plant surface microbiology.pdf

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Thomas F.C. Chin-A-Woeng et al.
and wilted. Cool soil temperatures favour the disease. The fungus lives over
winter and survives for many years in the soil as chlamydospores. Long distance
spread is caused by transplants and by soil on farm machinery. Spores
are air-borne in greenhouses. The disease causes losses in tomato cropping in
agricultural fields, glasshouses, and hydroponic growth. The fungus forms a
problem for hydroponic tomato growth in glasshouses in the Netherlands. In
the southwest of Florida it is one of the most important tomato diseases and
it is emerging at new locations in the United States. Until now only partially
resistant varieties have been identified and preplant fumigation with, e.g.
methylbromide, which is a management practice often used for many soilborne
diseases, does not completely control the fungus. This practice is also
deprecated in view of sustainable agricultural practices. Hence, an efficient
way to control the disease is important.
An alternative to chemical control of plant diseases is the use of bacteria
(biocontrol). They have the potential to displace or antagonise phytopathogenic
or deleterious microorganisms in the rhizosphere. Biocontrol bacteria
also produce chemicals, but these are degradable and only produced in low
amounts at targeted locations. The latter approach fits well in the worldwide
strategy to grow healthier plants in a sustainable way and, therefore produce
high quality food.
To use biocontrol strains efficiently, the molecular interactions between
plant, biocontrol agent, pathogen and their environment need to be understood.
Genetic engineering is an important tool in helping us to define the
molecular basis of pathogenicity and is also useful in helping us to identify
the mechanisms in the action of biocontrol strains. Molecular genetic modification
of microorganisms requires the development of plasmid-mediated
transformation systems that include: (1) introduction of exogenous DNA into
recipient cells, (2) expression of transformed genes, and (3) stable maintenance
and replication of the inserted DNA leading to expression of the
desired phenotypic trait. In this chapter, a practical approach to the analysis
of biocontrol strains including the isolation, testing, and tagging of these
strains, and transformation systems for pathogenic fungi to express reporter
genes to track and visualise them in the rhizosphere, are discussed in relation
to the pathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici.
3 Selection of Antagonistic Strains
3.1 Selection of Antagonistic Pseudomonas and Bacillus sp.
Pseudomonas and Bacillus species constitute, together with Streptomyces
species, a substantial fraction of the bacterial community isolated from the
rhizosphere. Their presence is sometimes correlated with disease suppression.
These beneficial bacteria can be exploited as biological pesticides to be