fate and effect of r-DNA biopesticides when released into the environment
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:
to view graphic (pdf)
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.
to view graphic (pdf)
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.
root colonization by P. fluorescens WCS365 and P. chlororaphis
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.
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.
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
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.
November 1998 November 2000
Leiden University (NL)
Katholieke Universiteit Leuven
University College Cork (IE)
Irish Sugar plc.
University of Oxford (UK)