-luxCDABE marker. Plasmid pPB111 contains genes encoding for a chlorobenzoate-1,2-dioxygenase that converts ortho-chlorobenzoates to their corresponding catechols. DNA hybridization experiments and cell-free extract assays with parental and recombinant P. fluorescens 2–79 RLD suggested that the reaction product of the plasmid pPB111 encoded chlorobenzoate dioxygenase was degraded by an endogenous catechol dioxygenase in P. fluorescens 2–79. After introduction of P. fluorescens 2–79 RLD into soil containing 10 mg kg−1 2,5-DCB, normally recalcitrant 2,5-DCB was degraded rapidly over a period of 2 to 4 days in soil with plants. In contrast, 2,5-DCB disappearance in nonplanted soil was significantly slower, requiring 7 days in one experiment, and more than 2 weeks in a second experiment. Population numbers of the degrader were similar in planted and nonplanted soil for the first 7 days, but declined in nonplanted soils by day 14. Physiological status, measured using an assay based on lag-phase duration, was similar in planted and nonplanted soils at day 2, but rapidly declined in nonplanted soil by day 7. At day 14, plasmid stability in P. fluorescens 2–79 RLD was significantly greater in rhizosphere soil; only 10% of P. fluorescens 2–79 RLD cells in rhizosphere soil had lost the ability to degrade 2,5-DCB, versus 94% of the cells in nonplanted soil. The plasmid also was transferred to indigenous bacteria in both planted and nonplanted soils, as determined by the appearance of novel degraders. The results demonstrate that the presence of plants promoted rapid degradation of DCB and provided a niche that enhanced maintenance of plasmid pPB111 in the degrader bacterium.

Gnotobiotic system for studying rhizosphere colonization by plant growth-promoting Pseudomonas bacteria.

A gnotobiotic system for studying tomato rhizosphere colonization by Pseudomonas bacteria was developed. The system is based on sterile seedlings that are inoculated with one or two strains and subsequently grown in a sterile glass tube containing quartz sand. After 7 days of growth in a climate-controlled growth chamber, the number of bacteria present on the root tip was analyzed. The system was optimized with respect to root morphology, inoculation of the seedling, and isolation of root tip bacteria. With this system, rhizosphere colonization on tomato, radish, wheat, and potato was analyzed. For detailed analysis of tomato rhizosphere colonization by some representative plant growth-promoting rhizo-bacteria, the colonization of known poor, moderate, and good potato root-colonizing Pseudomonas strains and of four Rhizobium strains was determined. All strains colonized the root tips when inoculated as single strains. When inoculated in competition with the efficient root colonizer P. fluorescens strain WCS365, many strains were outcompeted. Mutants of Pseudomonas biocontrol bacteria lacking flagella or the O-antigen of lipopolysaccharide (LPS), which were isolated in previous studies and shown to be impaired in potato rhizosphere colonization in field soil systems, showed a reduced colonization ability in the gnotobiotic system also. The gnotobiotic system was used to screen a collection of 300 random P. fluorescens WCS365::Tn5 mutants for colonization-impaired mutants. Three novel mutants were found that were outcompeted by the wild-type strain in tomato root tip colonization but were not impaired in known colonization traits such as motility, amino acid auxotrophy, and presence of the O-antigenic side chain of LPS. One strain appeared to be a thiamine auxotroph, suggesting that the root does not secrete a sufficient amount of thiamine to support growth of this strain. The other two mutants had a reduced growth rate in laboratory media, suggesting that growth rate is an important colonization factor. As the system is gnotobiotic and devoid of field-soil variables, it can also be used to study the effects of selected biotic and abiotic factors on colonization.

Construction of a bioinsecticidal rhizosphere isolate of Pseudomonas fluorescens

The cryIVB gene from Bacillus thuringiensis morrisoni PG-14 was cloned and expressed in Escherichia coli. A gene cassette was constructed that placed the gene under the control of the tac promotor. Three Pseudomonas ‘suicide’ vectors were made by cloning chromosomal DNA fragments from the root-colonizing Pseudomonas fluorescens strain P1 into plasmid pSUP202. The kanamycin resistance gene nptII and the cryIVB gene cassette were cloned within the Pseudomonas sequences. These constructs were introduced into the root-colonizing strain Pseudomonas fluorescens P1. Southern blot hybridizations demonstrated that the nptII and cryIVB genes were integrated into the chromosome whereas vector sequences were not. Expression of the cryIVB protein by transgenic Pseudomonas cells was demonstrated by Western blot analysis. Cell cultures of the transformed P. fluorescens were found to be toxic towards larvae of the malaria mosquito Anopheles stephensi and to leatherjackets, the larvae of Tipula oleracea.

Construction of a bioinsecticidal rhizosphere isolate ofPseudomonas fluorescens

The cryIVB gene from Bacillus thuringiensis morrisoni PG-14 was cloned and expressed in Escherichia coli. A gene cassette was constructed that placed the gene under the control of the tac promotor. Three Pseudomonas ‘suicide’ vectors were made by cloning chromosomal DNA fragments from the root-colonizing Pseudomonas fluorescens strain P1 into plasmid pSUP202. The kanamycin resistance gene nptII and the cryIVB gene cassette were cloned within the Pseudomonas sequences. These constructs were introduced into the root-colonizing strain Pseudomonas fluorescens P1. Southern blot hybridizations demonstrated that the nptII and cryIVB genes were integrated into the chromosome whereas vector sequences were not. Expression of the cryIVB protein by transgenic Pseudomonas cells was demonstrated by Western blot analysis. Cell cultures of the transformed P. fluorescens were found to be toxic towards larvae of the malaria mosquito Anopheles stephensi and to leatherjackets, the larvae of Tipula oleracea.