Bacterial-fungal interactions: hyphens between agricultural, clinical, environmental, and food microbiologists.

Bacteria and fungi can form a range of physical associations that depend on various modes of molecular communication for their development and functioning. These bacterial-fungal interactions often result in changes to the pathogenicity or the nutritional influence of one or both partners toward plants or animals (including humans). They can also result in unique contributions to biogeochemical cycles and biotechnological processes. Thus, the interactions between bacteria and fungi are of central importance to numerous biological questions in agriculture, forestry, environmental science, food production, and medicine. Here we present a structured review of bacterial-fungal interactions, illustrated by examples sourced from many diverse scientific fields. We consider the general and specific properties of these interactions, providing a global perspective across this emerging multidisciplinary research area. We show that in many cases, parallels can be drawn between different scenarios in which bacterial-fungal interactions are important. Finally, we discuss how new avenues of investigation may enhance our ability to combat, manipulate, or exploit bacterial-fungal complexes for the economic and practical benefit of humanity as well as reshape our current understanding of bacterial and fungal ecology.

Role of fungal trehalose and bacterial thiamine in the improved survival and growth of the ectomycorrhizal fungus Laccaria bicolor S238N and the helper bacterium Pseudomonas fluorescens BBc6R8.

The mycorrhiza helper bacterial strain Pseudomonas fluorescens BBc6R8 enhances the establishment of Laccaria bicolor S238N ectomycorrhizae by improving the pre-symbiotic growth and survival of the fungus. Nothing is known about the effect of the ectomycorrhizal fungus on the helper bacteria or the molecules that are involved in the interaction. In this study, we have monitored the population density of the helper strain P. fluorescens BBc6R8 in soils inoculated with L. bicolor and in control soils and found that the ectomycorhizal fungus improves the survival of the helper bacteria. We investigated the identity of the fungal and bacterial metabolites involved in this reciprocal growth-promoting effect using a combination of growth measurements, chemoattractant assays, HPLC and in silico genome analyses. We showed that trehalose, a disaccharide that accumulates to high levels in the fungal hyphae, chemoattracted and promoted the growth of the helper bacteria. Meanwhile, P. fluorescens BBc6R8 produced thiamine at concentrations that enhanced the fungal growth in vitro. Altogether our data indicate that the interaction between the two microorganisms is beneficial for both species and relies, at least in part, on trophic mutualism.

Rhizosphere bacterial communities associated with disease suppressiveness stages of take-all decline in wheat monoculture.

The decline of take-all disease (Gaeumannomyces graminis var. tritici), which may take place during wheat monocropping, involves plant-protecting, root-colonizing microorganisms. So far, however, most work has focused on antagonistic fluorescent pseudomonads. Our objective was to assess the changes in rhizobacterial community composition during take-all decline of field-grown wheat. The study was based on the development and utilization of a taxonomic 16S rRNA-based microarray of 575 probes, coupled with cloning-sequencing and quantitative PCR. Plots from one experimental field grown with wheat for 1 yr (low level of disease), 5 yr (high level of disease) or 10 yr (low level of disease, suppressiveness reached) were used. Microarray data discriminated between the three stages. The outbreak stage (5 yr) was mainly characterized by the prevalence of Proteobacteria, notably Pseudomonas (Gammaproteobacteria), Nitrosospira (Betaproteobacteria), Rhizobacteriaceae, Sphingomonadaceae, Phyllobacteriaceae (Alphaproteobacteria), as well as Bacteroidetes and Verrucomicrobia. By contrast, suppressiveness (10 yr) correlated with the prevalence of a broader range of taxa, which belonged mainly to Acidobacteria, Planctomycetes, Nitrospira, Chloroflexi, Alphaproteobacteria (notably Azospirillum) and Firmicutes (notably Thermoanaerobacter). In conclusion, take-all decline correlated with multiple changes in rhizobacterial community composition, far beyond the sole case of pseudomonads.

The plant pathogenic fungus Gaeumannomyces graminis var. tritici improves bacterial growth and triggers early gene regulations in the biocontrol strain Pseudomonas fluorescens Pf29Arp.

In soil, some antagonistic rhizobacteria contribute to reduce root diseases caused by phytopathogenic fungi. Direct modes of action of these bacteria have been largely explored; however, commensal interaction also takes place between these microorganisms and little is known about the influence of filamentous fungi on bacteria. An in vitro confrontation bioassay between the pathogenic fungus Gaeumannomyces graminis var. tritici (Ggt) and the biocontrol bacterial strain Pseudomonas fluorescens Pf29Arp was set up to analyse bacterial transcriptional changes induced by the fungal mycelium at three time-points of the interaction before cell contact and up until contact. For this, a Pf29Arp shotgun DNA microarray was constructed. Specifity of Ggt effect was assessed in comparison with one of two other filamentous fungi, Laccaria bicolor and Magnaporthe grisea. During a commensal interaction, Ggt increased the growth rate of Pf29Arp. Before contact, Ggt induced bacterial genes involved in mycelium colonization. At contact, genes encoding protein of stress response and a patatin-like protein were up-regulated. Among all the bacterial genes identified, xseB was specifically up-regulated at contact by Ggt but down-regulated by the other fungi. Data showed that the bacterium sensed the presence of the fungus early, but the main gene alteration occurred during bacterial-fungal cell contact.

The mycorrhiza helper Pseudomonas fluorescens BBc6R8 has a specific priming effect on the growth, morphology and gene expression of the ectomycorrhizal fungus Laccaria bicolor S238N.

The mycorrhiza helper Pseudomonas fluorescens BBc6R8 promotes the presymbiotic survival and growth of the ectomycorrhizal fungus Laccaria bicolor S238N in the soil. An in vitro fungal-bacterial confrontation bioassay mimicking the promoting effects of the bacteria on fungal growth was set up to analyse the fungal morphological and transcriptional changes induced by the helper bacteria at three successive stages of the interaction. The specificity of the P. fluorescens BBc6R8 effect was assessed in comparison with six other rhizobacterial strains possessing mycorrhiza helper or pathogen antagonistic abilities. The helper BBc6R8 strain was the only strain to induce increases in the radial growth of the colony, hyphal apex density and branching angle. These morphological modifications were coupled with pleiotropic alterations of the fungal transcriptome, which varied throughout the interaction. Early stage-responsive genes were presumably involved in recognition processes and transcription regulation, while late stage-responsive genes encoded proteins of primary metabolism. Some of the responsive genes were partly specific to the interaction with P. fluorescens BBc6R8, whereas others were mutually regulated by different rhizobacteria. The results highlight the fact that the helper BBc6R8 strain has a specific priming effect on growth, morphology and gene expression of its fungal associate L. bicolor S238N.

Sequence diversity of the oprI gene, coding for major outer membrane lipoprotein I, among rRNA group I pseudomonads.

The sequence of oprI, the gene coding for the major outer membrane lipoprotein I, was determined by PCR sequencing for representatives of 17 species of rRNA group I pseudomonads, with a special emphasis on Pseudomonas aeruginosa and Pseudomonas fluorescens. Within the P. aeruginosa species, oprI sequences for 25 independent isolates were found to be identical, except for one silent substitution at position 96. The oprI sequences diverged more for the other rRNA group I pseudomonads (85 to 91% similarity with P. aeruginosa oprI). An accumulation of silent and also (but to a much lesser extent) nonsilent substitutions in the different sequences was found. A clustering according to the respective presence and/or positions of the HaeIII, PvuII, and SphI sites could also be obtained. A sequence cluster analysis showed a rather widespread distribution of P. fluorescens isolates. All other rRNA group I pseudomonads clustered in a manner that was in agreement with other studies, showing that the oprI gene can be useful as a complementary phylogenetic marker for classification of rRNA group I pseudomonads.

The two-component regulators GacS and GacA influence accumulation of the stationary-phase sigma factor sigmaS and the stress response in Pseudomonas fluorescens Pf-5.

Three global regulators are known to control antibiotic production by Pseudomonas fluorescens. A two-component regulatory system comprised of the sensor kinase GacS (previously called ApdA or LemA) and GacA, a member of the FixJ family of response regulators, is required for antibiotic production. A mutation in rpoS, which encodes the stationary-phase sigma factor sigmaS, differentially affects antibiotic production and reduces the capacity of stationary-phase cells of P. fluorescens to survive exposure to oxidative stress. The gacA gene of P. fluorescens Pf-5 was isolated, and the influence of gacS and gacA on rpoS transcription, sigmaS levels, and oxidative stress response of Pf-5 was determined. We selected a gacA mutant of Pf-5 that contained a single nucleotide substitution within a predicted alpha-helical region, which is highly conserved among the FixJ family of response regulators. At the entrance to stationary phase, sigmaS content in gacS and gacA mutants of Pf-5 was less than 20% of the wild-type level. Transcription of rpoS, assessed with an rpoS-lacZ transcriptional fusion, was positively influenced by GacS and GacA, an effect that was most evident at the transition between exponential growth and stationary phase. Mutations in gacS and gacA compromised the capacity of stationary-phase cells of Pf-5 to survive exposure to oxidative stress. The results of this study provide evidence for the predominant roles of GacS and GacA in the regulatory cascade controlling stress response and antifungal metabolite production in P. fluorescens.

The sigma factor sigma s affects antibiotic production and biological control activity of Pseudomonas fluorescens Pf-5.

Pseudomonas fluorescens Pf-5, a rhizosphere-inhabiting bacterium that suppresses several soilborne pathogens of plants, produces the antibiotics pyrrolnitrin, pyoluteorin, and 2,4-diacetylphloroglucinol. A gene necessary for pyrrolnitrin production by Pf-5 was identified as rpoS, which encodes the stationary-phase sigma factor sigma s. Several pleiotropic effects of an rpoS mutation in Escherichia coli also were observed in an RpoS- mutant of Pf-5. These included sensitivities of stationary-phase cells to stresses imposed by hydrogen peroxide or high salt concentration. A plasmid containing the cloned wild-type rpoS gene restored pyrrolnitrin production and stress tolerance to the RpoS- mutant of Pf-5. The RpoS- mutant overproduced pyoluteorin and 2,4-diacetyl-phloroglucinol, two antibiotics that inhibit growth of the phytopathogenic fungus Pythium ultimum, and was superior to the wild type in suppression of seedling damping-off of cucumber caused by Pythium ultimum. When inoculated onto cucumber seed at high cell densities, the RpoS- mutant did not survive as well as the wild-type strain on surfaces of developing seedlings. Other stationary-phase-specific phenotypes of Pf-5, such as the production of cyanide and extracellular protease(s) were expressed by the RpoS- mutant, suggesting that sigma s is only one of the sigma factors required for the transcription of genes in stationary-phase cells of P. fluorescens. These results indicate that a sigma factor encoded by rpoS influences antibiotic production, biological control activity, and survival of P. fluorescens on plant surfaces.

Detection of the outer membrane lipoprotein I and its gene in fluorescent and non-fluorescent pseudomonads: implications for taxonomy and diagnosis.

The open reading frame of the OprI lipoprotein gene from Pseudomonas aeruginosa was amplified by polymerase chain reaction (PCR) starting from purified DNA or colony lysates. A fragment of the expected size (249 bp) was detected in all P. aeruginosa strains from various clinical and geographical origins. The gene could only be amplified in pseudomonads of rRNA group I which are considered to be the authentic genus Pseudomonas. Digestions with HaeIII, PvuII and SphI of the amplified fragments demonstrated a sequence variation in the oprI gene. Colony, dot and Western blots with two monoclonal antibodies (mAbs) against the lipoprotein I confirmed our PCR results. These findings open interesting perspectives for the molecular taxonomy of the genus Pseudomonas and the development of diagnostic tools.