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EC-sponsored Research on Safety of Genetically Modified Organisms - A Review of Results
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Micro-organisms replace
chemicals in crop pest and
disease control


B.J.J. Lugtenberg,
Institute of Molecular Plant Sciences,
Leiden University (NL)

Chemical pesticides are generally cost-effective in controlling pests and diseases and, as a consequence, have become an integral part of modern agriculture. However, since many of these chemicals are also implicated in ecological, environmental and human health problems there is an obvious need to find effective alternative approaches with minimal deleterious effects. Natural and genetically modified organisms (GMOs) provide such an alternative but this raises the new question of whether biological risks are associated with their use. The ideal microbial pesticide is sufficiently active to achieve control of the disease but should die soon afterwards. Such a trait would be good for biosafety as well as for the biopesticide producer who could then sell his product again the next season. Many traits concerning the efficacy of microbial pesticides as biocontrol agents appear also to be important for biological safety and vice versa. Hence, the research covered in this chapter, which deals with three types of biocontrol micro-organisms, focuses both on GMO biosafety and efficacy issues.

Biocontrol of model fungal diseases of plants through the application of beneficial Pseudomonas bacteria was the subject of four projects (BIO2-CT93-0196, BIO4-CT98-0254, BIO4-CT98-0283 and BIOT-CT91-0288). The diseases were tomato foot and root rot caused by the fungus Fusarium oxysporum f.sp. radicis lycopersici, damping off of sugar beet, and take-all disease of wheat, which are caused by the fungus Pythium ultimum.

Two traits contribute to the beneficial effects of the bacterial control agent. First, production of a (biodegradable) anti-fungal metabolite (AFM), such as phenazine-1-carboxamide (PCN) or 2,4 diacetyl phloroglucinol (Phl). Another mechanism, induced systemic resistance (ISR), the action mechanism of which is poorly understood, triggers the plant to respond faster and more aggressively towards pathogen attack. Second, root colonization by the beneficial microbe occurs as the delivery system of the AFM producing cells.

With reference to efficacy in the use of GMO Pseudomonas biocontrol strains, the objectives were increased AFM production and enhanced colonization. Using the mutant approach for PCN and Phl it appeared that increased AFM production could improve efficacy. Combining various mechanisms of biocontrol in one strain results in improved biocontrol in some cases but in decreased biocontrol in others, presumably through mutual interference of biosynthetic pathways. Production of a high level of AFMs poses a physiological burden on the producing cell and results in a slower growth rate and, consequently, in a less competitive strain. Using rhizosphere- or exudate-inducible promoters, the production of AFMs can be limited to the plant root, the only site where AFM is needed. This ensures minimal loss of energy, and therefore of competitive force, from the producing microbe. Improving the root colonising ability of the biocontrol agent appeared to be a realistic goal, and by using colonization-defective mutants various colonization traits have been identified; transgenic col(onization) genes enhanced the colonization ability of wild type Pseudomonas strains.

In parallel, the projects also tackled the biosafety considerations of the modified microbes. First, reporter genes such as lacZ or gfp are incorporated in the biocontrol strain to allow optimal tracking of the GMOs. Pseudomonas-based biocontrol strains persist for a limited period in soil and in rhizospheres. However, associated DNA persists longer. Successful containment strategies have been designed. In order to produce AFM at the right time and place (the root), exudate-induced promoters and rhizosphere-induced promoters have been isolated and characterized. The use of these promoters to control AFM production would guarantee minimal negative effects on indigenous beneficial microbes. If a biocontrol strain could only colonize a limited number of plants, crop rotation could be used to limit its life-span. Despite analysis of the host range of many colonization traits, none has been found so far that is not active on a particular host plant. The risk of killing indigenous beneficial microbes will be treated in chapter 4 on Plant Growth Promoting Micro-organisms. A microbe which is unable to utilise a major exudate component is unable to compete with indigenous microbes.

In consideration of the influence of environmental conditions on the behaviour of the GMO, it appears that oxygen concentration, and the presence of ions such as Fe3+ and Zn2+ and of organic compounds such as glycerol, dramatically influence the levels of AFM production, and therefore the growth rate and competitive abilities of biocontrol strains. Furthermore, promoter probe insertions have been used to identify genes responsive to environmental triggers such as exudate, the rhizosphere and soil conditions. One promoter, which was found to react to the presence of the exudate component proline, was studied in detail. Introduced Pseudomonas species respond quickly to nutrient scarcity in soil by producing stress-resistant cells. This occurred via the programmed induction of a myriad of stress-responsive genes. A mathematical model was developed to describe the dynamics of bacteria introduced in soil. Since environmental conditions strongly affect AFM production, better predictability is achieved by bringing AFM production under control of chosen promoters. Finally, combining biocontrol traits of several strains into one cell sometimes led to enhanced biocontrol. However, in a number of cases the results were negative, presumably because of metabolic interference of biosynthetic pathways.

In conclusion, it is clear that by constructing GMOs, biocontrol Pseudomonas strains with enhanced efficacy can be obtained. They live for a limited period of time in the soil and the rhizosphere and their life-span can be decreased by genetic containment strategies. A promising approach is the use of promoters which restrict AFM production to where it is needed.

As for future research on biocontrol Pseudomonas strains, the following topics are recommended. The use of gfp (green fluorescent protein)-labelled bacteria to study the behaviour of beneficial and pathogenic microbes, and their interactions, in the rhizosphere in a non-invasive way. The use of functional genomics (transcriptomics, proteomics) of beneficial microbes in order to enhance strain predictability. For example, to identify cascades of genes which are expressed in the seed and in the rhizosphere and to study their function, to produce chips dedicated to aspects relevant to biosafety, comparative genomics of biocontrol strains, to analyse the molecular basis of interference in attempts to produce two AFMs in one strain, and to understand the influence of environmental factors such as exudate compounds and metal ions on expression of relevant traits. Research should also aim to enrich strains with enhanced colonizing ability, examine molecular mechanisms of microbe-fungus interaction in the rhizosphere and to determine exudate composition and its role in microbe behaviour.

Another biocontrol agent studied is the endospore-forming bacterium Bacillus thuringiensis (Bt) which produces a pesticidal toxin commonly used to control insect larvae on commercially important plant species. This Bt toxin can bind on clays. Binding reduces the biodegradation of the toxin but does not eliminate toxicity to insect larvae. In the study reported here (IC18-CT97-0135), potential effects on tropical soils and water of Bt toxin produced by GMOs is evaluated, because if over-produced Bt can accumulate in the environment. The results indicate that adsorbed toxin has a higher toxicity than the free form, possibly because it escapes proteolysis. No negative effects against non-target organisms such as soil microbiota and earthworms were detected.

The third type of biocontrol examined concerns Baculoviruses which are pathogens for particular insects and which are being used as biological control agents of insect pests as alternatives to chemical insecticides. They cause no hazards to beneficial insects, animals or plants. The value of wild-type baculoviruses as sprayable, environmentally safe, biopesticides has been well established. The major drawback for the commercial use of wild-type baculoviruses is that they take several days to kill the target insect and during this time the insect can continue to damage the crop. Certain genetically modified viruses are assumed to stop feeding earlier and to reduce spreading of the virus from the insect cadaver. This would enhance efficacy and, at the same time, maintain and improve their safety.

In the projects reported here (BAP 0192/0201, BAP 0415/0416 and BIOT-CT91-0291), GM Baculoviruses were constructed with reduced ability, in comparison with the wild type, to spread from the dead caterpillar, and exhibiting a host range unaffected by the genetic modification.

In conclusion, these studies show that biocontrol micro-organisms can be modified to enhance efficacy, their fate can be followed and it is clear that they die. On the basis of more fundamental research, a start has been made on predicting their behaviour in the environment. Research lines which should be pursued to enhance their predictability have been indicated. It seems reasonable to forecast that in the next decade many biocontrol micro-organisms will be developed which are competitive with chemical pesticides in terms of efficacy but which are safer and whose effects are more predictable.

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