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EC-sponsored Research on Safety of Genetically Modified Organisms - A Review of Results
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image Predicting fate and effect of r-DNA biopesticides when released into the environment

Background and objectives (1)

There are various traits which contribute to the ability of a bacterium to control plant diseases caused by fungi. They are the production of antifungal factors (AFFs) such as 2,4-diacetyl phloroglucinol (DAPG) and phenazine-1-carboxamide (PCN); colonisation of the rhizosphere to deliver the AFFs at the right time and at the right sites along the root system; and what is called Induced Systemic Resistance (ISR), i.e. resistance of the plant induced by the beneficial bacterium. Promoters have been found which are specifically induced by exudate from the plant or in the rhizosphere. To understand their action, exudate analyses are required.

The genetic and functional mechanisms involved in microbial control of plant diseases are being studied to predict fate and effect of recombinant DNA biopesticides in the environment. European crop plants, sugar beet, tomato and wheat, are being used. Recombinant DNA biopesticides will be constructed with the aim of making more potent and more predictable biopesticides. By bringing AFF production under control of inducible promoters, the required amount of biodegradable AFF will be produced only at the site where it is required, namely in the rhizosphere. The results will contribute to a database for recombinant DNA biopesticides, which will underpin legislation.

(1) This project was a direct follow-on from EC project: BIO2-CT93-0196.

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Approach and methodology

Bacteria are being constructed which combine two of the three useful traits: production of DAPG and PCN; and ISR. The production of DAPG and PCN will be placed under the control of strong promoters or of exudate-induced or rhizosphere-induced promoters. A second topic is to test whether the mechanism behind the most important colonisation trait, bacterial motility, is in fact chemotaxis. This will lead to an analysis of whether colonisation genes can be used to improve colonisation. The efficacy and safety of the newly constructed strains will be tested in microcosms and in the greenhouse under semi-industrial conditions. Evaluation of the results in terms of biosafety will include an analysis of whether our mechanistic studies have provided sufficient know-how to predict fate and biosafety of the new constructs.

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Main findings and outcome

Recombinant DNA strains with greatly increased DAPG and PCN production have been constructed. Potentially valuable inducible promoters have been isolated. The colonisation traits and inducible promoters tested so far are not host-specific.

CheA mutants (cheA is a chemotaxis gene) are strongly impaired in root tip colonisation and colonisation can be improved by the presence of multiple copies of the sss/xerC gene. The role of colonisation in biocontrol was rigorously tested for the first time: it appeared to be an absolute requirement for biocontrol based on PCN production but less important when biocontrol was based on ISR. A patent based on PCN-based biocontrol has been submitted.

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Tomato root colonization by P. fluorescens WCS365 and P. chlororaphis PCL1391.
Germinated seedlings were inoculated with a 1:1 mixture of P. fluorescens WCS365 harbouring pMP4662 (DsRed) or pMP4641 (ecfp) (Bloemberg et al. submitted). P. chlororaphis PCL1391 harbouring pMP4655 (egfp) or pMP4658 (eyfp) (Bloemberg et al. submitted). 10 cm long root samples were inspected after 7 days of growth in the gnotobiotic system (Simons et al. 1996). Panels A and B show examples of the upper part of the tomato root system or root base (first 1-3 cm). Panels C and D show examples of the middle part of the root system (3-7 cm). Panel E represents the root hairs, and panel F is a close up of two micro-colonies on the middle part (3.5 cm) of the tomato root system. Panel A, D and E are a combination of DsRed-marked WCS365 and EGFP-marked PCL1391 cells. Panel B, C and F are a combination of ECFP-marked WCS365 and EYFP-marked PCL1391 cells. In all pictures WCS365 cells are depicted in red and PCL1391 cells are depicted in green. The sizebars in all panels represent 10 µm.


Conclusions

The results obtained concerning the feasibility of generating new bacterial strains for protecting crops against disease are very promising at this early stage of the work. The strains are currently being tested under semi-industrial conditions. Tests under true industrial conditions and complete safety analysis will follow.

 

Major publications

Chin-A-Woeng T.F., Bloemberg G.V., Mulders I.H., Dekkers L.C. and Lugtenberg B.J.J., “Root colonisation by phenazine-1-carboxamide-producing bacterium Pseudomonas chlororaphis PCL1391 is essential for biocontrol of tomato foot and root rot”.
Mol. Plant-Microbe Interact., 13, 2000, pp. 1340-1345.

Dekkers L.C., Mulders I.H.M., Phoelich C.C., Chin-A-Woeng T.F.C., Wijfjes A.H.M. and Lugtenberg B.J.J., “The sss colonization gene of the tomato-Fusarium oxysporum f.sp. radicis-lycopersici biocontrol strain Pseudomonas fluorescens WCS365 can improve root colonization of other wild type Pseudomonas bacteria”.
Mol. Plant-Microbe Interact., 13, 2000, pp. 1177-1183.

Lugtenberg B.J.J. and Dekkers L.C., “What makes Pseudomonas bacteria rhizosphere competent?”.
Environ. Microbiol., 1, 1999, p. 9.

Lugtenberg B.J.J., Dekkers L.C., Bansraj M., Bloemberg G.V., Camacho M., Chin-A-Woeng T., van den Hondel K., Kravchenko L., Kuiper I., Lagopodi A.L., Mulders I., Phoelich C., Ram A., Tikhonovich I., Tuinman S., Wijffelman C. and Wijfjes A., “Pseudomonas genes and traits involved in tomato root colonization”, in Biology of Plant-Microbe Interactions, Vol. 2, P.J.G.M. de Wit, T. Bisseling and W.J. Stiekema (eds.), International Society for MPMI, 2000, pp. 324-330.

Rainey P.B., “Adaptation of Pseudomonas fluorescens to the plant rhizosphere”.
Environ. Microbiol., 1, 1999, p. 243.
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imageResearch project
 

Contract number
BIO4-CT98-0254

Period
November 1998 – November 2000

Coordinator
B.J.J. Lugtenberg
Leiden University (NL)

Project website address
http://rulbim.leidenuniv.nl/
euproject/

 
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Partners


J. Vanderleyden
Katholieke Universiteit Leuven
Heverlee (BE)

F. O'Gara
University College Cork (IE)

D. Grogan
Irish Sugar plc.
Carlow (IE)

P. Rainey
University of Oxford (UK)

 
 
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