DYNAMIC OF BACTERIAL COMMUNITY COLONIZATION IN HIGH-ALTITUDE MOUNTAIN ENVIRONMENTS

Glacier forelands after glacier retreat are composed of harsh environmental niches characterized by severe climatic regimes and barren substrate with low total carbon and nitrogen content. Moraines represent ideal sites to study primary succession because glacier retreat releases an incoherent mineral substrate, where the primary succession and plant community establishment are key events in soil formation and fertilization. The underlying mechanisms driving this ecological succession remain still to be deepened. The physical and biogeochemical weathering processes provide soluble plant nutrient elements and when the plant colonization on parent materials occurs, the development of glacier foreland into fertile soils starts through rhizodeposition, exudation of nutritive substances, and decaying biomass. In these conditions, pioneer plants can select rhizosphere microbial communities able to promote plant growth thanks to the interactions with the system nutrient cycling. Moreover the rhizosphere community importantly contributes to the ecosystem functioning and carbon sequestration. Main purposes of this study were: i) to characterize bacterial communities involved in soil neo-genesis processes under the environmental harsh condition and nutrient scarcity typical of high mountain moraines; ii) to assess the diversity, structure and role of bacterial communities associated to pioneer plant grown in a deglaciated alpine environment. We studied first the Lobuche glacier moraines (Mount Everest area, Khumbu Valley, Nepal, about 5100 m a.s.l.), where we identified environmental niches characterized by different stages of biotic colonization, from bare mineral substrate to complex biological soil crusts (BSC). Seven sites of mineral proto-soil covered by BSC evidenced the ongoing soil development. The sites differed in several environmental parameters which could indicate different soil quality and developmental stages. Automated ribosomal intergenic spacer analysis (ARISA) showed distinctive bacterial community per each site and differences between the BSC and the below mineral substrate within the same site. The second studied site was the Weisskugel glacier foreland in the upper Matsch valley within the upper Vinschgau Valley (South Tyrol). Upper Matsch valley showed a wide range of ecosystems: grasslands, bogs and poor fens, vertical rocky walls colonized by lichens, rocky glaciers, isolated pioneer plants, loosely organized floristic proto-communities, transition and mature grassland stages. The study area is below a glacier foreland at 2400 m a.s.l. The vegetation colonization was evolved since 1840 when the Matschter glacier began to retreat. The bacterial communities associated to different environmental matrices i.e. rock surfaces, proto-soils, riparian sediments, lichen thalli, and water springs biofilms were investigated by three molecular techniques with different taxonomic resolutions: denaturing gradient gel electrophoresis (DGGE), length heterogeneity-PCR (LH-PCR), and ARISA. Bacterial communities were mainly composed of Acidobacteria, Proteobacteria, and Cyanobacteria but variations occurred among the sites. Proteobacteria were more represented in sediments, biofilms, and lichens whereas Acidobacteria were mostly found in proto-soils and Cyanobacteria on rocks. Firmicutes and Bacteroidetes were mainly found in biofilms. UniFrac P values confirmed a significant difference among different matrices. Significant differences (P < 0.001) in beta diversity were observed at the genus–species level, except for lichens and rocks which showed a more similar community structure, while two distinct bacterial communities between lichens and rocks were found at deep taxonomic resolution. In the same alpine ecosystem we investigated the effect of plant species on the rhizobacterial communities of 33 plant individuals belonging to 13 different pioneer species. We compared these bacterial communities to those from similar non vegetated patches as control. To obtain a culture-independent picture of the samples, metagenomic DNA was extracted from both rhizosphere and bulk soil samples and analyzed by ARISA and DGGE. ARISA fingerprinting showed different genetic structure per each plant species, extremely different from bulk soil bacterial communities, and the DGGE analysis showed rhizosphere bacterial communities mainly composed by Acidobacteria and Proteobacteria. Unifrac P values of DGGE results confirmed the rhizosphere effect exerted by the different plant species (P < 0.05). We concluded that in early primary succession pioneer plant species can select distinct rhizobacterial communities. Moreover, the rhizosphere effect on the bacterial communities associated to 21 alpine plants belonging to 14 pioneer species within three floristic communities (RW, FI, M sites) was studied. When little sites surrounded by big stones (safe sites) are filled up of stone debris or mud, opportunistic pioneer plant species settle down and form new floristic consortia strongly influenced by the chemistry of the lytic materials. While RW site was a safe site of early developmental stage, FI site represented an intermediate stage and M site could be considered a later stage where floristic consortia are matured. ARISA fingerprints showed different bacterial genetic structure per each patchy floristic communities which differed also from the bulk soil (BS site) bacterial communities. ANOSIM and PERMANOVA analyses indicated as significant the differences among the ARISA profiles of the sites (P < 0.0001). When plants of the same species occurred within the same site, almost all their rhizosphere bacterial communities clustered together. The Unifrac significance value (P < 0.05) revealed significant differences between BS site and the vegetated sites with a weak similarity to the RW site. The intermediate plant colonization stage, FI site, did not differ significantly from the RW and the M vegetated sites. These results highlighted the peculiar effect of the different floristic communities rhizospheres on their soil bacterial communities. The percentage of N and C in the four soils confirmed a different developmental stage per each of the sites with a kind of gradient from BS through RW, FI up to the M site. The biodiversity, structure and role of both the overall and the active rhizobacterial communities of the most common floristic associations in the valley (Cetrario Loiseleurenion, Nardion strictae, Festucetum halleri) were studied during the early and late growing season at two different time points of glacier retreat characterized by particular environmental parameters. ARISA and 16S rRNA gene pyrosequencing were applied to metagenomic DNA and RNA. The presence of nitrogen cycle key-players was detected with specific 16S rRNA gene PCR-DGGE and nifH gene pyrosequencing. The overall rhizobacterial community structure of the two transects in the early and late growing seasons were significantly different from the correspondent active rhizobacterial communities (PERMANOVA P < 0.0001). Within the overall bacterial communities, each plot differed significantly according first to the sampling season and then to the soil age, whereas within the active bacterial communities the plots clustered mainly according to the season. A marked shift in active Proteobacteria, Acidobacteria, Planctomycetes highlighted the different between the different vegetation plots, growing seasons and soil ages. Moreover, most of the rhizobacterial communities involved to N-cycle exhibited specific diversity according to the growing season. In high-altitude mountain environment, niches with different soil developmental stages coexist in the same area and different environmental constraints (growing season, site position, plant species) lead to the selection of specific pioneer bacterial communities characterized by peculiar taxonomic patterns and functional diversity.