Anthropization of Palaeolithic caves open for tourism may favour collembola invasion and result in the formation of black stains attributed to pigmented fungi. However, ecological processes ...underpinning black stain formation are not fully understood. Here, we tested the hypotheses that black stains from the Apse room of Lascaux Cave display a specific microbiota enriched in pigmented fungi, and that collembola thriving on the stains have the potential to consume and disseminate these black fungi. Metabarcoding showed that the microbiota of black stains and neighbouring unstained parts strongly differed, with in black stains a higher prevalence of Ochroconis and other pigmented fungi and the strong regression of Pseudomonas bacteria (whose isolates inhibited in vitro the growth of pigmented fungi). Isotopic analyses indicated that Folsomia candida collembola thriving on stains could feed on black stain in situ and assimilate the pigmented fungi they were fed with in vitro. They could carry these fungi and disseminate them when tested with complex black stains from Lascaux. This shows that black stain formation is linked to the development of pigmented fungi, which coincides with the elimination of antagonistic pseudomonads, and points towards a key role of F. candida collembola in the dynamics of pigmented fungi.
During evolution, plants have become associated with guilds of plant-growth-promoting rhizobacteria (PGPR), which raises the possibility that individual PGPR populations may have developed mechanisms ...to cointeract with one another on plant roots. We hypothesize that this has resulted in signaling phenomena between different types of PGPR colonizing the same roots. Here, the objective was to determine whether the Pseudomonas secondary metabolite 2,4-diacetylphloroglucinol (DAPG) can act as a signal on Azospirillum PGPR and enhance the phytostimulation effects of the latter. On roots, the DAPG-producing Pseudomonas fluorescens F113 strain but not its phl-negative mutant enhanced the phytostimulatory effect of Azospirillum brasilense Sp245-Rif on wheat. Accordingly, DAPG enhanced Sp245-Rif traits involved in root colonization (cell motility, biofilm formation, and poly-β-hydroxybutyrate production) and phytostimulation (auxin production). A differential fluorescence induction promoter-trapping approach based on flow cytometry was then used to identify Sp245-Rif genes upregulated by DAPG. DAPG enhanced expression of a wide range of Sp245-Rif genes, including genes involved in phytostimulation. Four of them (i.e., ppdC, flgE, nirK, and nifX-nifB) tended to be upregulated on roots in the presence of P. fluorescens F113 compared with its phl-negative mutant. Our results indicate that DAPG can act as a signal by which some beneficial pseudomonads may stimulate plant-beneficial activities of Azospirillum PGPR.
Cave anthropization related to rock art tourism can lead to cave microbiota imbalance and microbial alterations threatening Paleolithic artwork, but the underpinning microbial changes are poorly ...understood. Caves can be microbiologically heterogeneous and certain rock wall alterations may develop in different rooms despite probable spatial heterogeneity of the cave microbiome, suggesting that a same surface alteration might involve a subset of cosmopolitan taxa widespread in each cave room. We tested this hypothesis in Lascaux, by comparing recent alterations (dark zones) and nearby unmarked surfaces in nine locations within the cave.
Illumina MiSeq metabarcoding of unmarked surfaces confirmed microbiome heterogeneity of the cave. Against this background, the microbial communities of unmarked and altered surfaces differed at each location. The use of a decision matrix showed that microbiota changes in relation to dark zone formation could differ according to location, but dark zones from different locations displayed microbial similarities. Thus, dark zones harbor bacterial and fungal taxa that are cosmopolitan at the scale of Lascaux, as well as dark zone-specific taxa present (i) at all locations in the cave (i.e. the six bacterial genera Microbacterium, Actinophytocola, Lactobacillus, Bosea, Neochlamydia and Tsukamurella) or (ii) only at particular locations within Lascaux. Scanning electron microscopy observations and most qPCR data evidenced microbial proliferation in dark zones.
Findings point to the proliferation of different types of taxa in dark zones, i.e. Lascaux-cosmopolitan bacteria and fungi, dark zone-specific bacteria present at all locations, and dark zone-specific bacteria and fungi present at certain locations only. This probably explains why dark zones could form in various areas of the cave and suggests that the spread of these alterations might continue according to the area of distribution of key widespread taxa.
Aims The plant-beneficial bacterium Pseudomonas fluorescens F113 harbours an acdS gene, which enables deamination of 1-aminocyclopropane-1-carboxylate. The impact of abiotic and biotic factors on the ...expression of this gene was assessed, as well as the plant-beneficial properties of F113 under different soil moistures. Methods An acdS-egfp biosensor was constructed in F113, validated in vitro and used to analyse, by microscopy, its expression on roots of Zea mays comparatively to Beta vulgaris. An acdS mutant was constructed and compared with the wild-type to characterize plant-beneficial effects of F113 on maize lines EP1 and FV2, under well-watered and water deficit conditions. Results Different patterns of root colonization and acdS expression were observed according to plant genotype. acdS rhizoplane expression was higher on Beta vulgaris, and on maize line FV2 and hybrid PR37Y15 than on maize line EP1 and teosinte. Strain F113 but not its acdS mutant promoted root growth of EP1 under well-watered conditions and germination of FV2 under water deficit conditions. Conclusions Maize lines differed in their ability to induce acdS expression and to respond to P. fluorescens F113. The maize line leading to higher acdS expression, FV2, was the one benefiting from inoculation under water deficit.
Plant Growth-Promoting Rhizobacteria (PGPR) enhance plant health and growth using a variety of traits. Effective PGPR strains typically exhibit multiple plant-beneficial properties, but whether they ...are better adapted to the rhizosphere than PGPR strains with fewer plant-beneficial properties is unknown. Here, we tested the hypothesis that strains with higher numbers of plant-beneficial properties would be preferentially selected by plant roots. To this end, the co-occurrence of 18 properties involved in enhanced plant nutrition, plant hormone modulation, or pathogen inhibition was analyzed by molecular and biochemical methods in a collection of maize rhizosphere and bulk soil isolates of fluorescent Pseudomonas. Twelve plant-beneficial properties were found among the 698 isolates. Contrarily to expectation, maize preferentially selected pseudomonads with low numbers of plant-beneficial properties (up to five). This selection was not due to the predominance of strains with specific assortments of these properties, or with specific taxonomic status. Therefore, the occurrence of only few plant-beneficial properties appeared favorable for root colonization by pseudomonads.
The World-famous UNESCO heritage from the Paleolithic human society, Lascaux Cave (France), has endeavored intense microclimatic perturbations, in part due to high touristic pressure. These ...perturbations have resulted in numerous disturbances of the cave ecosystem, including on its microbial compartment, which resulted in the formation of black stains especially on the rock faces of the passage. We investigated the cave microbiome in this part of Lascaux by sampling three mineral substrates (soil, banks, and inclined planes) on and outside stains to assess current cave microbial assemblage and explore the possibility that pigmented microorganisms involved in stain development occur as microbial consortia.
Microbial abundance and diversity were assessed by means of quantitative PCR and high-throughput sequencing (Illumina MiSeq) of several DNA and cDNA taxonomic markers. Five sampling campaigns were carried out during winter and summer to embrace potential seasonal effect in this somewhat stable environment (based on measurements of temperature and CO
concentration).
While the season or type of mineral substrate did not affect the abundances of bacteria and micro-eukaryotes on or outside stains, mineral substrate rather than stain presence appears to be the most significant factor determining microbial diversity and structuring microbial community, regardless of whether DNA or cDNA markers were considered. A phylogenetic signal was also detected in relation to substrate types, presence of stains but not with season among the OTUs common to the three substrates. Co-occurrence network analyses showed that most bacterial and fungal interactions were positive regardless of the factor tested (season, substrate, or stain), but these networks varied according to ecological conditions and time. Microorganisms known to harbor pigmentation ability were well established inside but also outside black stains, which may be prerequisite for subsequent stain formation.
This first high throughput sequencing performed in Lascaux Cave showed that black stains were secondary to mineral substrate in determining microbiome community structure, regardless of whether total or transcriptionally active bacterial and micro-eukaryotic communities were considered. These results revealed the potential for new stain formation and highlight the need for careful microbiome management to avoid further cave wall degradation.
Actinobacteria are important cave inhabitants, but knowledge of how anthropization and anthropization‐related visual marks affect this community on cave walls is lacking. We compared Actinobacteria ...communities among four French limestone caves (Mouflon, Reille, Rouffignac, and Lascaux) ranging from pristine to anthropized, and within Lascaux Cave between marked (wall visual marks) and unmarked areas in different rooms (Sas‐1, Passage, Apse, and Diaclase). In addition to the 16S rRNA gene marker, 441 bp fragments of the hsp65 gene were used and an hsp65‐related taxonomic database was constructed for the identification of Actinobacteria to the species level by Illumina‐MiSeq analysis. The hsp65 marker revealed higher resolution for species and higher richness (99% operational taxonomic units cutoff) versus the 16S rRNA gene; however, more taxa were identified at higher taxonomic ranks. Actinobacteria communities varied between Mouflon and Reille caves (both pristine), and Rouffignac and Lascaux (both anthropized). Rouffignac displayed high diversity of Nocardia, suggesting human inputs, and Lascaux exhibited high Mycobacterium relative abundance, whereas Gaiellales were typical in pristine caves and the Diaclase (least affected area of Lascaux Cave). Within Lascaux, Pseudonocardiaceae dominated on unmarked walls and Streptomycetaceae (especially Streptomyces mirabilis) on marked walls, indicating a possible role in mark formation. A new taxonomic database was developed. Although not all Actinobacteria species were represented, the use of the hsp65 marker enabled species‐level variations of the Actinobacteria community to be documented based on the extent of anthropogenic pressure. This approach proved effective when comparing different limestone caves or specific conditions within one cave.
hsp65 Marker as a complement to 16S rRNA gene in Actinobacteria community detection with the metabarcoding approach. Some Actinobacteria species were detected for the first time in caves.Taxa related to rare opportunistic pathogens detected in anthropized caves. Streptomyces were detected in marked areas of Lascaux caves. The in‐house hsp65‐based database is available to the scientific community
The genetic and evolutionary relationship among 2,4-diacetylphloroglucinol (Phl)-producing pseudomonads that protect plants from soil-borne pathogens were investigated by multilocus sequence typing. ...A total of 65 pseudomonads consisting of 58 Phl-positive biocontrol strains of worldwide origin and seven Phl-negative representatives of characterized Pseudomonas species were compared using 10 housekeeping genes (i.e. rrs, dsbA, gyrB, rpoD, fdxA, recA, rpoB, fusA, rpsL and rpsG). Multilocus sequence typing differentiated 51 strains among 58 Phl-positive pseudomonads and proved to be as discriminative as enterobacterial repetitive intergenic consensus polymerase chain reaction profiling. As phylogenetic trees inferred from each locus were rather incongruent with one another, we derived the topology from all concatenated loci, which led to the identification of six main groups of Phl-producing Pseudomonas spp. Taxonomically, these groups could correspond to at least six different species. Linkage disequilibrium analysis pointed to a rather clonal structure, even when the analysis was restricted to Phl-producing pseudomonads from a same geographic location or a same phylogenetic group. Intragenic recombination was evidenced for gyrB, rpoD and fdxA, but was shown to be a weaker force than mutation in the origin of intragenetic diversity. This is the first multilocus assessment of the phylogeny and population structure of an ecologically important bacterial group involved in plant disease suppression.
Natural suppressiveness of Swiss soils to Thielaviopsis basicola-mediated tobacco black root rot is thought to depend mainly on fluorescent pseudomonads producing the antimicrobial compound ...2,4-diacetylphloroglucinol. However, the relation between these phl+Pseudomonas populations and both the T. basicola population and disease suppressiveness in these soils is unknown, and real-time PCR tools were used to address this issue. Significant rhizosphere levels of phl+ pseudomonads had been evidenced before in suppressive as well as conducive soils, but this was done using culture-based approaches only. Here, a phlD-based real-time PCR method targeting all phlD+ genotypes, unlike the strain-specific real-time PCR methods available so far, was developed and validated (detection limit around 4 log cells g−1 soil and amplification efficiency >80%). When implemented on Swiss soils suppressive or conducive to black root rot, it clarified the hypothesis that suppressiveness does not require higher levels of phlD+ pseudomonads. The parallel assessment of T. basicola population by real-time PCR (method of Huang and Kang, 2010) suggested that suppressiveness was not due to the inability of the pathogen to colonize the rhizosphere and tobacco roots in suppressive soils, but rather that phl+ pseudomonads might act by limiting root penetration by the pathogen in suppressive soils. In conclusion, an effective real-time PCR method was achieved for phlD+ pseudomonads and can be used to monitor this key functional group in various environmental conditions, including here to better understand the ecology of suppressive soils.
► A phlD-based real-time PCR tool is designed for phloroglucinol-producing pseudomonads. ► Thielaviopsis suppressiveness is independent from phlD+Pseudomonas density in soil. ► Thielaviopsis suppressiveness does not entail pathogen inhibition in the rhizosphere. ► phlD+ strains probably act by limiting pathogen root penetration in suppressive soils.
The root disease take-all, caused by Gaeumannomyces graminis var. tritici, can be managed by monoculture-induced take-all decline (TAD). This natural biocontrol mechanism typically occurs after a ...take-all outbreak and is believed to arise from an enrichment of antagonistic populations in the rhizosphere. However, it is not known whether these changes are induced by the monoculture or by ecological rhizosphere conditions due to a disease outbreak and subsequent attenuation. This question was addressed by comparing the rhizosphere microflora of barley, either inoculated with the pathogen or noninoculated, in a microcosm experiment in five consecutive vegetation cycles. TAD occurred in soil inoculated with the pathogen but not in noninoculated soil. Bacterial community analysis using terminal restriction fragment length polymorphism of 16S rRNA showed pronounced population shifts in the successive vegetation cycles, but pathogen inoculation had little effect. To elucidate rhizobacterial dynamics during TAD development, a 16S rRNA-based taxonomic microarray was used. Actinobacteria were the prevailing indicators in the first vegetation cycle, whereas the third cycle--affected most severely by take-all--was characterized by Proteobacteria, Bacteroidetes, Chloroflexi, Planctomycetes, and ACIDOBACTERIA: Indicator taxa for the last cycle (TAD) belonged exclusively to Proteobacteria, including several genera with known biocontrol traits. Our results suggest that TAD involves monoculture-induced enrichment of plant-beneficial taxa.
PDF
