chemicals in crop pest and
Institute of Molecular Plant Sciences,
Leiden University (NL)
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
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
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
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.