Partner und Internationale Organisationen
(Englisch)
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Uni. College Cork(I), Irish Sugar (I), Uni. Padova (I), Agronomica (I), Uni Bielefeld (D), Uni. Politec. Madrid (E), CSIC (E), Leiden Uni. (NL), S&G Seeds (NL), Uni. of York (UK), Uni.Torino (I), CSIC (E), Uni. Surrey (UK), TÜV Sw (D), Kath. Uni Leuven (B), seedsNovartis (NL).
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Abstract
(Englisch)
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IMPACT 2 is a 17-partner project with the objective to assess the ecological impact of genetically-modified bacterial inoculants and crops in the soil/rhizosphere. Several partners including the Swiss group apply root-colonizing bacteria to protect plants from soil-borne pathogenic fungi. By genetic engineering, the traits conferring biocontrol activity or plant growth-promoting activity can be overexpressed in these bacteria. For biosafety assessment, monitoring the effects of such wild-type (WT) or modified (GM) bacterial inoculants in the environment, especially their potential ecological impact on the non-target microbiota and on plants, becomes an important issue. Arguably, certain ecological effects of a GM microbial inoculant may be difficult to predict from the results of short term experiments in which a single inoculation is performed.
The Swiss group has evaluated the influence of environmental factors and regulatory mechanisms on the survival in soil and the expression of relevant biocontrol traits of Pseudomonas fluorescens strain CHA0, a well-characterised bacterium which protects plants from a variety of soilborne diseases. In addition, derivatives of strain CHA0, genetically engineered for increased biocontrol and plant growth-promoting activity, were monitored for their ecological impact on the resident microbial community in the plant rhizosphere. Strain CHA0 is an effective root coloniser and excretes the antimicrobial compounds 2,4-diacetylphloroglucinol (2,4-DAPG), pyoluteorin (Plt), and hydrogen cyanide (HCN) which contribute to its plant protective capacity. The large-scale and repeated release into soil of bacteria producing such antibiotics potentially might have adverse effects on antibiotic-sensitive resident bacterial populations and might favour the emergence of resistance to these antibiotics. Soil microcosm experiments with cucumber plants showed that even repeated inoculations of strain CHA0 or a 2,4-DAPG- and Plt-overproducing GM derivative with improved biocontrol activity did not modify the diversity of the resident bacterial community and did not lead to an enrichment of bacterial populations resistant to 2,4-DAPG and/or Plt. In contrast, repeated growth of cucumber in the same soil resulted in significant modifications in the bacterial community. Strain CHA0 was also genetically engineered to overproduce the plant hormone indole-3-acetic acid (IAA) to test the possibility whether enhanced production of the compound on roots may be beneficial to plant growth. Indeed, the IAA overproducer was able to improve root growth of cucumber by up to >30%, in natural soil. However, the GM derivative was not superior to the parental strain in protecting the plants against root rot caused by pathogenic oomycete Pythium ultimum, indicating that IAA is not a major biocontrol component of strain CHA0.
A variety of environmental signals and regulatory mechanisms may influence the production of relevant biocontrol compounds and thus disease suppressive capacity in P. fluorescens. The effect of environmental signals on 2,4-DAPG and HCN production by strain CHA0 was monitored by using lacZ reporter fusions in the corresponding structural genes. Expression of 2,4-DAPG biosynthetic genes was higher on the roots of monocotyledonous (maize, wheat) than on those of dicotyledonous (cucumber, bean) plants and varied depending on plant age. The 2,4-DAPG reporter was expressed at a markedly higher level in presence of the pathogen P. ultimum on all plant species tested. In contrast, fusaric acid, a pathogenicity factor of Fusarium oxysporum specifically repressed 2,4-DAPG production and gene expression. The production of HCN, 2,4-DAPG and other secondary metabolites is tightly controlled by the GacS-GacA two-component regulatory system. GacA was found to exert its control on the HCN biosynthetic genes indirectly via a novel post-transcriptional mechanism, involving the global translational repressor RsmA and a diffusible factor which is produced by strain CHA0. In addition, oxygen limitation and iron were identified as environmental stimuli which strongly induce the expression of the HCN genes, via a mechanism requiring the anaerobic regulator ANR. Based on this knowledge, an oxygen-sensing reporter derivative of CHA0 was constructed and used to monitor the distribution of low-oxygen habitats in soil.
Following introduction into field soil, an important fraction of P. fluorescens CHA0 may enter a viable but nonculturable (VBNC) state. The environmental factors and regulatory mechanisms involved are virtually unknown. It was therefore of interest to see whether regulatory genes of biocontrol metabolites be involved in the control of survival, persistence and the VBNC state. The VBNC state could be induced by low pH in vitro and by a combination of low oxygen availability and low redox potential in soil microcosms. In agricultural and forest bulk soils, strain survival and formation of VBNC cells was influenced by the global regulatory system GacA/GacS and the stress sigma factor RpoE, but not by the anaerobic regulator ANR. GacA-, GacS- or RpoE-deficient mutants formed higher numbers of VBNC cells, as compared with the wild-type CHA0. On plant roots, however, no such differences could be observed, suggesting that these regulators contribute to strain persistence only when environmental stress increases.
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