Saskia Gerards

Plants release a wide set of secondary metabolites including volatile organic compounds (VOCs). Many of those compounds are considered to function as defense against herbivory, pests, and pathogens. However, little knowledge exists about the role of belowground plant VOCs for attracting beneficial soil microorganisms. We developed an olfactometer system to test the attraction of soil bacteria by VOCs emitted by Carex arenaria roots. Moreover, we tested whether infection of C. arenaria with the fungal pathogen Fusarium culmorum modifies the VOCs profile and bacterial attraction. The results revealed that migration of distant bacteria in soil towards roots can be stimulated by plant VOCs. Upon fungal infection, the blend of root VOCs changed and specific bacteria with antifungal properties were attracted. Tests with various pure VOCs indicated that those compounds can diffuse over long distance but with different diffusion abilities. Overall, this work highlights the importance of plant VOCs in belowground long-distance plant–microbe interactions.

Recent studies indicated that the production of secondary metabolites by soil bacteria can be triggered by interspecific interactions. However, little is known to date about interspecific interactions between Gram-positive and Gram-negative bacteria. In this study, we aimed to understand how the interspecific interaction between the Gram-positive Paenibacillus sp. AD87 and the Gram-negative Burkholderia sp. AD24 affects the fitness, gene expression and the production of soluble and volatile secondary metabolites of both bacteria. To obtain better insight into this interaction, transcriptome and metabolome analyses were performed. Our results revealed that the interaction between the two bacteria affected their fitness, gene expression and the production of secondary metabolites. During interaction, the growth of Paenibacillus was not affected, whereas the growth of Burkholderia was inhibited at 48 and 72 h. Transcriptome analysis revealed that the interaction between Burkholderia and Paenibacillus caused significant transcriptional changes in both bacteria as compared to the monocultures. The metabolomic analysis revealed that the interaction increased the production of specific volatile and soluble antimicrobial compounds such as 2,5-bis(1-methylethyl)-pyrazine and an unknown Pederin-like compound. The pyrazine volatile compound produced by Paenibacillus was subjected to bioassays and showed strong inhibitory activity against Burkholderia and a range of plant and human pathogens. Moreover, strong additive antimicrobial effects were observed when soluble extracts from the interacting bacteria were combined with the pure 2,5-bis(1-methylethyl)-pyrazine. The results obtained in this study highlight the importance to explore bacterial interspecific interactions to discover novel secondary metabolites and to perform simultaneously metabolomics of both, soluble and volatile compounds.

Rhizogenic Agrobacterium biovar 1 is the causative agent of hairy root disease (HRD) in the hydroponic cultivation of tomato and cucumber causing significant losses in marketable yield. In order to prevent and control the disease chemical disinfectants such as hydrogen peroxide or hypochlorite are generally applied to sanitize the hydroponic system and/or hydroponic solution. However, effective control of HRD sometimes requires high disinfectant doses that may have phytotoxic effects. Moreover, several of these chemicals may be converted to unwanted by-products with human health hazards. Here we explored the potential of beneficial bacteria as a sustainable means to control HRD. A large collection of diverse bacterial genera was screened for antagonistic activity against rhizogenic Agrobacterium biovar 1 using the agar overlay assay. Out of more than 130 strains tested only Paenibacillus strains showed antagonistic activity. Strikingly, phylogenetic analysis showed that antagonistic activity was restricted to a particular Paenibacillus clade, representing the species P. illinoisensis, P. pabuli, P. taichungensis, P. tundrae, P. tylopili, P. xylanexedens and P. xylanilyticus. Assessment of the spectrum of activity revealed that some strains were able to inhibit the growth of all 35 rhizogenic agrobacteria strains tested, while others were only active against part of the collection, suggesting a different mode of action. Preliminary characterization of the compounds involved in the antagonistic activity of two closely related Paenibacillus strains, tentatively identified as P. xylanexedens, revealed that they are water-soluble and have low molecular weight. Application of a combination of these strains in greenhouse conditions resulted in a significant reduction of HRD, indicating the great potential of these strains to control HRD.

There is increasing evidence that volatile organic compounds (VOCs) play an important role in the interactions between fungi and bacteria, two major groups of soil inhabiting microorganisms. Yet, most of the research has been focused on effects of bacterial volatiles on suppression of plant pathogenic fungi whereas little is known about the responses of bacteria to fungal volatiles. In the current study we performed a metabolomics analysis of volatiles emitted by several fungal and oomycetal soil strains under different nutrient conditions and growth stages. The metabolomics analysis of the tested fungal and oomycetal strains revealed different volatile profiles dependent on the age of the strains and nutrient conditions. Furthermore, we screened the phenotypic responses of soil bacterial strains to volatiles emitted by fungi. Two bacteria, Collimonas pratensis Ter291 and Serratia plymuthica PRI-2C, showed significant changes in their motility, in particular to volatiles emitted by Fusarium culmorum. This fungus produced a unique volatile blend, including several terpenes. Four of these terpenes were selected for further tests to investigate if they influence bacterial motility. Indeed, these terpenes induced or reduced swimming and swarming motility of S. plymuthica PRI-2C and swarming motility of C. pratensis Ter291, partly in a concentration-dependent manner. Overall the results of this work revealed that bacteria are able to sense and respond to fungal volatiles giving further evidence to the suggested importance of volatiles as signaling molecules in fungal–bacterial interactions.

There is increasing evidence that organic volatiles play an important role in interactions between micro-organisms in the porous soil matrix. Here we report that volatile compounds emitted by different soil bacteria can affect the growth, antibiotic production and gene expression of the soil bacterium Pseudomonas fluorescens Pf0–1. We applied a novel cultivation approach that mimics the natural nutritional heterogeneity in soil in which P. fluorescens grown on nutrient-limited agar was exposed to volatiles produced by 4 phylogenetically different bacterial isolates (Collimonas pratensis, Serratia plymuthica, Paenibacillus sp., and Pedobacter sp.) growing in sand containing artificial root exudates. Contrary to our expectation, the produced volatiles stimulated rather than inhibited the growth of P. fluorescens. A genome-wide, microarray-based analysis revealed that volatiles of all four bacterial strains affected gene expression of P. fluorescens, but with a different pattern of gene expression for each strain. Based on the annotation of the differently expressed genes, bacterial volatiles appear to induce a chemotactic motility response in P. fluorescens, but also an oxidative stress response. A more detailed study revealed that volatiles produced by C. pratensis triggered, antimicrobial secondary metabolite production in P. fluorescens. Our results indicate that bacterial volatiles can have an important role in communication, trophic - and antagonistic interactions within the soil bacterial community.

It is increasingly recognized that volatile organic compounds play an import role during interactions between soil microorganisms. Here, we examined the possible involvement of volatiles in the interaction of Collimonas bacteria with soil fungi. The genus Collimonas is known for its ability to grow at the expense of living fungi (mycophagy), and antifungal volatiles may contribute to the attack of fungi by these bacteria. We analyzed the composition of volatiles produced by Collimonas on agar under different nutrient conditions and studied the effect on fungal growth. The volatiles had a negative effect on the growth of a broad spectrum of fungal species. Collimonas bacteria did also produce volatiles in sand microcosms supplied with artificial root exudates. The production of volatiles in sand microcosms was enhanced by the presence of fungi. The overall picture that we get from our study is that antifungal volatiles produced by Collimonas could play an important role in realizing its mycophagous lifestyle. The current work is also interesting for understanding the ecological relevance of volatile production by soil bacteria in general as we found strong influences of root exudates composition and incubation conditions on the spectrum of volatiles produced.

Through a combinatorial approach of plasposon mutagenesis, genome mining, and heterologous expression, we identified genes contributing to the chitinolytic phenotype of bacterium Collimonas fungivorans Ter331. One of five mutants with abolished ability to hydrolyze colloidal chitin carried its plasposon in the chiI gene coding for an extracellular endochitinase. Two mutants were affected in the promoter of chiP-II coding for an outer-membrane transporter of chitooligosaccharides. The remaining two mutations were linked to chitobiose/N-acetylglucosamine uptake. Thus, our model for the Collimonas chitinolytic system assumes a positive feedback regulation of chitinase activity by chitin degradation products. A second chitinase gene, chiII, coded for an exochitinase that preferentially released chitobiose from chitin analogs. Genes hexI and hexII showed coding resemblance to N-acetylglucosaminidases, and the activity of purified HexI protein towards chitin analogs suggested its role in converting chitobiose to N-acetylglucosamine. The hexI gene clustered with chiI, chiII, and chiP-II in one locus, while chitobiose/N-acetylglucosamine uptake genes colocalized in another. Both loci contained genes for conversion of N-acetylglucosamine to fructose-6-phosphate, confirming that C. fungivorans Ter331 features a complete chitin pathway. No link could be established between chitinolysis and antifungal activity of C. fungivorans Ter331, suggesting that the bacterium's reported antagonism towards fungi relies on other mechanisms.

The isolation and annotation of an 8994-bp DNA fragment from Pseudomonas putida 1290, which conferred upon P. putida KT2440 the ability to utilize the plant hormone indole 3-acetic acid (IAA) as a sole source of carbon and energy, is described. This iac locus (for indole 3-acetic acid catabolism) was identified through analysis of a plasposon mutant of P. putida 1290 that was no longer able to grow on IAA or indole 3-acetaldehyde and was unable to protect radish roots from stunting by exogenously added IAA. The iac locus consisted of 10 genes with coding similarity to enzymes acting on indole or amidated aromatics and to proteins with regulatory or unknown function. Highly similar iac gene clusters were identified in the genomes of 22 bacterial species. Five of these, i.e. P. putida GB-1, Marinomonas sp. MWYL1, Burkholderia sp. 383, Sphingomonas wittichii RW1 and Rhodococcus sp. RHA1, were tested to confirm that bacteria with IAA-degrading ability have representatives in the Alpha-, Beta- and Gammaproteobacteria and in the Actinobacteria. In P. putida 1290, cat and pca genes were found to be essential to IAA-degradation, suggesting that IAA is channeled via catechol into the β-ketoadipate pathway. Also contributing to the IAA degrading phenotype were genes involved in tricarboxylate cycling, gluconeogenesis, and carbon/nitrogen sensing.

Plasposons are modified mini-Tn5 transposons for random mutagenesis of Gram-negative bacteria. Their unique design allows for the rescue cloning and sequencing of DNA that flanks insertion sites in plasposon mutants. However, this process can be laborious and time-consuming, as it involves genomic DNA isolation, restriction endonuclease treatment, subsequent religation, transformation of religated DNA into an Escherichia coli host, and re-isolation as a plasmid, which is then used as a template in sequencing reactions with primers that read from the plasposon ends into the flanking DNA regions. We describe here a method that produces flanking DNA sequences directly from genomic DNA that is isolated from plasposon mutants. By eliminating the need for rescue cloning, our protocol dramatically reduces time and effort, typically by 2 to 3 working days, as well as costs associated with digestion, ligation, transformation, and plasmid isolation. Furthermore, it allows for a high-throughput automated approach to analysis of the plasposome, i.e. the collective set of plasposon insertion sites in a plasposon mutant library. We have tested the utility of genomic flank-sequencing on three plasposon mutants of the soil bacterium Collimonas fungivorans with abolished ability to degrade chitin. [KEYWORDS: Chitinase ; Collimonas fungivorans ; Genomic sequencing ; Mini-Tn5 transposon ; Plasposome ; Plasposon]

We assessed the utility of fluorescent in situ hybridization (FISH) in the screening of clone libraries of (meta)genomic or environmental DNA for the presence and expression of bacterial ribosomal RNA (rRNA) genes. To establish proof-of-principle, we constructed a fosmid-based library in Escherichia coli of large-sized genomic DNA fragments of the mycophagous soil bacterium Collimonas fungivorans, and hybridized 768 library clones with the Collimonas-specific fluorescent probe CTE998-1015. Critical to the success of this approach (which we refer to as large-insert library FISH or LIL-FISH) was the ability to induce fosmid copy number, the exponential growth status of library clones in the FISH assay and the use of a simple pooling strategy to reduce the number of hybridizations. Twelve out of 768 E. coli clones were suspected to harbour and express Collimonas 16S rRNA genes based on their hybridization to CTE998-1015. This was confirmed by the finding that all 12 clones were also identified in an independent polymerase chain reaction-based screening of the same 768 clones using a primer set for the specific detection of Collimonas 16S ribosomal DNA (rDNA). Fosmids isolated from these clones were grouped by restriction analysis into two distinct contigs, confirming that C. fungivorans harbours at least two 16S rRNA genes. For one contig, representing 1-2% of the genome, the nucleotide sequence was determined, providing us with a narrow but informative view of Collimonas genome structure and content.

The dynamics of culturable chitin-degrading microorganisms were studied during a 16-week incubation of chitin-amended coastal dune soils that differed in acidity. Soil samples were incubated at normal (5% Why) and high (15% w/w) moisture levels. More than half of the added chitin was decomposed within 4 weeks of incubation in most soils. This rapid degradation was most likely due to fast-growing chitinolytic fungi (mainly Mortierella spp. and Fusarium spp.) at both moisture levels, as dense hyphal networks of these fungi were observed during the first 4 weeks of incubation. Chitin N mineralization was inhibited by cycloheximide, and fast-growing fungal isolates were capable of rapid chitin decomposition in sterile sand, further suggesting that these fungi play an important role in initial chitin degradation. The strong increase in fast-growing fungi in chitin-amended dune soils was only detected by direct observation. Plate counts and microscopic quantification of stained hyphae failed to reveal such an increase. During the first part of the incubation, numbers of unicellular chitinolytic bacteria also increased, but their contribution to chitin degradation was indicated to be of minor importance. During prolonged incubation, colony forming units (CFU) of chitinolytic streptomycetes and/or slow- growing fungi increased strongly in several soils, especially at the 5% moisture level. Hence, the general trend observed was a succession from fast-growing fungi and unicellular bacteria to actinomycetes and slow-growing fungi. Yet, the composition of chitinolytic CFU over time differed strongly between chitin- amended dune soils, and also between the two moisture levels. These differences could not be attributed to pH, organic matter or initial microbial composition. The possible consequence of such unpredictable variation in microbial community composition for the use of chitin-amendments as a biocontrol measure is discussed. [KEYWORDS: chitin degradation; succession; fungi; bacteria; actinomycetes Forest soil; nematodes; bacteria; mode]

Ammonia-oxidising bacteria are poor competitors for limiting amounts of ammonium. Hence, starvation for ammonium seems to be the regular condition for these bacteria in natural environments. Long-term survival in the absence of ammonium will be dependent on the ability to maintain large population sizes at the expense of endogenous energy sources and on the preservation of a relatively large capacity for ammonium oxidation. The effect of freshly added ammonium on the performance of ammonia-oxidising bacteria was studied in ammonium-enriched slurries consisting of samples taken from non-water-saturated soil and sand columns inoculated with Nitrosomonas europaea and Nitrobacter winogradskyi and continuously percolated with mineral medium containing ammonium. Immediately after introduction of the nitrifying bacteria to the columns, ammonium oxidation started and nitrate leached from the columns. After 6 weeks of incubation of the columns, 94% of the ammonium supplied was recovered as nitrate in the effluent and net cell growth had ceased. In slurries with freshly added ammonium, ammonium oxidation decreased after an initial period of relatively high oxidation rates, which lasted 6 at the most. This indicated that the cells had been starved for ammonium in the columns. After 3 days of slurry incubation the ammonium-oxidising activity restarted, bur not in the presence of chloramphenicol, indicating de novo synthesis of enzyme systems. Restart of activity after 3 days could not be attributed to the release of free-living cells from the sand particles or to the presence of organotrophic bacteria in the slurries. [KEYWORDS: nitrification; ammonium oxidation; inhibition Nitrosomonas europaea; Nitrobacter winogradskyi Heterotrophic bacteria; competition; nitrification; hydroxylamine; nitrobacter; oxidation; columns; growth; roots; ph]

The dissimilatory nitrate-reducing bacterial community in the rhizosphere of aerenchymatous plant species such as Glyceria maxima, consists of oxidative. denitrifying and fermentative nitrate-ammonifying bacteria. To study the respective ecological niches of both types of nitrate-reducing bacteria, competition for nitrate or glucose between the representative denitrifier Pseudomonas fluorescens and the representative fermentative nitrate-ammonifying Bacillus licheniformis under continuous or fluctuating anoxic conditions were performed in continuous culture. Competition started by mixing the separate, steady-slate mono-cultures of the two species at different ratios. All the experiments were performed at a dilution rate of 0.05 h(-1). The competition was followed by measuring concentrations of nitrogen, glucose and fatty acids and by determining the cell numbers of P. fluorescens and B. licheniformis. Under continuous anoxic nitrate-limited conditions and under certain fluctuating anoxic conditions (8 h 10% and 16 h 0% air saturation), B. licheniformis was able to maintain itself in the chemostat at a low percentage of 4-7%. Under continuous anoxic glucose-limited conditions and under specific fluctuating anoxic (16 h 10% and 8 h 0% air saturation) conditions, B. licheniformis washed out. The outcome of the competition was explained by a higher affinity of P. fluorescens for nitrate arid glucose compared to B. licheniformis. B. licheniformis was able to maintain itself in the chemostat under continuous anoxic nitrate-limited conditions and under certain fluctuating anoxic conditions (8 h 10% and 16 h 0% air saturation) due to the fer;mentation of the remaining glucose. [KEYWORDS: competition; nitrate reduction; chemostat; Pseudomonas fluorescens; Bacillus licheniformis Denitrification; oxygen; rhizosphere; transient; culture]

Marram grass (Ammophila arenaria L.), a sand stabilizing plant species in coastal dune areas, is affected by a specific pathosystem thought to include both plant-pathogenic fungi and nematodes, To study the fungal component of this pathosystem, we developed a method for the cultivation-independent detection and characterization of fungi infecting plant roots based on denaturing gradient gel electrophoresis (DGGE) of specifically amplified DNA fragments coding for 188 rRNA (rDNA), A nested PCR strategy was employed to amplify a 569-bp region of the 188 rRNA gene, with the addition of a 36-bp GC clamp, from fungal isolates, from roots of test plants infected in the laboratory, and from field samples of marram grass roots from both healthy and degenerating stands from coastal dunes in The Netherlands, PCR products from fungal isolates were subjected to DGGE to examine the variation seen both between different fungal taxa and within a single species, DGGE of the 18S rDNA fragments could resolve species differences from fungi used in this study yet was unable to discriminate between strains of a single species, The 18S rRNA genes from 20 isolates of fungal species previously recovered from A. arenaria roots were cloned and partially sequenced to aid in the interpretation of DGGE data, DGGE patterns recovered from laboratory plants showed that this technique could reliably identify known plant-infecting fungi. Amplification products from field A. arenaria roots also were analyzed by DGGE, and the major bands were excised, reamplified, sequenced, and subjected to phylogenetic analysis, Some recovered 18S rDNA sequences allowed for phylogenetic placement to the genus level, whereas other sequences were not closely related to known fungal 18S rDNA sequences, The molecular data presented here reveal fungal diversity not detected in previous culture-based surveys. [KEYWORDS: 16s ribosomal-rna; polymerase chain-reaction; polymorphic dna; genetic diversity; pcr; identification; soil; differentiation; amplification; populations]

The effects of nitrate availability and the presence of Glyceria maxima on the composition and activity of the dissimilatory nitrate-reducing bacterial community were studied in the laboratory. Four different concentrations of NO3-, 0, 533, 1434, and 2,905 mu g of NO3--N g of dry sediment(-1), were added to pots containing freshwater sediment, and the pots were then incubated for a period of 69 days. Upon harvest, NH4+ was not detectable in sediment that received 0 or 533 mu g of NO3-- N g of dry sediment(-1). Nitrate concentrations in these pots ranged from 0 to 8 mu g of NO3--N g of dry sediment(-1) at harvest. In pots that received 1,434 or 2,905 mu g of NO3--N g of dry sediment(-1), final concentrations varied between 10 and 48 mu g of NH4+-N g of dry sediment(-1) and between 200 and 1,600 mu g of NO3--N g of dry sediment(-1), respectively. Higher input levels of NO3- resulted in increased numbers of potential nitrate-reducing bacteria and higher potential nitrate-reducing activity in the rhizosphere. In sediment samples from the rhizosphere, the contribution of denitrification to the potential nitrate-reducing capacity varied from 8% under NO3--limiting conditions to 58% when NO3- was in ample supply. In bulk sediment with excess NO3-, this percentage was 44%. The nitrate-reducing community consisted almost entirely of NO2--accumulating or NH4+-producing gram- positive species when NO3- was not added to the sediment. The addition of NO3- resulted in an increase of denitrifying Pseudomonas and Moraxella strains. The factor controlling the composition of the nitrate-reducing community when NO3- is limited is the presence of G. maxima. In sediment with excess NO3-, nitrate availability determines the composition of the nitrate-reducing community. [KEYWORDS: Denitrifying bacteria; denitrification; soil; sediments; populations; rhizosphere; reduction; acetylene; plants; roots]