Distribution, diversity, and activity of antibiotic-producing Pseudomonas spp.
Bacteria of the genus Pseudomonas are potential biocontrol agents of plant diseases caused by various fungi and oomycetes. Antibiotic production is an important trait responsible for the activity of several Pseudomonas strains against plant pathogens. Despite the amount of information obtained during the past decades on biosynthesis and regulation of antibiotics, little is known about the distribution and diversity of antibiotic-producing Pseudomonas spp. in natural environments. Knowledge about the diversity of naturally occurring populations of antibiotic-producing Pseudomonas spp. could contribute to improve biological control by the identification of new strains that are ecologically more competent. The main objectives in this thesis were to study the distribution, diversity, and activity of antibiotic-producing Pseudomonas spp. in rhizosphere environments.In Chapter 1, an overview is given on detection, distribution and diversity of antibiotic-producing Pseudomonas spp. Special attention is paid to the biosynthesis and regulation of the antibiotics 2,4-diacetylphloroglucinol (2,4-DAPG), phenazines (PHZ), pyrrolnitrin (PRN), pyoluteorin (PLT), and biosurfactant antibiotics, which are subject of the experiments described in this thesis.In Chapter 2, the diversity, phylogenetic relationships, and frequency of Pseudomonas and Burkholderia species able to produce the antibiotics PRN or PLT were studied. Primers were developed from conserved sequences within the biosynthetic loci for each of the two antibiotics and allowed detection of multiple Pseudomonas and Burkholderia spp. that either produce PRN or PLT or both. Subsequent RFLP (Restriction Fragment Length Polymorphisms) analysis of the amplified pltC fragment showed no polymorphisms among PLT-producing Pseudomonas strains. Polymorphisms within the amplified prnD fragment, however, allowed the assessment of the diversity among PRN-producing Pseudomonas and Burkholderia spp. to a level similar to that obtained by RAPD (Random Amplified Polymorphic DNA) analysis. Phylogenetic analyses further revealed that the prn genes of B. pyrrocinia DSM10685 were more closely related to those of PRN-producing Pseudomonas strains, suggesting that lateral gene transfer may have occurred. PRN- and PLT-producing Pseudomonas and Burkholderia spp. were not detected on roots of wheat grown in five agricultural soils collected in the Netherlands, two of them being naturally suppressive to the take-all pathogen Gaeumannomyces gramminis var. tritici . These results suggested that these bacteria do not contribute to the natural suppressiveness found in Dutch take-all suppressive soils.Chapter 3 focused on the response regulator gene gacA in Pseudomonas species. This gene influences the production of several antibiotics in antagonistic Pseudomonas spp. We developed primers and a probe for the gacA gene of Pseudomonas species and sequenced gacA from ten Pseudomonas strains isolated from different plant-associated environments. PCR analysis and Southern hybridization showed that gacA is highly conserved within the genus Pseudomonas and indicated that gacA can be used as a complementary genetic marker for detection of Pseudomonas spp. in environmental samples. Phylogenetic relationships inferred from the newly sequenced gacA genes and gacA homologs present in the databases, showed that gacA may also serve as a marker for phylogenetic studies of Pseudomonas spp. and Gram-negative bacteria other than Pseudomonas . Estimations of nonsynonymous to synonymous substitution rates (Ka/Ks ratios) showed that purifying selection is acting on gacA , indicating that there is a selective pressure to avoid substitutions leading to functional changes in the GacA protein.Chapter 4 focused on the role of antibiotic-producing Pseudomonas spp. in Dutch take-all suppressive soils. Natural suppressiveness of soils to take-all disease of wheat, referred to as take-all decline (TAD), occurs worldwide. It has been postulated that different microbial genera and mechanisms are responsible for TAD in soils from different geographical regions. Based on population density studies and the use of antibiotic-deficient mutants, we demonstrated that fluorescent Pseudomonas spp. that produce 2,4-DAPG play a key role in the natural suppressiveness of two Dutch TAD soils. Our results showed that in addition to the physicochemically different TAD soils from Washington State (USA), 2,4-DAPG-producing fluorescent Pseudomonas spp. are also a key component of the natural suppressiveness found in Dutch TAD soils. Furthermore, it is the first time since the description of Dutch TAD soils, that at least part of the mechanisms and microorganisms operating in their suppressiveness are identified. In spite of similarities in population densitis and activity, 2,4-DAPG-producing Pseudomonas spp. found in the Dutch TAD soils are genotypically different from those found in TAD soils from Washington State.
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| Format: | Doctoral thesis biblioteca |
| Language: | English |
| Subjects: | antibiotics, biological control, distribution, diversity, plant pathogenic fungi, pseudomonas, antibiotica, biologische bestrijding, distributie, diversiteit, plantenziekteverwekkende schimmels, |
| Online Access: | https://research.wur.nl/en/publications/distribution-diversity-and-activity-of-antibiotic-producing-pseud |
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| Summary: | Bacteria of the genus Pseudomonas are potential biocontrol agents of plant diseases caused by various fungi and oomycetes. Antibiotic production is an important trait responsible for the activity of several Pseudomonas strains against plant pathogens. Despite the amount of information obtained during the past decades on biosynthesis and regulation of antibiotics, little is known about the distribution and diversity of antibiotic-producing Pseudomonas spp. in natural environments. Knowledge about the diversity of naturally occurring populations of antibiotic-producing Pseudomonas spp. could contribute to improve biological control by the identification of new strains that are ecologically more competent. The main objectives in this thesis were to study the distribution, diversity, and activity of antibiotic-producing Pseudomonas spp. in rhizosphere environments.In Chapter 1, an overview is given on detection, distribution and diversity of antibiotic-producing Pseudomonas spp. Special attention is paid to the biosynthesis and regulation of the antibiotics 2,4-diacetylphloroglucinol (2,4-DAPG), phenazines (PHZ), pyrrolnitrin (PRN), pyoluteorin (PLT), and biosurfactant antibiotics, which are subject of the experiments described in this thesis.In Chapter 2, the diversity, phylogenetic relationships, and frequency of Pseudomonas and Burkholderia species able to produce the antibiotics PRN or PLT were studied. Primers were developed from conserved sequences within the biosynthetic loci for each of the two antibiotics and allowed detection of multiple Pseudomonas and Burkholderia spp. that either produce PRN or PLT or both. Subsequent RFLP (Restriction Fragment Length Polymorphisms) analysis of the amplified pltC fragment showed no polymorphisms among PLT-producing Pseudomonas strains. Polymorphisms within the amplified prnD fragment, however, allowed the assessment of the diversity among PRN-producing Pseudomonas and Burkholderia spp. to a level similar to that obtained by RAPD (Random Amplified Polymorphic DNA) analysis. Phylogenetic analyses further revealed that the prn genes of B. pyrrocinia DSM10685 were more closely related to those of PRN-producing Pseudomonas strains, suggesting that lateral gene transfer may have occurred. PRN- and PLT-producing Pseudomonas and Burkholderia spp. were not detected on roots of wheat grown in five agricultural soils collected in the Netherlands, two of them being naturally suppressive to the take-all pathogen Gaeumannomyces gramminis var. tritici . These results suggested that these bacteria do not contribute to the natural suppressiveness found in Dutch take-all suppressive soils.Chapter 3 focused on the response regulator gene gacA in Pseudomonas species. This gene influences the production of several antibiotics in antagonistic Pseudomonas spp. We developed primers and a probe for the gacA gene of Pseudomonas species and sequenced gacA from ten Pseudomonas strains isolated from different plant-associated environments. PCR analysis and Southern hybridization showed that gacA is highly conserved within the genus Pseudomonas and indicated that gacA can be used as a complementary genetic marker for detection of Pseudomonas spp. in environmental samples. Phylogenetic relationships inferred from the newly sequenced gacA genes and gacA homologs present in the databases, showed that gacA may also serve as a marker for phylogenetic studies of Pseudomonas spp. and Gram-negative bacteria other than Pseudomonas . Estimations of nonsynonymous to synonymous substitution rates (Ka/Ks ratios) showed that purifying selection is acting on gacA , indicating that there is a selective pressure to avoid substitutions leading to functional changes in the GacA protein.Chapter 4 focused on the role of antibiotic-producing Pseudomonas spp. in Dutch take-all suppressive soils. Natural suppressiveness of soils to take-all disease of wheat, referred to as take-all decline (TAD), occurs worldwide. It has been postulated that different microbial genera and mechanisms are responsible for TAD in soils from different geographical regions. Based on population density studies and the use of antibiotic-deficient mutants, we demonstrated that fluorescent Pseudomonas spp. that produce 2,4-DAPG play a key role in the natural suppressiveness of two Dutch TAD soils. Our results showed that in addition to the physicochemically different TAD soils from Washington State (USA), 2,4-DAPG-producing fluorescent Pseudomonas spp. are also a key component of the natural suppressiveness found in Dutch TAD soils. Furthermore, it is the first time since the description of Dutch TAD soils, that at least part of the mechanisms and microorganisms operating in their suppressiveness are identified. In spite of similarities in population densitis and activity, 2,4-DAPG-producing Pseudomonas spp. found in the Dutch TAD soils are genotypically different from those found in TAD soils from Washington State. |
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