Download II International Symposium on Tomato Diseases

Biological Control of Tomato Foot and Root Rot Caused by Fusarium
oxysporum f.sp. radicis-lycopersici by Pseudomonas Bacteria
F. Kamilova, S. Validov and B. Lugtenberg
Leiden University, Institute of Biology
Wassenaarseweg 64, 2333 AL Leiden
The Netherlands
Keywords: biocontrol, antibiosis, induced systemic resistance, competition for nutrients
and niches, Pseudomonas, tomato foot and root rot, Fusarium oxysporum f.sp. radicislycopersici
Abstract
Rhizobacteria are a natural and most suitable source for the isolation of
potential microbiological control agents that can protect plants from soilborne
pathogens and consequently improve crop quality and yield. The beneficial effect of
such bacteria on plant health depends in many cases on their ability to aggressively
colonize the rhizosphere and compete with the indigenous, including pathogenic,
microflora for nutrients and niches on the plant root. Bacterial strains Pseudomonas
chlororaphis PCL1391 and P. fluorescens WCS365 employ antibiosis and induced
systemic resistance, respectively, to control tomato foot and root rot (TFRR) caused by
phytopathogenic fungus Fusarium oxysporum f.sp. radicis-lycopersici (Forl). For the
selection of biocontrol bacteria acting via the mechanism “competition for nutrients
and niches” we have developed an enrichment method for enhanced tomato root tip
colonizers, starting from a crude mixture of rhizobacteria coated on the seed, using a
sterile quartz sand/plant nutrient solution gnotobiotic system. As a result of this
enrichment procedure, and subsequent tests on competitive tomato root tip
colonization, the strongly competitive biocontrol strains P. fluorescens PCL1751 and P.
putida PCL1760 were isolated. Both strains effectively suppress TFRR under soil and
hydroponic cultivation conditions.
INTRODUCTION
Successful management of plant diseases caused by phytopathogenic organisms
includes many factors such as development of resistant plant cultivars, application of
chemical pesticides, crop rotation, improvements of the qualities of the growth substrate
as well as rational use of irrigation. Biological control is one of the important components
of integrated pest control and sustainable agricultural practice. Biocontrol of plant
pathogens is based on application of their natural enemies such as beneficial antagonistic
microorganisms producing antimicrobial components, or microbes which can induce
systemic resistance (ISR) by triggering defense mechanisms in the plant, or microbes
which can out compete pathogens by faster and more efficient occupation of niches and
consequently quicker consumption of nutrients provided by plants. In this article we
describe a few plant growth promoting rhizosphere pseudomonads which use different
mechanisms of control of TFRR caused by the phytopathogenic fungus Forl.
P. chlororaphis PCL1391, a Phenazine-Producing Biocontrol Strain Acting through
Antibiosis
Screening of a large collection of tomato rhizosphere isolates resulted in selection
of strain P. chlororaphis PCL1391, which has a strong in vitro antagonistic activity
against Forl as well as against many other phytopathogenic fungi. This strain produces the
antibiotic phenazine-1-carboximide (PCN), the volatile HCN, and enzymes such as
protease and chitinase (Chin-A-Woeng et al., 1998). P. chlororaphis PCL1391 shows
stable efficient biocontrol of TFRR in soil. It appeared that PCN is a crucial factor
contributing to this activity. Mutants impaired in the biosynthesis of PCN are lacking
biocontrol activity (Chin-A-Woeng et al., 1998). In addition to the production of PCN,
Proc. IInd Intl. Symposium on Tomato Diseases
Eds.: H. Saygili et al.
Acta Hort. 808, ISHS 2009
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also efficient colonization of tomato roots is crucial for the biocontrol ability of PCL1391.
Motility-impaired mutants that are one thousand-fold impaired in competitive tomato root
tip colonization cannot control TFRR, although the level of production of PCN is the
same as that of the parental strain (Chin-A-Woeng et al., 2000). This finding indicates that
root colonization by antagonistic strain P. chlororaphis PCL1391 acts as the delivery
mechanism of antifungal compounds along the root to control phytopathogens.
P. fluorescens WCS365 Induces Systemic Resistance against TFRR
P. fluorescens WCS365 was isolated by Geels and Schippers (1983) and causes a
reduction of the narrow rotation effect of potato. This strain does not inhibit growth of
phytopathogenic fungi in vitro, but has a profound suppressive effect on TFRR
(Lugtenberg et al., 2001; Kamilova et al., 2005). P. fluorescens WCS365 was used as a
model strain to study bacterial competitive colonization traits because of its excellent
colonization of the root systems of various plants (Lugtenberg et al., 2001). In contrast to
P. chlororaphis PCL1391, colonization mutants of P. fluorescens WCS365 are not or
hardly impaired in biological control of TFRR (Lugtenberg et al., 2001). Apparently a
high efficiency of colonization of the complete root system is not important for the
biocontrol ability of this bacterium. This finding excluded the notion that WCS365 acts
through the mechanism “competition for nutrients and niches”. We subsequently tested
whether WCS365 induces systemic resistance (ISR) in the plant. Indeed when WCS365 is
used for inoculation of tomato plants with a split-root system, in which the beneficial and
pathogenic strains are spatially separated, this strain controls TFRR as good as in regular
biocontrol experiments with an intact root system (Kamilova et al., 2005).
P. fluorescens PCL 1751 and P. putida PCL1760 Control TFRR via Competition with
the Pathogen for Nutrients and Niches
1. Selection of Biocontrol Strains by Enrichment of Enhanced Tomato Root Tip
Colonizers. In a gnotobiotic system (Simons et al., 1996) sterile tomato seedlings are
grown in quartz sand or in stonewool moistured with plant nutrient solution. After
bacterization of seeds with a mixture of thousands of rhizobacteria, and subsequent
growth of the plant, good root colonizers move along the root towards the root tip. The
distribution of individual strains along the root is a result of at least two characteristics:
their abilities to swim and to utilize root exudate nutrients. Those strains which grow fast
on root exudate and can move fast along the root will colonize the whole root system and
will be the first to reach the root tip. They will be excellent candidates to compete with
pathogens for nutrients and niches along the root. Based on this assumption we applied an
enrichment strategy for the isolation of the best tomato root colonizers from a crude
mixture of rhizobacteria. The majority of the resulting isolates did not show antagonistic
activity against Forl in vitro. After identification of the best colonizing isolates, only
strains belonging to safety group 1 (Anonymous, 1998) were compared with P.
fluorescens strain WCS365 or P. fluorescens PCL 1285, a Km-resistant derivative of P.
fluorescens WCS365, as a reference strain, in a competitive tomato root tip colonization
assay. Those isolates that appeared as good as or better than the reference root colonizing
strain were tested in biocontrol experiments. As a result, the excellent tomato root
colonizing strains with strong biocontrol ability P. fluorescens PCL 1751 (Kamilova et al.,
2005) and P. putida PCL1760 (Validov et al., 2007) were selected. To our knowledge this
is the only published method for direct selection of biocontrol strains.
2. Selected Biocontrol Strains Suppress Forl by Competition for Nutrients and
Niches. Growth tests of the enhanced colonizers in tomato root exudate revealed that
these strains out compete randomly chosen rhizobacteria and suppress the development of
Forl propagules. These results indicate that the selected bacteria are highly competitive
for the nutrients provided by the plant root. These data are in agreement with results of
experiments with P. putida PCL1760 mutants impaired in the uptake of dicarboxylic
acids, major exudate compounds. These mutants are significantly worse in the control of
TFRR than the parental strain. Motility mutants of P. fluorescens PCL1751 are also
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inefficient in biocontrol. Based on these results we claim that the selected strains control
TFRR using the mechanism “competition for nutrients and niches”.
CONCLUSIONS
Many bacterial strains are described as biocontrol agents (see reviews Tomashow
and Weller, 1995; Bloemberg and Lugtenberg, 2001; Defago and Haas, 2005). Most of
this strains act via antibiosis. Antagonistic strains can be isolated by means of a rather
simple and quick screening procedure in vitro. But practice shows that very often
antagonistic strains fail in field and greenhouse experiments or show unstable results
(Handelsman and Stabb, 1996). One of the reasons is that production of antimicrobial
metabolites is regulated in a very complex way. Due to this complexity, small changes in
pH, temperature, salinity and other environmental factors can cause a negative effect on
the biosynthesis of antimicrobials and consequently on biocontrol ability of these strains
(Duffy and Defago, 1999; van Rij et al., 2004). Moreover, production of antibiotics by a
potential biocontrol agent is an extra burden for its registration as a product. For strains
which do not produce antibiotics and act through the mechanisms ISR or competition for
nutrients and niches, registration would not be a problem. However, screening of strains
for ISR is a very laborious task and direct selection is not possible. From this point of
view biocontrol bacteria using the mechanism “competition for nutrients and niches” have
a big advantage: their direct selection is easy and can be adjusted to the required plant
species, growth substrate and environmental conditions.
ACKNOWLEDGEMENTS
This presentation was supported by the Technology Foundation Stichting voor de
Technische Wetenschappen, Applied Science Division of the Nederlandse Organisatie
voor Wetenschappelijk Onderzoek, and the Technology Programme of the Ministry of
Economic Affairs (LBI.5884).
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