Distribution, diversity, and activity of antibiotic-producing Pseudomonas spp.

J.T. de Souza

Research output: Thesisinternal PhD, WU

Abstract

<strong></strong><p>Bacteria of the genus <em>Pseudomonas</em> 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 <em>Pseudomonas</em> 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 <em>Pseudomonas</em> spp. in natural environments. Knowledge about the diversity of naturally occurring populations of antibiotic-producing <em>Pseudomonas</em> 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 <em>Pseudomonas</em> spp. in rhizosphere environments.</p><p>In <strong>Chapter 1,</strong> an overview is given on detection, distribution and diversity of antibiotic-producing <em>Pseudomonas</em> 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.</p><p>In <strong>Chapter 2,</strong> the diversity, phylogenetic relationships, and frequency of <em>Pseudomonas</em> and <em>Burkholderia</em> 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 <em>Pseudomonas</em> and <em>Burkholderia</em> spp. that either produce PRN or PLT or both. Subsequent RFLP (Restriction Fragment Length Polymorphisms) analysis of the amplified <em>pltC</em> fragment showed no polymorphisms among PLT-producing <em>Pseudomonas</em> strains. Polymorphisms within the amplified <em>prnD</em> fragment, however, allowed the assessment of the diversity among PRN-producing <em>Pseudomonas</em> and <em>Burkholderia</em> spp. to a level similar to that obtained by RAPD (Random Amplified Polymorphic DNA) analysis. Phylogenetic analyses further revealed that the <em>prn</em> genes of <em>B. pyrrocinia</em> DSM10685 were more closely related to those of PRN-producing <em>Pseudomonas</em> strains, suggesting that lateral gene transfer may have occurred. PRN- and PLT-producing <em>Pseudomonas</em> and <em>Burkholderia</em> 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 <em>Gaeumannomyces gramminis</em> var. <em>tritici</em> . These results suggested that these bacteria do not contribute to the natural suppressiveness found in Dutch take-all suppressive soils.</p><strong><p>Chapter 3</strong> focused on the response regulator gene <em>gacA</em> in <em>Pseudomonas</em> species. This gene influences the production of several antibiotics in antagonistic <em>Pseudomonas</em> spp. We developed primers and a probe for the <em>gacA</em> gene of <em>Pseudomonas</em> species and sequenced <em>gacA</em> from ten <em>Pseudomonas</em> strains isolated from different plant-associated environments. PCR analysis and Southern hybridization showed that <em>gacA</em> is highly conserved within the genus <em>Pseudomonas</em> and indicated that <em>gacA</em> can be used as a complementary genetic marker for detection of <em>Pseudomonas</em> spp. in environmental samples. Phylogenetic relationships inferred from the newly sequenced <em>gacA</em> genes and <em>gacA</em> homologs present in the databases, showed that <em>gacA</em> may also serve as a marker for phylogenetic studies of <em>Pseudomonas</em> spp. and Gram-negative bacteria other than <em>Pseudomonas</em> . Estimations of nonsynonymous to synonymous substitution rates (Ka/Ks ratios) showed that purifying selection is acting on <em>gacA</em> , indicating that there is a selective pressure to avoid substitutions leading to functional changes in the GacA protein.</p><strong><p>Chapter 4</strong> focused on the role of antibiotic-producing <em>Pseudomonas</em> 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 <em>Pseudomonas</em> 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 <em>Pseudomonas</em> 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 <em>Pseudomonas</em> spp. found in the Dutch TAD soils are genotypically different from those found in TAD soils from Washington State.
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
Supervisors/Advisors
  • de Wit, Pierre, Promotor
  • Raaijmakers, J.M., Promotor
Award date1 Oct 2002
Place of PublicationS.l.
Print ISBNs9789058087270
Publication statusPublished - 2002

Keywords

  • pseudomonas
  • antibiotics
  • distribution
  • diversity
  • biological control
  • plant pathogenic fungi

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