Research project C3/013 (Research action C3)
Context
The genus Pseudomonas has been a dumping ground for respiratory, Gram negative rods, motile by polar flagellae. As a consequence of this vague definition, the genus encompassed a number of species that turned out to be phylogenetically very diverse. Indeed, the introduction of 16S rRNA based similarity studies revealed that at least 5 different rRNA groups with separate genus or even family status existed within the original genus Pseudomonas. The genus Pseudomonas that is considered as the ‘authentic genus Pseudomonas’ is limited to rRNA group 1 species and contains the type species P. aeruginosa. However, on itself this authentic genus Pseudomonas is still very heterogeneous and contains various groups of species that are not very well delineated. One of these, the so-called group of fluorescent pseudomonads is composed of a continuously growing number of species of great importance. Indeed, some of them are plant or human (nosocomial infections) pathogens while others exhibit antifungal activities with biocontrol possibilities. These fluorescent species are often only weakly described and the delineation at the species level is far from clear. Hence identifications of new isolates are unreliable.
Description of the project
This project aims at improving the taxonomic frame of this group of fluorescent pseudomonads by applying molecular techniques in a comparative analysis of housekeeping genes such as oprI, rpoD, gyrA, gyrB etc but also 16S rRNA. The resulting individual and combined phylogenetic trees will allow to delineating groups of which the taxonomic status needs to be evaluated. It is known from the 16S rDNA sequence analysis that this method does not allow discrimination at the species level, but it is also known that sequences of at least some of the other genes do discriminate at the species level. Apart from this sequence analysis, FAME analyses of methylated fatty acids and SDS-PAGE profiling of cell proteins will support the sequence-based taxon delineation and will also provide preliminary identifications retrieved from commercialized and in house (Laboratory for Microbiology) databases. Pyoverdine profiling, a technique known to allow discrimination at a very fine taxonomic level will also be applied.
It is to be expected that new taxa will be discovered and reference strains of these freshly isolated fluorescent pseudomonads will be deposited in the BCCM/LMG bacteria culture collection. In a first preliminary step to introduce new molecular identification tools for this group of bacteria, an extensive search of the newly determined sequences will be undertaken to retrieve specific sub-sequences that may be applied for PCR-based identification tests. It is however to be expected that the evaluation of these subsequences will not be possible in the frame of this project. Finally, additional tests for plantpathogenic characteristics and/or eventual production of new secondary metabolites will be performed merely on the VUB collection of isolates.
The following Work packages (WPs) and Tasks are defined:
WP 1: Molecular and phenotypic characterization of fluorescent pseudomonads – Phylogenetic analysis and identification
Task 1: Molecular characterization (oprI sequencing)
Task 2: Molecular analysis – multilocus sequence analysis
Task 3: Chemotaxonomic characterisation via SDS-PAGE of whole cell protein profiles
Task 4: Phenotypic characterization (siderotyping)
Task 5: Individual and combined numerical analysis of the data with the BioNumerics software
WP 2: Detection of strains producing secondary metabolites active against phytopathogens
Task 1: In vitro screening
WP 3: Detection of strains with phytopathogenic potential
Task 1: Inoculation of Belgian endives and Roman lettuce
The research results expected from this work concern:
1. Improved taxonomic frame for the genus Pseudomonas in particular the fluorescent
pseudomonads
2. Description of new species
3. Molecular identification tool
4. Valorisation of BCCM/LMG Bacteria Collection
5. Detection of new bio-active metabolites
6. Evaluation of plant pathogenic potential of environmental isolates
Partners
Partner 1: BCCM/LMG, Ghent University
Promotor: Prof. Paul De Vos
Laboratory of Microbiology
K.L. Ledeganckstraat 35
9000 Gent
Tel: 09 264 5110
Fax: 09 264 5092
Email: Paul.DeVos@ugent.be
http://lmg.UGent.be; http://bccm.belspo.be/
Partner 2: Vrije Universiteit Brussel
Promotor: Prof. Pierre Cornelis
MINT
Paardenstraat 65,
1640 Sint-Genesius-Rode
Tel: 02 359 0221
Fax: 02 359 0399
Email: Pierre.Cornelis@imol.vub.ac.be
User Committee
Member 1:
Institutute voor Landbouw- en Visserijonderzoek (ILVO)
Departement Gewasbescherming
Burgemeester van Gansberghelaan, 96
B-9820 Merelbeke
Member 2:
Provinciaal onderzoeks- en voorlichtingscentrum voor Land- en Tuinbouw (POVLT)
Iepersesteenweg 87
B-8800 Rumbeke
Member 3:
Puracor n.v./S.a.
Industrielaan, 25
B-1702 Groot-Bijgaarden.
Member 4:
Institution : La Clinique des Plantes
UCL-Faculté d’ingénerie biologique, agronomique et envrionnementale
Unité de phytopathologie
Croix du Sud, 2bte 3
B-1348 Louvain-la-Neuve
Member 5:
Applied Maths BVBA
Keistraat120
9830 St-Martens-Latem
Member 6 :
Vakgroep gewasbescherming LA03
Coupure Links 653
9000 Gent
