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EC-sponsored Research on Safety of Genetically Modified Organisms - A Review of Results
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Plant microbes
Microbes that help
plants to grow


Introduction


M.P. Nuti,
Dipartimento di Chimica e Biotecnologie Agrarie,
Università di Pisa (IT)

Plant growth promoting micro-organisms are very important in agriculture. They include bacteria and microfungi which can have either direct beneficial effects on crops, for example, by producing substances which help protect plants from pathogens or increase nutrient uptake in roots, or indirect effects, for example, by enhancing nutrient mobility in soil. Some of them can contribute to pollution bioremediation through their ability to degrade xenobiotics.

Because of the relatively widespread use of these microbes in agriculture and the possibility to improve useful traits via both conventional genetics and rDNA techniques, it was important to develop tools to expand our knowledge on how to track them in the environment, and to assess their fate, particularly when destined for large-scale environmental releases. The experimental approach was necessarily multidisciplinary, starting from a better definition of the biodiversity of selected microbial groups, then improving methods to identify strains and species, and developing tagging strategies and environmentally friendly methods for monitoring genetically modified microbial inoculants.
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Identification of microbial isolates can be achieved via phenotypic traits as well as by molecular fingerprints. In the former, samples from colonies are thermally degraded under vacuum (pyrolysis), and the fragments produced are ionised and analysed by mass spectrometry. Strain or species fingerprinting is also possible using arbitrary primers annealing at low stringency to the template DNA from the regions between closely spaced primers. In addition, high-resolution automated microbial identification methods were developed, allowing reliable and reproducible identification of several bacterial species. Other genomic fingerprinting techniques have been developed or adapted, including AFLP, rep-PCR analysis of DNA profiles by pulsed-field gel electrophoresis in a contour-clamped homogeneous electric field (PFGE-CHEF). All the above methods successfully monitored bacteria which interact directly with plant roots, i.e. rhizobia-forming nitrogen-fixing nodules, Azospirilli, and beneficial Pseudomonas. Some techniques can also be adapted to vesicular-arbuscular mycorrhizal fungi.

Particular emphasis was devoted to developing marker genes other than those for resistance to antibiotics, and catabolic genes such as lac SY were used as appropriate reporter genes for Rhizobium and Bradyrhizobium. The sensitivity of detection can be improved substantially when strong promoters are used, or by replacing the lac promoter with inducible promoters responding to unrelated environmental signals. Among the marker systems, gus and lux were also used; however, it appears that gfp has greater potential and is easier to use.
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By using the above techiniques, alone or in combination, the fate of genetically modified microbial inoculants was assessed upon release into the environment. The following results were obtained:

- In general, GMM behave in a similar manner with respect to the non-modified parental strains;

- Some agronomic factors, such as the depth of water-table, can affect the survival of introduced inoculant;

- The persistence of Rhizobium, Azospirillum and Pseudomonas, monitored over several years, and different crop cycles, remained around 102 - 103 per gram of soil for rhizobia, and below detection limit for the other two;

- The genetically modified plants and microbial inoculants used do not have a negative impact on the indigenous microbial population (soil microbial biomass, selected soil micro-organisms, microbial activities);

- Gene transfer from GM bacteria to soil microbiota is possible, although in some cases the occurence appeared to be rare and often limited to non-functional DNA fragments; the time-scale of the genetic flux is unclear; and

- New information is now available on bacterial population behaviour and factors affecting the signalling among members (quorum-sensing); new information is also available on the viable-nonculturable state of microbes, as opposed to the viable and culturable state. Factors such as oxygen or nitrate can affect modulation between the two states.

 
 
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