Collimonas is a genus belonging to the class of Betaproteobacteria and consists mostly of soil bacteria with the ability to exploit living fungi as food source (mycophagy). Collimonas strains differ in a range of activities, including swimming motility, quorum sensing, extracellular protease activity, siderophore production, and antimicrobial activities. In order to reveal ecological traits possibly related to Collimonas lifestyle and secondary metabolites production, we performed a comparative genomics analysis based on whole-genome sequencing of six strains representing 3 recognized species. The analysis revealed that the core genome represents 43.1 to 52.7 % of the genomes of the six individual strains. These include genes coding for extracellular enzymes (chitinase, peptidase, phospholipase), iron acquisition and type II secretion systems. In the variable genome, differences were found in genes coding for secondary metabolites (e.g. tripropeptin A and volatile terpenes), several unknown orphan polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS), nonribosomal peptide synthetase (NRPS) gene clusters, a new lipopeptide and type III and type VI secretion systems. Potential roles of the latter genes in the interaction with other organisms were investigated. Mutation of a gene involved in tripropeptin A biosynthesis strongly reduced the antibacterial activity against Staphylococcus aureus, while disruption of a gene involved in the biosynthesis of the new lipopeptide had a large effect on the antifungal/oomycetal activities. Overall our results indicated that Collimonas genomes harbour many genes encoding for novel enzymes and secondary metabolites (including terpenes) important for interactions with other organisms and revealed genomic plasticity, which reflect the behaviour, antimicrobial activity and lifestylesof Collimonas spp.

The ability to colonize the host plants’ rhizospheres is a crucial feature to study in the case of Plant Growth Promoting Rhizobacteria (PGPRs) with potential agricultural applications. In this work, we have created GFP-tagged derivatives of three candidate PGPRs: Bacillus subtilis MB73/2, Pseudomonas sp. P482 and Ochrobactrum sp. A44. The presence of these strains in the rhizosphere of soil-grown potato (Solanum tuberosum L.) was detected with a classical fluorescence microscope and a confocal laser scanning microscope (CLSM). In this work, we have used a broad-field-of-view CLMS device, dedicated to in vivo analysis of macroscopic objects, equipped with an automated optical zoom system and tunable excitation and detection spectra. We show that features of this type of CLSM microscopes make them particularly well suited to study root colonization by microorganisms. To facilitate the detection of small and scattered bacterial populations, we have developed a fast and user-friendly enrichment method for root sample preparation. The described method, thanks to the in situ formation of mini-colonies, enables visualization of bacterial colonization sites on large root fragments. This approach can be easily modified to study colonization patterns of other fluorescently tagged strains. Additionally, dilution plating of the root extracts was performed to estimate the cell number of MB73/2, P482 and A44 in the rhizosphere of the inoculated plants.

Dickeya solani and Pectobacterium carotovorum subsp. brasili¬ense are recently established species of bacterial plant pathogens causing black leg and soft rot of many vegetables and ornamental plants. Pseudomonas sp. strain P482 inhibits the growth of these pathogens, a desired trait considering the limited measures to combat these diseases. In this study, we determined the genetic background of the antibacterial activity of P482, and established the phylogenetic position of this strain. Pseudomonas sp. P482 was classified as Pseudomonas donghuensis. Genome mining revealed that the P482 genome does not contain genes determining the synthesis of known antimicrobials. However, the ClusterFinder algorithm, designed to detect atypical or novel classes of secondary metabolite gene clusters, predicted 18 such clusters in the genome. Screening of a Tn5 mutant library yielded an antimicrobial negative transposon mutant. The transposon insertion was located in a gene encoding an HpcH/HpaI aldolase/citrate lyase family protein. This gene is located in a hypothetical cluster predicted by the ClusterFinder, together with the downstream homologues of four nfs genes, that confer production of a nonfluorescent siderophore by P. donghuensis HYST. Site-directed inactivation of the HpcH/HpaI aldolase gene, the adjacent short chain dehydrogenase gene, as well as a homologue of an essential nfs cluster gene, all abolished the antimicrobial activity of the P482, suggesting their involvement in a common biosynthesis pathway. However, none of the mutants showed a decreased siderophore yield, neither was the antimicrobial activity of the wild type P482 compromised by high iron bioavailability. A genomic region comprising the nfs cluster and three upstream genes is involved in the antibacterial activity of P. donghuensis P482 against D. solani and P. carotovorum subsp. brasiliense. The genes studied are unique to the two known P. donghuensis strains. This study illustrates that mining of microbial genomes is a powerful approach for predicting the presence of novel secondary-metabolite encoding genes especially when coupled with transposon mutagenesis.

Possibilities to protect potato tubers from rotting caused by Soft Rot Pectobacteriaceae (SRP) under disease favoring conditions were investigated using compatible mixtures of bacterial antagonists and tested with a newly developed stepwise efficacy-based screening protocol. Twenty-two bacterial antagonists were evaluated against a combination of five Pectobacterium and Dickeya strains representing species and subspecies most often associated with potato soft rot in Europe. To enable potential synergistic activity, the antagonists were initially tested against the combination of pathogens in 15 random mixtures containing up to 5 antagonists each. Three mixtures (M2, M4, and M14) out of 15 tested reduced tuber tissue maceration due to soft rot. The individual antagonists derived from M2, M4, and M14 mixtures were tested on potato slices and whole tuber injection assays. These five strains (S. plymuthica strain A294, E. amnigenus strain A167, R. aquatilis strain H145, S. rubidaea strain H440, and S. rubidaea strain H469) were combined to develop a tailored biological control mixture against potato soft rot. The new mixture, designated the Great Five (GF), was tested on seed potato tubers vacuum infiltrated with antagonists and subsequently with the combination of five SRP pathogens. In these experiments, the GF mixture provided stable protection of inoculated potato tubers, reducing soft rot by 46% (P = 0.0016) under high disease pressure conditions. The A294, A167, H145, H440, and H469 antagonists were characterized for features important for viable commercial applications including growth at different temperatures, resistance to antibiotics, and potential toxicity toward Caenorhabditis elegans. The implications for control of soft rot caused by SRP with the use of the GF mixture of antagonists are discussed.