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Interactions between Microbial inoculants and resident Populations in the rhizosphere of Agronomically important Crops in Typical soils


the use of microbial inoculants in agriculture ha major potential for environmentally friendly crop production. However, our knowledge of the ecological interactions of these inoculants with target and resident picrobial populations is often limited. The research objectives set out within the IMPACT project have utilised molecular approaches to microbial ecology that will allow us to understand, manipulate, and predict a range of agronomically important processes in the rhizosphere of crop plants. The overall objective of the project was to evaluate the ecological impact of wildtype (WT) and genetically modified (GM) strains on key biological components of the rhizosphere. the project was organized into a set of worktasks/sectors that addressed the following key issues.

1 Monitoring the population dynamics of introduced and specific resident microbial strains.

2. Measurement of rhizosphere and soil for changes in biochemical parameters as a result of introduction of microbial inoculants.

3. Development, optimization and evaluation of novel methods for improved microbial detection from environmental locations.

4. The design and construction of inoculant strains for monitoring purposes and improved function.
1. Monitoring the population dynamics of introduced and specific resident microbial strains. This was subdivided into five specific worktasks (1.1-1.5).
1.1: Effects of wild type and genetically modified biocontrol Pseudomonas, Azospirillum and Rhizobium strains on the resident soil microbial community as a whole. Effects on resident microbial populations were measured using several methods, which included plant performance (i.e. yield, crop quality), measurements of the microbial biomass of soil, and biochemical characteristics of soil (nutrient content and soil enzyme activities) Results employing both WT and GM inoculants of these three microbial groups has revealed an overall absence of significatn advere effects on any parameter examined.
1.2: Evaluation of the persistence of microbial inoculants and their effect on similar resident strains. The ability of WT and GM inoculant strains of Pseudomonas, Azospirillum, R. leguminosarum and R. meliloti to colonise and persist in the rhizosphere of inoculated plants constituted a major portion of this worktask. The overall results from these studies has indicated that no significant differences in colonisation andHor persistence between WT and GM inoculant strains was evident. Field trials involving marked Pseudomonas biocontrol strains indicated that levels of these strains declined steadily over time and one year after release, these strains had declined close to or below detection levels. The inability of inoculant stains to persist in soil for a long period may be a desirable characteristic for microbial inoculants. One study examined the effect of hydrogen uptake (HUP) phenotype of R. leguminosarum on the resident population of R. Meliloti. This phenotype is one potential target for genetic modification in rhizobial inoculants. No significant alterations on the level of diversity of the resident population of R. meliloti were observed. The impact of biocontrol Pseudomonas inoculants on resident P. fluorescens strains is summarised below under Worktask 1.3.
1.3: Effect of inoculants on selected beneficial microbial groups. Beneficial microbial groups choesen for evaluation included fluorescent pseudomonads, Rhizobium, Bradyrhizobium and Arbuscular Mycorrhizal (AM) fungi. Studies involving WT and GM Rhizobium inoculants have demonstrated no adverse effects on AM fungi. A GM Rizobium strain with improved ability to outcompete indigenous, strains for nodulation appears to have a stimulatory effect on AM fungi and results in improved plant performance. Strains of Azospirillum which were venetically modified to overproduce the plant growth promoting hormone IAA or which were deficient in its synthesis did not adversely affect either plant performance or selected resident microbial strains. Studies involving WT and GM biocontrol Pseudomonas strains have indicated an overll lack of anysignificant adverse effects on Bradyrhizobium, Rhizobium, AM fungi and resident strains of fluorescent pseudomonads. Of particular importance are studies in which P. fluorescens strains engineered to overproduce antifungal metabolites such as 2,4-dicaetylphloroglucinol (Phl) and pyoluterorin (Plt) were shown not to adversely affect the resident community of fluorescent pseudomonads, or selected AM fungal strains. Biocontrol P. fluorecens strains overproducing antifungal metabolites (Phl, Plt) have been demonstrated to stimulate AM fungal growth and development and improve plant performance. An important observation from these studies has been that the natural biological diversithy of fluorescent pseudomonads in soil appears to be much greater than pregiously believed. This was achieved using sensitive molecular meghods (PCR) adapted specifically within this project (Worktask 3).
1.4: Effect of biocontrol inoculants on deleterious microbial groups (i.e. pathogenic fungi). The evaluation of WT and GM biocontrol Pseudomonas strains with regard to effects on pathogenic microbial groups has received considerable attention within the project. Evaluation WT strains in greenhouse studies has demonstrated the effectiveness of these strains in suppressing certain fungal plant root diseases. genetically modified biocontrol strains sith increased production of antifungal metabolites such as Phl, Plt, and salicylic acid (Worktask 4.1) have been shown to provide increased protection against fungal pathogens compared to the WT strain in soil microcosms. The inability to accurately predict disease pressure in field trials prior to sowing has been a limiting factor in the critical evaluation of biocontol strains.
1.5: Effect inoculants on crop rotational system. The purpose of these studies was to examine the effect(s) of biocontrol inoculants within normal agricultural practice. Specifically, the effect of inoculant strains on the performance of an alternative rotational crop planted one year following release at the same site was evaluated. No significant effects were observed with regard to clover performance (crop yield, nodulation) at a site which had been sown with sugarbeet and inoculated the previous year sith a WT biocontrol Pseudomonas strain. Preliminary results indicate that the diversity of rhizobial strains isolated from nodules obtained in these studies is significantly less than what was observed for the resident fluorescent pseudomonad community. This will facillitate the detection of alterations in the rhizobial community as a consequence of inoculation with the biocontrol strain, and this analysis is continuing. In a separate study a field inoculated in 1994 with a biocontrol strain was evaluated for effects on sorghum planted in 1995 as a rotation crop. No significant effects on plant performance were observed in plots inoculated with the biocontrol strain (strain F113) compared to uninoculated control plots.

2. Measurement of the rhizosphere and soil for changes in biochemical parameters as a result of introduction of microbial inoculants.
One of the main objectives of this worktask was the design of assays to assess the impact of microbial inoculation on nutrient cycling potential in the rhizosphere. This specific objective has been achieved since accurate and reliable methodology was developed in the project and is now available to assay up to 10 enzymes from soil salples as small as 2 grams. Assays for all major nutrient cycles, including carbon, nitrogen, sulphur, and phosphorous have been designed. The direct measurements of specific nutrient levels in soil are also important. Both of these methods have been used to evaluate soil samples from field trials involving release of microbial inoculants from a number of the partners, as well as from soil used in greenhouse evaluations. The overall conclusion from evaluations using these sensitive procedures is that the use of microbial inoculants do not adversely affect the resident microbial population of soil shoch is directly responsible for the fertility of soil.

3. Development, optimisation and evaluation of novel methods for improved microbial detection from environmental locations.
The use of PCR-based methods (ARDRA and RAPD) for the identification and typing of environmental microbial isolates provided by several of the partners has been achieved. The optimisation of commercial software for the input, retrieval and analysis of results obtained from these sensitive methods and the generation of a comprehensive information database was accomplished. a large database of PCR fingerprints was developed and is available for a diverse group of microbial species, including field-isolated fluorescent pseudomonads, and R. leguminosarum biovars viceae and trifolii, which will prove useful in future studies. The development within the project of primers specific for AM fungi has resulted in the extension of PCR-based (ARDRA and RAPD) methods for the detection of AM fungi from environmental locations. This constitutes a novel approach for future ecological studies of AM fungi which provides improvements over conventional methods currently in use. The discovery of a bacterial population related to Pseudomonas (Burkholderia) hosted within AM fungal cells has been an intriguing discovery. The development and application of these sensitive molecular detection methods has proven to be extremely useful in evaluating the effects of microbial inoculants on resident microbial strains whithin this project.

4. The design and contruction of inoculant strains for monitoring purposes and improved function. This was further subdivided into three worktasks outlined below (4.1-4.3).
4.1: Development of inoculant strains with genetic markers and altered biocontrol and biofertilizer functions. A number of specific genes have been employed within IMPACT to generate marked inoculant strains to facilitate detection of released inoculant strains in the invironment. Emphasis was placed on the use of marker genes which did not encode resistance to antibiotics to address concerns regarding lateral spread of antibiotic resistance genes within the environment. These marker genes included those encoding a biochemical function (lacZY, gusA, xylE), bioluminescence (luc, lux), and resistance to mercury (mer). These alternative marker genes have proven to be as sensitive as traditional antibiotic resistance marker genes and do not affect the function of the marked strain, or adversely affeect resident microbial species. In addition, GM derivatives have been developed with altered production of antifungal metabolites (Phl, Plt, salicylate; Pseudomonas), plant-growth promoting compounds such as indole-3-acetic-acid (IAA; Azospirillum), and symbiotic performance by Rhizobium.
4.2: Development of strategies for the biological containment of microbial (i.e. Rhizobium) inoculants. Two strategies were employed, one involved the use of mutants in recA, and the other relies on mutations in thyA. A RecA mutant of R. meliloti has been constructed and exhibits increased sensitivity to UV radiation and is deficient in recombination. This mutation has a negative effect on symbiotic performance and reduced plasmid stability compared to the WT strain. In addition, the RecA strain exhibited an increased sensitivity to heat stress compared to the WTstrain. The survival of a RecA- strain insoil microcosms was not significantly affected compared to the WT RecA+ strain, however. The effectiveness of a RecA-based containment system is primarily a consequence of defects in recombination, which act to limit gene transfer. The alternative containment system is based on the essential gene thyA, which encodes thymidylate synthase. Thy mutants are unable to survive in the absence of an external source of thymidine. Previous studies utilising spontaneous Thy mutants have demonstrated the effectiveness of this system. A drawback to that study was the use of spontaneous mutants, which are able to revert back to a Thy+ phenotype. The development of a stable thyA mutant which should presumably solve this problem has been generated within this project. This strain will be used in combination with an autoselective plasmid containing the thyA gene under the control of a symbiotic (nifH) promoter also developed within this project. Expression of the thyA gene on this plasmid should occur only under symbiotic (microaerobic) conditions and not outside of the nodule, thereby resulting in an inability of the inoculant to persist in the absence of the host plant. This plasmid construct has been demonstrated to successfully complemet the thyA mutation in this strain.
4.3: Gene transfer - Effect of genetic transfer of recombinant genes to the resident microbial population. One study in this project has determined that conjugal gene transfer between microbial strains appears to be related to nutritional status, and that conditions required for optimal gene transfer may differ between various soil types and potential recipients in the soil. In long-term soil microcom experiments, no transfer o a gusA marked nonsymbiotic plasmid from R. meliloti to the indigenous icrobial community has been detected. This indicates that if conjugal transfer of plasmids from inoculant strains to the resident microbial population occurs, it is below detection levels. A number of resident rhizobial strains have ben isolated from a Spanish soil which proved to be good recipient strains under laboratory conditions. These strains have been evaluated in soil microcosms in studies designed to determine what conditions are required for gene transfer to take place in soil. Transfer of the marked nonsymbiotic plasmid was detected for only one resident strain tested, and only when the recipient strain was present at levels much greater than what would be expected in natural soil (i.e. >1E6/gm). In a separate study designed to estimate the impact of gene transfer in soil, strains were generated under laboratory conditions which would occur as a result of gene transfer in soil. These strains were evaluated for phenotypic changes in the rhizosphere of plants. One R. meliloti transconjugant exhibited increase nodulation competitivenesson alfalfa. In other instances the transferred plasmids were either unstable or underwent plasmid rearrangements, which did not significantly affect their symbiotic phenotype. these studies have identified factors which influence transfer efficiencies such as the nutritional status of the cells and the number of potential recipients in soil. In addition, it appears that differences exist in the ability of the resident microbial community to act as recipients in conjugal transfer.

1. Deliberate release of wild type and genetically modified microbial inoculant strains under EU Directive 90/220/EEC in a number of EU countries has been achieved.
2. Data from field experiments indicates the lack of significant detrimental effects associated with the use of wild type (WT) or genetically modified (GM) microbial inoculants on crop yield, soil biomass, soil fertility, and selected soil microorganisms, including taxonomically similar strains and beneficial soil microorganisms.
3. Genetically modified Pseudomonas strains with altered production of antifungal secondary metabolites have been constructed and have been demonstrated to possess increased antifungal activity against several pathogenic fungi. Preliminary results indicate these strains provide increased protection against a variety of fungal pathogens.
4. Several marker genes (lacZ, gusA, luc, lux, xylE, etc) have been used successfully to mark inoculant strains. Data utilising these marked strains from laboratory and field trials are vailable. No significant adverse effects were associated whith the use of these marked strains in comparison to the WT unmarked strains with regard to plant performance and resident soil microorganisms.
5. A fast semi-quantification method for monitoring root colonisation using two marker genes has been developed and evaluated. The marker gene gusA has been utilised on both wheat and tomato, while lux has been employed with tomato. These methods have proven to be reliable and sensitive (c.a. 1E3/cm root).
6. No significant adverse effects associated with the use of WT or GM Rhizobium or Pseudomonas inoculants on the beneficial interaction between mycorrhizal fungi and plants were observed. On the contrary, under some circumstances a significant stimulation of this association was observed.
7. Identification of key resident strains from the rhizospheres of tomato, sugarbeet, pea, alfalfa, bean, have been acomplished. Reconstruction of the microbial rhizosphere of tomato in a gnotobiotic system has been accomplished.
8. Molecular methodology for identification and quantification of arbuscular mycorrhizal (AM) fungi using competitive PCR and specific primers has been developed and successfully tested.
9. The discovery and characterisation of a bacterial endosymbiotic population associated with abuscular mycorrhizal fungi has been achieved. Differences in colonisation of AM fungal structures by microbial inoculants have been identified.
10. Methodology to detect biochemical peturbation in soils (i.e. soil enzymes) has been developed and successfully applied in the evaluation of field releases of microbial inoculants.
11. Optimisation of PCR-based methods for identification (ARDRA) and typing (RAPD) of bacteria from environmental locations has been achieved.
12. The generation of a database of PCR-generated fingerprints has been established for rhizosphere and soil bacteria provided by several of the IMPACT partners.
13. Commercial software for the input, retrieval and analysis of PCR-generated fingerprints has been assessed, refined, and is available for future studies.
14. A containment system for Rhizobium inoculants based on thyA has been developed.
15. Persostence and dissemination of microbial inoculants in microcosm, lysimeters and field studies were investigated. Levels of inoculants remaining in a culturable state decline to or below detection limits whithin one year. Estimates of the populations of viable but non-culturable and dormant cells of P. fluorescens strain CHAO in soil and rhizosphere from field lysimeter studies are available.

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