Molecular-based strategies to exploit Pseudomonas biocontrol strains for environmental biotechnologyapplications

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1 Molecular-based strategies to exploit biocontrol strains for environmental biotechnologyapplications Genevieve L. Mark, John P. Morrissey, P. Higgins & Fergal O Gara The BIOMERIT Research Centre, Department of Microbiology, National University of Ireland (University College Cork), Cork, Ireland Correspondence: Fergal O Gara, The BIOMERIT Research Centre, Department of Microbiology, National University of Ireland (University College Cork), Cork, Ireland. Tel.: ; fax: ; f.ogara@ucc.ie Received 16 December 2004; revised 22 September 2005; accepted 23 September First published online 31 January doi: /j x Editor: Angela Sessitsch Keywords biocontrol; genomics; plant microbe interactions. Abstract Exploitation of beneficial plant microbe interactions in the rhizosphere can result in the promotion of plant health and have significant implications for low input sustainable agriculture applications such as biocontrol. Bacteria such as Bacillus and, and fungi such as Trichoderma, have been developed as commercial biocontrol products. Registration of microbial inocualants as biocontrol agents in either the European Union or the United States requires production of extensive dossiers covering efficiacy, safety and risk assessment. Despite the fact that a number of biocontrol products have been marketed there are still some limitations hampering the development of this technology for widespread use in agriculture. Although many strains show good performance in specific trials, this is often not translated into consistent, effective biocontrol in diverse field situations. Advances in Omics technology and the publication of complete genome sequences of a number of plant-associative bacterial strains, has facilitated investigations into the molecular basis underpinning the establishment of beneficial plant microbe interactions in the rhizosphere. The understanding of these molecular signalling processes and the functions they regulate is fundamental to promoting beneficial microbe plant interactions, to overcome existing limitations and to designing improved strategies for the development of novel Pseudmonas biocontrol inoculant consortia. Introduction Conventional agriculture is heavily dependent on the application of chemical inputs, including fertilizers and pesticides, to maintain consistent high yields. There is, however, a growing desire for alternatives to this system. This comes from two perspectives: (1) environmental protection; and (2) human health considerations. With regard to the environment, issues such as contamination of soil and water with chemical residues, habitat damage and loss, negative impact of chemicals on nontarget species, and impacts on biodiversity are to the fore. Consumers and health authorities are also expressing concerns regarding the potential effects on human health of chemical residues in food products and in the environment. There is, of course, precedent for negative effects, with many fungicides and pesticides that were previously permitted now banned. Because of these environmental and health issues, the EU monitors chemicals approved under directive 91/414/EC. This is a continuous process and has included a requirement to defend some previously approved chemicals (COM1112/2002). There has been strong pressure from some member states to ban some fungicides (e.g. Sweden, thiabenzadole), and it is likely that there will be further prohibitions at EU level on fungicide use in agriculture. Viable alternatives to chemicals must be provided, however, if fungicide reduction is to be achieved. It has long been recognized that there are many naturally occurring bacteria and fungi that are antagonistic to crop pathogens, and consequently have the potential to provide an alternative to chemical fungicides. The best-known example is probably the control of Lepidoptera by the bacterium Bacillus thuringiensis (Bt toxin), but there are also many well-documented examples of bacteria and fungi that can inhibit the growth of soil-borne bacterial and fungal diseases of crops. These diseases, such as root rot, damping off, and take-all, are currently controlled, at least in part, by the application of chemical fungicides as seed coatings or liquid formulations. The possibility of replacing these fungicides with microbes possessing natural intrinsic activity against fungal pathogens is very appealing.

2 168 G.L. Mark et al. Microbial biocontrol inoculants A number of recent reviews have discussed the potential applications of microbial inoculants for sustainable agriindustry, such as phytostimulation, biofertilization, bioremediation and biocontrol (Haas et al., 2000; Bloemberg & Lugtenberg, 2001; de Lorenzo, 2001; Walsh et al., 2000, 2001a; Mark et al., 2003). Biocontrol is a multitrait phenomenon with success depending on many factors. These include the ability of the microbial inoculant to survive in the rhizosphere and to compete with the resident microbial populations, as well as protecting the plant host against pathogens at both the time and site of infection (Chin-A-Woeng et al., 2000). It has long been recognized that there are many naturally occurring bacteria and fungi that are antagonistic to crop pathogens, and consequently have the potential to provide an alternative to chemical fungicides. Bacteria such as those belonging to the Bacillus genera, and fungi in the Trichoderma genus have been cited as potential biological control agents (Chet & Inbar, 1994; Walsh et al., 2001b). A number of biocontrol products based on Bacillus have been marketed (mainly in the US). More recently, bacteria have attracted interest and a number of -based biocontrol inoculants have now been commercially developed (mainly in the US) (described in Table 1). Genotypic and phenotypic characteristics of the soil-borne fluorescent pseudomonads have highlighted this group of rhizobacteria as potential candidates for use in biocontrol applications (Bloemberg & Lugtenberg, 2001; Walsh et al., 2001a; Morrissey et al., 2004a). bacteria are of particular interest because of the intrinsic ability of certain strains to colonize the rhizosphere at a high density, to compete successfully with microorganisms, and to produce secondary metabolites with powerful antifungal activity such as 2,4-diacetylphloroglucinol (Phl). Phl is a broad-spectrum antimicrobial produced by a range of fluorescens spp. and has biological activity against a range of fungal and bacterial plant pathogens (Morrissey et al., 2004a). Recombinant DNA technology has established that biocontrol efficacy of some strains of P. fluorescens is linked to the production of antifungal secondary metabolites such as Phl (Fenton et al., 1992; Keel et al., 1992). inoculants that produce Phl have also been recently implicated in inducing systemic disease resistance in plants (Van Loon et al., 1998). Complex regulatory systems govern the production of Phl and occur at both the transcriptional and posttranscriptional level (Delany et al., 2000; Abbas et al., 2002). The GacS/GacA system is an environmentally responsive regulatory system necessary for the production of Phl, where a mutation in either component results in the abolition of Phl biosynthesis (Corbell & Loper, 1995; Dunne et al., 1996). By uncoupling transcriptional regulatory controls, P. fluorescens F113 can be genetically modified to overproduce Phl (Fenton et al., 1992; Delany et al., 2001). Other extracellular signals can also have an influence on the metabolites produced by bacterial strains, and the addition of molecular signals to P. fluorescens strains has been shown to enhance the production of antifungal metabolites (Duffy & Defago, 1999; Abbas et al., 2004). Registration and marketing of microbial inoculants (including antimicrobial metabolites) In the USA, pesticides are categorized as antimicrobials, biopesticides and conventional pesticides. Microbial inoculants and the antimicrobial metabolites they produce fall under the category of biopesticides. Registration and authorization of biopesticides is regulated by the Environmental Protection Agency under the authority of the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA, 1996). In 1994, the Biopesticide and Pollution Prevention Division was established under the EPA umbrella to aid in the registration of biopesticides and promotion of the utilization of these products particularly as part of an Integrated Pest Management (IPM) agricultural approach. Biopesticides can be registered in 1 year as opposed to the 3 years generally taken for conventional chemical-based pesticides, and data must be included on the composition, toxicity, degradation and other properties of the active substance present. Rigorous risk assessment must be carried out to confirm that the microbial-based biopesticide has no deleterious effects on human health and/or the environment. Registration costs of microbial-based products are lower than those required for conventional pesticides, and there is a growing trend for commercial companies to prefer to develop (in collaboration with the scientific community) microbial inoculants that produce antimicrobial compounds as biopesticides. Utilization and release of inoculants in Europe requires approval under the relevant directives, and these directives have been reviewed by Morrissey et al. (2002) and Walsh et al. (2001a). Plant protection products (PPPs) are defined as products that protect against damagecausing organisms, influence plant-life processes (excluding nutrients), and can be used against unwanted plant species (i.e. herbicides). European legislation concerned with the registration and use of PPPs is addressed in Council Directive 91/414/EEC ( entitled the The Plant Protection Directive, and its adoption (15 July 1991) is under the control of the European Food Safety Authority (EFSA). This directive provides a framework for the establishment of an approved list of active substances that show no risk to human health and/or the environment. These active substances are then authorized for use and can be subsequently marketed. Council Directive 91/414/EEC

3 Molecular-based strategies to exploit strains 169 Table 1. A list of commercially available (in the USA) -based biocontrol products Product name Biocontrol organism Target pathogen Crop Formulation/application Manufacturer BioJect Spot-Less aureofaciens Tx-1 EPA registered 1999 Collectotrichum graminicola (Anthracnose) Bio-Save 10LP, 110 syringae ESC-10 & ESC-11 Sclerotinia homeocarpa (Dollar spot) Turf Liquid applied as spray Eco Soil Systems Inc. (San Diego, CA) Pythium aphanidermatum (Pythium) Michrodochium nivale (Pink snow mold) Botrytis cinerea, Penicillium spp., Mucor pyroformis, Geotrichum candidum Pome fruit, citrus, cherries and potatoes Lyophilized product, frozen cell concentrated pellets added to water to form a liquid suspension and applied postharvest to fruit as drench, drip or spray EcoScience Corp. (Longwoord, FL) EPA registered BlightBan A506 EPA registered fluorescens A506 Cedemon chlororaphis strain Erwinia amylovora and russetinducing bacteria Leaf stripe, net blotch, Fusarium spp., spot blotch, leaf spot Almond, apple, apricot, blueberry, cherry, peach, pear, potato, strawberry and tomato Wettable powder applied as spray at bloom time Barley and oats Seed treatment applied as seed dressing NuFarm Inc. (Burr Ridge, IL) BioAgri (Uppsala, Sweden) EPA registered Potential for use on cereals Liquid applied as drench Eco Soil Systems Inc. (San Diego, CA) AtEze chlororaphis Pythium spp., Rhizoctonia solani, Fusarium oxysporum Ornamentals and greenhouse grown vegetables EPA registered Frost Technology Corporation FROSTBAN fluorescens A506, 1629RS Frost-forming bacteria Fruit crops, almond, potato and tomato Liquid applied as spray early in growing season EPA registered 1992 P. syringae Plant Health Technologies (Lathrop, CA) 742RS Information regarding products highlighted is adapted from a list compiled by Dr D. Fravel, USDA-ARS 2000, and updated by the APS Biological Control Committee, USA, 2005 ( Information regarding products highlighted is adapted from product registration lists compiled by the US Environmental Protection Agency (

4 170 G.L. Mark et al. was amended with Council Directive 2001/36/EC to include microorganisms and the antimicrobial metabolites they produce as PPPs and came into effect on 16 May Annex I of Council Directive 91/414/EEC details the criteria required for the approval and authorization of a microbial biocontrol inoculant and/or the antimicrobial metabolite they produce as an active substance. In 2004 chlororaphis (2004/71/EC) was added as an active substance to Annex 1 of 91/414/EEC. A dossier must be submitted in which the active substance is identified, the physical and chemical attributes documented, the mode of action described, and the target organisms specified. Rigorous risk assessment must be permitted by the applicant to investigate the impact of the microbial-derived active substance on human health and the environment. The dossier is then submitted to the member states for evaluation and a report sent to the EFSA from where it is forwarded to the Standing Committee on Plant Health. The committee forms an opinion on whether the active substance should be included in Annex I. If approval is successful, the microbial inoculant and/or the metabolite produced will then be added as an active substance to Annex I, authorized in Annex II, and added as a PPP in Annex III of Council Directive 91/414/ EEC. Despite the costs for the registration of microbial biocontrol inoculants being similar to those for chemical pesticides, the extensive data requirements can deter companies from developing and marketing such active substances as PPPs. The biological properties of the microbial inoculant and identity of the target organism need to be specified, and detailed information on the metabolite involved and its production need to be included. The nature, structure and regulation of the metabolite, whether it is present intracellularly of extracellularly, its stability, mode of action, what conditions are needed for its production, and whether it has any toxic effects on human health and/or the environment have to be addressed in detail before approval can be granted. Postregulation monitoring of the microbial inoculant and the metabolite it produces can also be required, where focus is placed on fate and behaviour in the environment and toxicity to nontarget organisms. Only PPPs that are authorized and included in Annex 1 can be commercially marketed for use in agriculture. Despite a high application frequency of patents for microbial-based pesticides, only a small number have been approved for agricultural use. The reasons for this involve (1) the specific action and efficacy of products, and (2) biosafety concerns (Montesinos, 2003). Risk assessment of biocontrol inoculants In Europe the deliberate release into the environment of genetically modified organisms is legislated by Council Directive 2001/18/EC, which repeals Council Directive 90/ 220/EEC. Before market approval, consideration must be given to (1) case-by-case environmental risk assessment prior to any release, (2) deliberate release at the research stage as a necessary part of new product development, (3) progressive authorized release step by step, and (4) satisfactory fieldtesting at the research stage in ecosystems that could be affected. In North America release into the environment of genetically modified organisms is under the authority of the USDA, FDA and EPA. The successful utilization of microbial inoculants with improved biocontrol traits such as the ability to overproduce secondary metabolites relies on their introduction to the rhizosphere posing no risk to the environment (Mark et al., 2003). Prior to assessing the impact of these genetically modified strains on nontarget microorganisms, the influence of wild-type strains must be investigated to establish appropriate comparative baselines. In independent field-based trials, the influence of P. fluorescens F113, a Phlproducing wild-type strain, on sugarbeet yield parameters (Moënne-Loccoz et al., 1998) and on the ecologically significant resident fluorescent population in the rhizosphere of sugarbeet was assessed (Moënne-Loccoz et al., 2001). Plant parameters such as germination, root yield/ quality and sugar yield were not reduced by the addition of P. fluorescens F113. Inoculation with this strain also had no influence on size, genetic diversity or distribution of phylogenetic groups (based on amplified ribosomal DNA restriction analysis, ARDRA) of the resident fluorescent population in the rhizosphere of sugarbeet. Shifts (based on carbon utilization) in this resident population occurred following inoculation, but these were spatially limited to the rhizoplane and did not result in a deleterious alteration of ecosystem function (Moënne-Loccoz et al., 2001). The residual impact of this P. fluorescens F113 Phl-producing strain was measured in field-based studies by investigating the effect of a previous inoculation of sugarbeet on the resident rhizobia population nodulating a subsequent red clover crop rotation (Walsh et al., 2003). Addition of the P. fluorescens F113 Phl-producing strain did not reduce genetic diversity, but resulted in a shift in the population where an enrichment of Rhizobium leguminosarum bv. trifoli strain isolates that belonged to the dominant rapid amplified polymorphic DNA profile occurred. However, this perturbation did not result in the ability of the Rhizobium clover symbiosis to function or reduce clover yield. As expected, in some cases inoculation with P. fluorescens F113 resulted in perturbations in the soil microbial community structure; however, this did not translate into deleterious consequences for plant yield. A rigorous assessment of the impact of P. fluorescens F113 genetically modified to overproduce Phl, on both nontarget microbial communities in the rhizosphere and plant yield parameters, was carried out within the framework of two multidisciplinary EU collaborative networks, IMPACT 1/II

5 Molecular-based strategies to exploit strains 171 and ECO-SAFE. Notification for field release of P. fluorescens F113 inoculants genetically modified to overproduce Phl occurred in November 1997 and May 1998 for field sites cropped to maize (Granada, Spain) and sugarbeet (Ravenna, Italy), respectively. In the field trials carried out in Granada, Spain, the impact of these strains on mycorrhizal fungal communities in the rhizosphere of maize was investigated. Inoculation had a beneficial influence on mycorrhizal symbiosis where it stimulated mycelial development from germinated Glomus mosseae spores and the degree of overall mycorrhizal colonization of maize roots. In addition, inoculation resulted in an increase in both maize shoot and root yield compared with wild-type-inoculated and uninoculated control plants (Barea et al., 1998). In the parallel study carried out in Ravenna, Italy, inoculation with the Phloverproducing P. fluorescens F113 strain had no influence on the population sizes of resident microbial populations, namely microfungi, Streptomycetes and fluorescent pseudomonads, in the rhizosphere of sugarbeet (Resca et al., 2001) with respect to the wild-type control. In addition, inoculation by this strain did not have a harmful effect on crop yield or quality, with germination, root length and biomass comparable to sugarbeet plants inoculated with the wildtype strain and the uninoculated control plants (Resca et al., 2001). Other key studies have been conducted to investigate the impact of other inoculants with a range of genetically modified biocontrol relevant traits on nontarget microrganisms in the rhizosphere (Table 2). In some instances, the scientific literature suggests that the addition of a microbial inoculant genetically modified for improved biocontrol traits such as secondary metabolite production can cause perturbations in the microbial community structure in the rhizosphere; however, this did not translate into deleterious consequences for plant yield. Future directions for the exploitation of biocontrol inoculants Internationally, there is a large body of work investigating the detailed mechanisms by which control phytopathogenic fungi, and a number of biocontrol products based on these microbes have been marketed (mainly in the US). Despite this, there are still some major factors that limit the development of this technology for widespread use in agriculture (Walsh et al., 2001b; Morrissey et al., 2002), one example being the efficacy of the product. Although many strains show good performance in specific trials, this is often not translated into consistent, effective biocontrol in diverse field situations. Some of this is as a result of external factors such as soil or climatic conditions, but a major part is the result of intrinsic traits of the microbe, such as variable production of required metabolites or poor colonization under certain conditions. The rhizosphere is the site of intense interactions between microbes and plants. A large body of scientific knowledge already exists on the biology and biochemistry of this ecologically significant niche; however, little is known about the molecular interactions that take place between plants and microbes in the rhizosphere (Morrissey et al., 2004b). The exchange of signals between bacteria and plant hosts influences the outcome of the diverse associations between these biological systems in nature. In plant bacteria interactions, signal molecules exuded by the plant influence both the primary initiation and subsequent behaviour of the bacteria in these complex associations. These signalling pathways play a key role in the initiation of beneficial interactions, such as biocontrol. Aggressive colonization and the ability to compete with resident microorganisms are prerequisites for the establishment of effective biocontrol strains (Chin-A-Woeng et al., 2000). Molecules exuded by the plant root may act as signals to influence the ability of microbial strains to colonize the root and to survive in the rhizosphere (Simons et al., 1996; Gough et al., 1997; Kuiper et al., 2001). Microbes can influence the composition of these root exudates by affecting root cell leakage, cell metabolism, and plant nutrition status (Yang & Crowley, 2000). The plant host-specific responses of several plant pathogens have been well documented. Symbiotic microbes such as mycorrhizal fungi and Rhizobium can also form associations where a high degree of host specificity can exist (Mark & Cassells, 1996; Vandenkoornhuyse et al., 2003; Mutch & Young, 2004). It is now evident that many other beneficial associations between microbes and plants exhibit a certain degree of host specificity. Different plant species (Yang & Crowley, 2000; Smalla et al., 2001; Wieland et al., 2001; Kowalchuk et al., 2002; Kuske et al., 2002; Landa et al., 2002; Press & Phoenix, 2005) can actively select for distinct microbial populations in the rhizosphere. Using a cultureindependent approach, Sessitsch et al. (2004) also reported the existence of plant-host-specific endophytic bacterial communities in field-based trials. This plant host selection can also occur within varieties or cultivars of the same plant species. In field-based studies we have established that different varieties of sugarbeet can select for genetically and functionally diverse populations of resident culturable fluorescent pseudomonads in the rhizosphere (G. L. Mark, unpublished data). This plant-mediated selection relies in part on the activation of bacterial gene expression in response to molecular signals exuded by the host roots. Despite the general acceptance that plant-derived extracellular signals can influence bacterial behaviour in the rhizosphere, very little is known about the influence of these signals on the patterns of bacterial gene expression and about the role of those genes with altered expression in the plant microbe interaction.

6 172 G.L. Mark et al. Table 2. Key studies investigating the impact of wild-type and genetically modified inoculants on nontarget microorganisms in the rhizosphere and plant yield WT strain GM trait (GM derivative) Impact on nontarget microbial communities in the rhizosphere and plant yield Reference putida WCS358 fluorescens F113 fluorescens F113 fluorescens CHA0 fluorescens CHA0 fluorescens CHA0 fluorescens CHA0 fluorescens CHA0 Produce Phz and Phl (WCS358r::phz) (WCS358r::phl) Overproduce Phl Overproduce Phl Overproduce Phl and Plt (CHA0[pME3424]) Overproduce Phl and Plt (CHA0-Rif[pME342]) Overproduce IAA (CHA0[pME3468]) Overproduce Phl and Plt (CHA0-Rif[pME3424]) Overproduce Phl and Plt (CHA0[pME3424]) Inoculation with the GM derivatives resulted in transient shifts in composition of bacteria and fungal communities in the rhizosphere of wheat. However, they had no effect on soil metabolic activity Inoculation with the GM derivative had no influence on population sizes of resident microbial populations in the rhizosphere of sugarbeet with respect to the WT control. Inoculation had no deleterious impact on sugarbeet yield Inoculation with the GM derivative had a beneficial influence on mycorrhizal symbiosis in maize WT strain and GM derivative altered the structure of the culturable fungal community fraction in the rhizosphere of mungbean GM derivative had a detectable influence on fungal community in the rhizosphere of cucumber at day 32 of growth cycle. Repeated cropping of cucumber, however, had a greater impact than GM derivative In natural soil the GM derivative increased root yield; however, in autoclaved soil, despite WT increasing root and shoot yield, inoculation with GM resulted in root stunting and subsequent reduction in plant biomass WT strain and GM derivative inoculation altered the metabolic activity of the bacterial community in the cucumber rhizosphere but this was transient. Plant growth had a more pronounced impact on bacterial metabolic activity. WT and GM inoculants had no impact on the genetic structure of the bacterial community or on the proportion of bacteria that were tolerant or sensitive to Phl or Plt present WTstrain and GM derivative inoculation decreased the population size of the nitrogen-fixing symbiotic Sinorhizobium meliloti in soil microcosms. Inoculants also altered the diversity of this population. Alteration to the ability of Sinorhizobium meliloti strains to nodulate alfalfa roots also occurred following inoculation with WT and GM CHA0. However, addition of the WT strain increased plant yield Glandorf et al. (2001), Bakker et al. (2002), Viebahn et al. (2003) Resca et al. (2001) Barea et al. (1998) Shaukat & Siddiqui (2003) Girlanda et al. (2001) Beyler et al. (1999) Natsch et al. (1998) Niemann et al. (1997) Abbreviations are as follows: WT, wild-type strain; GM, genetically modified derivative; Phz, phenazine; Phl, 2,4 diacetylphloroglucinol; Plt, pyoluteorin; IAA, indole-3-acetic acid; Rif, Rifampicin resistance. Other related reviews include Raaijmakers et al. (2002). Advances in gene fusion technology such as in vivo expression technology (IVET) can provide a powerful approach to studying bacterial gene expression in the rhizosphere (Rainey, 1999; Rediers et al., 2005). By adopting an IVET approach, Rainey s group identified 20 genes with elevated levels of expression in the rhizosphere in P. fluorescens SBW25. Of the 20 genes identified, 14 showed homology to published gene sequences with functions involved in nutrient acquisition, stress response, and the type-three secretion system (Preston et al., 2001). More recently, this technology was used for another beneficial P. fluorescens strain Pf0-1 in order to identify genes that may play a role in bacterial survival (Silby & Levy, 2004). IVET strategies have also been constructed for several pathogenic bacteria such as P. aeruginosa (Wang et al., 1996), Yersinia entercolitica (Young & Miller, 1997) and Staphylococcus aureus (Lowe et al., 1998). One limitation of IVET technology is that global bacterial gene expression cannot be profiled. Omics technologies such as transcriptomics allow for the analysis of global gene expression. Microarray-based experiments have focused on model organisms whose genomes have been completely sequenced and for which commercial GeneChips are available. Schenk et al. (2000) reported on changes in gene expression in Arabidopsis infected by an incompatible fungal pathogen or treated with defence-related signalling molecules and identified genes involved in signalling and complex regulatory pathways involved in the interaction. This reiterates the scope of this technology in providing a more complete picture of gene regulation in a plant microbe interaction. Scheideler et al. (2002) showed using microarray technology that infection of this model plant with the pathogen syringae pv. tomato caused shifts in plant-host metabolism. Other studies have concentrated on global gene expression in P. aeruginosa in response to certain factors such as quorum sensing (Schuster et al., 2003), iron

7 Molecular-based strategies to exploit strains 173 Plant host Biological solutions for control of crop disease Microbial behaviour in soil Adaptation to stress Host selection Colonisation Secondary metabolitie production Fig. 1. A systems-based approach to investigate molecular-based signalling mechanisms in plant interactions. Microbial signals Other Environmental stresses Root exudates plant signals GeneChip global gene expression Identify novel genes Comparative Genomics Functional Genomics Genomics Targeted mutagenesis In situ imaging (Ochsner et al., 2002; Palma et al., 2003), hydrogen peroxide (Palma et al., 2004; Salunkhe et al., 2005), and airway fluid from cystic fibrosis patients (Wolfgang et al., 2004). The advances in commercial microarray technology together with the publication of complete genome sequences of a number of plant-associative bacterial strains (Pühler et al., 2004) has facilitated investigations into profiling bacterial gene expression on a global scale. Complete genomes for three strains: P. aeruginosa PAO1 (Stover et al., 2000, putida KT2440 (Nelson et al., 2002, and P. syringae pv. tomato DC3000 (Buell et al., 2003, are available. Two other P. syringae pathovars are being sequenced, and there are genome sequencing programmes for further isolates of P. aeruginosa (including P. aeruginosa PA-14). Three genome programmes are underway for beneficial plant-associative bacterial strains belonging to the P. fluorescens lineage, namely SBW25 (the Sanger Institute, Wellcome Trust Genome Campus, Cambridge, UK, PF5 (TIGR, The Institute for Genomic Research, Rockville, MD, and Pf-01 (US Department of Energy/Joint Genome Institute Genomic Facility, Walnut Creek, CA). The availability of these genomic resources has made it possible to investigate in depth the molecular basis of beneficial plant microbe interactions at both the comparative and functional genomic level (Morrissey et al., 2004b). Advances in microarray technology have allowed pilot microarrays to be developed for the plant-associative bacteria Sinorhizobium meliloti, and gene expression has been analysed under a range of symbiotic and nonsymbiotic conditions (Ampe et al., 2003; Berges et al., 2003) to identify the genes involved. More recently, a dual-genome Chip housing S. meliloti and plant host Medicago truncatula (Barnett et al., 2004) has been used to elucidate changes in gene expression in the plant and prokaryotic partner simultaneously. Proteomic-based technology can be used to complement transcriptomics (Wilkins et al., 1996) and has been used to investigate several plant-growth-promoting rhizobacteria. In Rhizobium leguminosarum bv. trifoli, proteins have been identified that have a role in the initial stages of nodulation (Morris & Djordjevic, 2001). Proteins that are implicated in the endomycorrhizal symbiosis have also been identified (Bestel-Corre et al., 2003). Recently directed proteomics has been used, where the focus was on a subset of phosphorylated plant proteins. This has been successfully used to identify Arabidopsis proteins that are divergently expressed in response to fungal elicitors such as chitosan (Ndimba et al., 2003). By adopting a systems biology approach (Fig. 1) with the application of omics technology a better understanding of the regulation of bacterial gene expression and of the role of those genes with altered expression in microbe plant interactions can be achieved. Our approach to identifying bacterial genes that may play a role in microbial behaviour in the rhizosphere has been to examine the influence of plant-root exudates on the entire transcriptome of a model strain using Gene- Chip technology (P. aeruginosa PA01Affymetrix Gene- Chip s ). Our model system (Mark et al., 2006) to identify bacterial genes that may play a role in microbial behaviour in the rhizosphere exploits the isolate P. aeruginosa strain PA01, which has been shown successfully to colonize the rhizosphere (Walker et al., 2004) and profiles the effects of root exudates from varieties of sugarbeet. Subsets of genes have been identified and characterized as genes with functions known to be implicated in plant microbe interactions,

8 174 G.L. Mark et al. genes not yet shown to have a role and genes encoding hypothetical proteins. By adopting genomic/functional genomic, proteomic and in situ imaging technologies we can now elucidate the role of these novel genes in the plant interactome in the rhizosphere. The understanding of the molecular signalling processes and the functions they regulate within the rhizosphere will play a pivotal role in promoting beneficial microbe plant interactions in the rhizosphere, in overcoming existing limitations, and in designing strategies for the generation of novel inoculant consortia with applications in sustainable environmental biotechnology such as biocontrol. Note in Proof Since original submission of this paper, the genome sequence of P. fluorescens Pf5 has been published (Paulsen et al., 2005). Acknowledgements We acknowledge Claire Adams and Max Dow for useful advice and discussions. We acknowledge the IMPACT, ECO- SAFE and PSEUDOMICS consortia for fruitful discussion and valuable scientific comment. The research presented was supported in part by grants awarded by the European Union, namely BIO4-CT (IMPACT 11), QLK3-CT (ECO-SAFE), QLRT (PSEUDO- MICS), and by the Higher Education Authority of Ireland (PRTI2, PRTI3) and Science Foundation of Ireland (SFI). References Abbas A, Morrissey JP, Marquez PC, Sheehan MM, Delany IR & O Gara F (2002) Characterization of interactions between the transcriptional repressor PhlF and its binding site at the phla promoter in fluorescens F113. J Bacteriol 184: Abbas A, McGuire JE, Crowley D, Baysse C, Dow M & O Gara F (2004) The putative permease PhlE of fluorescens F113 has a role in 2,4-diacetylphloroglucinol resistance and in general stress tolerance. 150: Ampe F, Kiss E, Sabourdy F & Batut J (2003) Transcriptome analysis of Sinorhizobium meliloti during symbiosis. Genome Biol 4: Bakker PA, Glandorf DC, Viebahn M, Ouwens TW, Smit E, Leeflang PK, Wernars K, Thomashow LS, Thomas-Oates JE & van Loon LC (2002) Effects of putida modified to produce phenazine-1-carboxylic acid and 2,4- diacetylphloroglucinol on the microflora of field grown wheat. Antonie Van Leeuwenhoek 81: Barea JM, Andrade G, Bianciotto V, Dowling D, Lohrke S, Bonfante P, O Gara F & Azcon-Aguilar C (1998) Impact on arbuscular mycorrhiza formation of strains used as inoculants for biocontrol of soil-borne fungal plant pathogens. 64: Barnett J, Toman J, Fisher F & Long S (2004) A dual-genome Symbiosis Chip for coordinate study of signal exchange and development in a prokaryote host interaction. Proc Natl Acad Sci 101: Berges H, Lauber E, Liebe C, Batut J, Kahn D, de Bruijn FJ & Ampe F (2003) Development of Sinorhizobium meliloti pilot macroarrays for transcriptome analysis. Appl Environ Microbiol 69: Bestel-Corre G, Dumas-Gaudot E & Gianinazzi S (2003) Proteomics as a tool to monitor plant microbe endosymbioses in the rhizosphere mycorrhiza. 14: Beyler M, Keel C, Michaux P & Haas D (1999) Enhanced production of indole-3-acetic acid by a genetically modified strain of fluorescens CHA0 affects root growth of cucumber, but does not improve protection of the plant against Pythium root rot. FEMS Microbiol Ecol 28: Bloemberg GV & Lugtenberg BJ (2001) Molecular basis of plant growth promotion and biocontrol by rhizobacteria. Curr Opin Plant Biol 4: Buell CR, Joardar V, Lindeberg M, et al. (2003) The complete genome sequence of the Arabidopsis and tomato pathogen syringae pv. tomato DC3000. Proc Natl Acad Sci USA 100: Chet I & Inbar J (1994) Biological control of fungal pathogens. Appl Biochem Biotechnol 48: Chin-A-Woeng TF, Bloemberg GV, Mulders IH, Dekkers LC & Lugtenberg BJ (2000) Root colonization by phenazine-1- carboxamide-producing bacterium chlororaphis PCL1391 is essential for biocontrol of tomato foot and root rot. Mol Plant Microbe Interact 13: Corbell N & Loper JE (1995) A global regulator of secondary metabolite production in fluorescens Pf-5. J Bacteriol 177: Delany I, Sheehan MM, Fenton A, Bardin S, Aarons S & O Gara F (2000) Regulation of production of the antifungal metabolite 2,4-diacetylphloroglucinol in fluorescens F113: genetic analysis of PhlF as a transcriptional repressor. Microbiology 146: Delany IR, Walsh UF, Ross I, Fenton AM, Corkery DM & O Gara F (2001) Enhancing the biocontrol efficacy of fluorescens F113 by altering the regulation and production of 2,4-diacetylphloroglucinol. Plant Soil 232: Duffy BK & Defago G (1999) Environmental factors modulating antibiotic and siderophore biosynthesis by fluorescens biocontrol strains. Appl Environ Microbiol 65: Dunne C, Delany I, Fenton A & O Gara F (1996) Mechanisms involved in biocontrol by microbial inoculants. Agronomie 16: Fenton AM, Stephens PM, Crowley J, O Callaghan M & O Gara F (1992) Exploitation of gene(s) involved in 2,4- diacetylphloroglucinol biosynthesis to confer a new biocontrol

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