The plant microbiome refers to the composite community of microbes associated with plants (Mendes et al., 2013). Plants constantly feed the adjacent rhizosphere, phyllosphere, endosphere, and spermosphere by depositing assimilated carbon thereby modulating microbial composition and activities. Complex communities of plant-associated microbes are an untapped reservoir that promotes plant health and productivity (Bisseling et al., 2009). By providing rhizodeposits viz; exudates, mucilage, border cells, the host plants diversify the members of bacteria, fungi, oomycetes, nematodes, protozoa, algae, viruses, archaea, and arthropods in the rhizosphere (Cook et al., 1995; Pierret et al., 2007; Raaijmakers et al., 2009). Moreover, rhizosphere specific differential selection of bacterial communities is displayed by different pioneer plant species: Festuca halleri All, Gnaphalium supinum (L.), Leucanthemopsis alpina (L.), Linaria alpina (L.) Mill, Minuartia sedoides (L.) Hiern, Potentilla aurea (L.), Saxifraga bryoides (L.), Sedum alpestre Vill., Senecio carniolicus (Willd.) Braun-Blanq, Sibbaldia procumbens (L.), Silene acaulis (L.) Jacq, and Veronica bellidioides (L.) in high mountain ecosystem and well documented (Ciccazzo et al., 2014).

Rhizospheric microbial communities are pivotal to natural ecosystems, maintenance of ecosystem diversity and in restoration projects through means of transplantation practices. Such practices apart from repairing landscapes also act as conservation tool for relocating plant communities, saving species from condemned locations, enhancing dwindling populations or creating new ones (Fahselt, 2007). Nevertheless, microbial communities are poorly understood and ignored while considering such practices. Lau and Lennon (2011) have shown that below ground rhizospheric microbial community has the power of modulating plant evolutionary process, thereby affecting patterns of natural selection on plant traits. By altering composition of below-ground microbial community with treatments, the authors showed Brassica rapa plants associated with simplified, less diverse microbial community compared to plants with highly diverse complex microbial community, were smaller, had reduced chlorophyll content, produced less flower and had less fecundity. Such reports relate to the reciprocal interactions that exist between the plant and associated microbes in modulating each other. With a significant fraction of global plant taxas classified as endangered, endemic, threatened or rare, long term sustainability and conservation strategies involving transplantation practices may be strengthened with an in-depth understanding of the plant microbiome as outlined and exemplified by global organizations. Fate of rare, threatened or endangered plants is often tied to undisturbed habitat, intact communities and relocation difficulties arise in moving such single species (Fahselt, 2007). Reports on the rhizosphere microbes are concentrated mostly on the complex mycorrhizal communities in the domesticated plants from soil samples to which these plants were once introduced. Shen and Wang (2011) showed that transplanted seedlings of endemic and critically endangered Euryodendron excelsum with native arbuscular mycorrhizal fungi enhanced survivability by 80% compared to 46% on their own. Such studies suggest that below ground microbial communities may play important role in determining diversity, productivity and composition of above ground plant community. Alternatively, phylochip, pyrosequencing approaches are limited to maize crop, oat in microcosm/forest soil, sugar beet, potato, Arabidopsis thaliana to mention a few (Mendes et al., 2013). Often overlooked, microbiome studies of endemic medicinal plants are still primitive.

The Eastern Himalayan (EH) biodiversity hotspots (an amalgamation of Indo Malayan, Palaearctic and Sino-Japanese region) covers an area of 5,25,000 km², spreads from the Kaligandaki Valley, Kosi Basin, Mechi Basin (central Nepal) to Yunnan province (northwest China) (Shrestha et al., 2010) and incubates 200 Global ecoregions (Sharma et al., 2010). Of the Global Biodiversity Hotspots (34) EH encompasses 25 ecoregions, of which 19 have high conservation significance in terms of their global conservation value (Shrestha et al., 2010). Myers et al. (2000) predicted that the Indo-Burma Hotspot alone shelters 2.3% of global endemic plants and 1.9% of global endemic vertebrates with a species density of 7.0 and 0.5 per 100 km² area, respectively.

The Tawang district (27.58 N 91.86 E, elevation 3048 m) in Arunachal Pradesh (hereafter AP) falls in the alpine shrub bioclimatic zone, experiences a high number of frost/ice days and high precipitation. Toward the extreme north in Pankang Thang, Nagula mostly conifer trees, rhododendron shrubs, alpine meadows are prevalent. The vegetation above the tree line entertains a community of dense juniper, Ponerorchis spp., Boschniakia spp., Saussurea obvallata and Rhododendron shrubberies that extend to about 4,500 m (Sekar and Srivastava, 2010). Many high altitude medicinal plants such as Fritillaria cirrhosa, Aconitum, and Rheum are also found in this region. Rhododendron genera are widely distributed at higher elevations in the Sino-Himalayan regions. A maximum concentration of 86% species is observed in AP. Pharmacologically, anti-inflammatory, anti-nociceptive, anti-diabetic, hepatoprotective, antioxidant, and anti-diarrhoeal activity of Rhododendron arboreum is reported in potential animal models (Hariharan and Rangaswami, 1966; Swaroop et al., 2005; Verma et al., 2011; Srivastava, 2012).

Although, EH is documented as biodiversity hotspot, microbial communities of cold adapted endemic plants of this region was not previously assessed. Such mountain ecosystems are under constant pressure of loss in biological diversity and are vulnerable due to climate change. Such issues are addressed by the special programs under the Convention on Biological Diversity (CBD) which aims at reducing the loss at global, regional, or national levels in the EH region.

The aim of the present work was to assess the colonization pattern of the rhizospheric bacteria in medicinally important endemic R. arboreum Sm. Subsp. delavayi occurring at high altitude acidic soils using Illumina based 16S sequencing. Plant specific selection of rhizospheric bacterial community is reported for field grown agricultural plants (Smalla et al., 2001) and different pioneer plants in high mountain ecosystem (Ciccazzo et al., 2014) but variable results were reported in non-agricultural perennial bunch grasses, herbaceous plants. We used DGGE fingerprints to describe whether the plant species effect on rhizospheric bacterial community composition exists in endemic medicinal R. arboreum plant in natural high mountain ecosystem along altitudinal blocks. To find out any influence of edaphic factors, elevational gradients variables were scaled on to dominating groups, sub-groups that were identified by amplicon sequencing. Efforts in understanding the rhizosphere of medicinal, endemic plant species will be vital for bioprospection of ecto- and endophytic microbes and in selecting suitable receptor sites for ex situ conservation, transplantation models.

We assessed the rhizospheric bacterial community of endemic medicinal plant R. arboreum Sm. subsp. delavayi and its association with soil properties at a high altitude region upstream of Tawang, India situated in the eastern slope of the Himalaya. DGGE profiles of bulk soil and rhizospheric soil ruled out the possibility for specific bacterial group selection by the dry, cold Himalayan soils and emphasized on the phenomenon of “rhizosphere effect.” In order to understand the mechanisms involved in specificity (adherence, recognition) and rhizosphere enrichment processes, microbial ecology studies of rhizosphere from native plants in natural ecosystem other than agricultural or model crops will be important. Comparative, biogeographic studies will help in assessing the possible role of rhizospheric microbial communities as selective constraints for the diversification of more than 420000 plants (Berg and Smalla, 2009). Many previous studies on rhizosphere bacterial community have identified soil type and plant specificity as one of the major factor in shaping microbial communities. Significant differences among the community composition based on both DGGE fingerprint and pyrosequencing sequencing analysis of 16SrRNA amplicons from bulk and rhizosphere soil of lettuce (cv. “Tizian”) grown in three soils diluvial, alluvial and lowess was identified (Schreiter et al., 2014). In addition a strong partitioning effect between bulk and rhizospheric soil of lettuce was also observed at two different time points (3 and 7 weeks post planting). Similarly differing relationship between C concentrations (and in some experiments N) along the root and the distribution and abundance of oligotrophic and copiotrophic bacteria in the bulk and rhizospheres of lettuce and tomato was reported by Maloney et al. (1997). Soil type dependent composition of bacterial communities in the rhizosphere of A. thaliana grown under greenhouse conditions in different soils was also observed (Bulgarelli et al., 2012; Lundberg et al., 2012). Although the same phyla were reported in the rhizosphere of different plants viz; oak, A. thaliana, Bt- and conventional maize variety (Uroz et al., 2010; Bulgarelli et al., 2012; Lundberg et al., 2012; Dohrmann et al., 2013) their relative abundances substantially differed to that of bulk soil.

We observed a significant difference in bacterial community structure at different altitudes but could not find a difference in diversity values along the altitudinal trend, as opposed to other studies supporting the Baas-becking hypothesis (Finkel et al., 2012). This might be due to significant intervals between sampling sites and requires considerably large sampling effort at short incremental intervals to comment on it. In another study pertaining to the biogeographic distribution of soil archaeal and bacterial communities across three vertical climate zones (3,106–4,479 m.a.s.l.) in Mt. Shegyla using pyrosequencing approaches the authors showed altitudinal distribution patterns of soil bacterial and archaeal assemblages with increase in elevation (Wang et al., 2014). Similar altitudinal trends were found in Northern Slope of Changbai Mountain, China (Zhang et al., 2013). According to another study in the Colorado Rocky Mountains, phylogenetic β-diversity of bacterial lineages were not randomly distributed, but rather had a gradient dependent spatial structure (Bryant et al., 2008).

The Mantel’s statistic identified pH, OM and TN in the soil were reshaping the community composition differences as observed by Illumina sequencing. The pattern of specific bacterial phylum responses to pH gradients is well documented (Lauber et al., 2009). In a gradient dependent study comprising soil bacterial communities across six elevations with vegetation types ranging from forest to alpine tundra in Mt. Changbai, China, pH was established to be the best predictor for community composition (Sheng et al., 2013). Acidobacteria responses to decreasing pH gradient uphill as observed in our current study (Figure 2) co-aligns with that of studies from diverse array of 87 soils across North and South America by Jones et al. (2009), where they combined pyrosequencing and clone library to arrive at the conclusion. The significant negative correlation of Acidobacteria abundance to TN in our study was also corroboratory to finding by Foesel et al. (2014). The influence of pH, however, in the grassland soils was found positively correlated to Acidobacteria abundance which is in contrast to our current finding and to that of Jones et al. (2009). Comparative genomics involving three genomes of Acidobacteria suggests their role in nitrogen cycling in soils and sediments is the reduction of nitrate, nitrite, and possibly nitric oxide (Ward et al., 2009). The soil microbial communities across vertical climate zones on the Tibetan Plateau also showed an elevational zonation feature, and distribution patterns of soil microbes were regulated mainly by soil pH, SOC and TN (Finkel et al., 2012). Within this phylum family of Coriobacteriaceae, Solibacteraceae, and Acidobacteriaceae were highly dominant with genus level classification of Candidatus koribacter and C. solibacter. A clone library approach (Shivaji et al., 2011) to understand the bacterial diversity in the Pindari glacier in the Himalayan mountain ranges India, showed 15.9% of the clones were represented by Acidobacteria in the library with genera identified as Acidobacterium, Bryobacterium, Geothrix, Koribacter, and Solibacter. Comparing three genome of Acidobacteria showed large class of high-molecular weight excreted proteins suggesting important factors for desiccation resistance, biofilm formation and soil structure modeling. Apart from presence of polyketide synthase and macrolide synthase, which shows high propensity of novel antimicrobial sources, exhibition of slow metabolic rates under low nutrient conditions equip them to tolerate fluctuations in soil hydration (Ward et al., 2009). Colonization of Acidobacteria in Rhododendron rhizosphere might shed light on adaption biology and plant–microbe interactions.

Actinobacteria occurred substantially at all four sites; however, the proportion in the rhizosphere was more at the upper region than lower. None of the measured parameters accounted for the changes in actinobacterial community differences, suggesting other factors not studied in the current study might account for the change. In a culture-independent survey of high elevation mineral soils from the dry valleys of Himalaya, the diversity of bacteria varied similar to the dry valleys of Antarctica (Costello et al., 2009). They reported the dominant groups in high altitude Himalayas were Actinobacteria, Acidobacteria, and Cyanobacteria, whereas in high Andes were found to be Actinobacteria, Chloroflexi. It is noteworthy that the Chloroflexi phylum does not represent most of its phylotypes in low-elevation habitats but inhabits cold and plant dominated high elevation ecosystems (Costello and Schmidt, 2006; Freeman et al., 2009). A previous detailed study in cool temperate mountain in Northwest of Yunnan, China at 3000–3900 m with annual temperature of 5°C reported Streptomyces, Micromonospora, Nocardia, and Thermoactinomyces strains where 97% constituted of Streptomyces genera. Also the snowy mountain in the same region at 3900–4500 which experiences average annual temperature below 0°C and represented by vegetation of R. primulaeflorum, R. nivale among others showed Streptomyces populations of 83% (Xu et al., 1996). Psychrophilic Actinomycetes genera identified were Streptomyces, Micromonospora, Actinomadura, Saccharopolyspora, and Nocardia. A recent study pertaining to the deglaciated high altitude soils of Himalaya along transacts from 5000 to 6000 m corresponded to equally likely phylogenetically distant and closely related communities at 50 m short distances and no significant difference in community structure was observed in response to time and season. Acidobacteria, Proteobacteria, Verrucomicrobia, and Actinobacteria were encountered at 21, 19.8, 14.9, and 10.3% average frequency, respectively (Stres et al., 2013).

It is interesting to notice the prevalence of Rhodococcus, an important genus of the family Nocardiaceae, phylum Actinobacteria with higher abundance in the lower altitudinal region. Rhodococcus is an endophytic bacterium which represents an enormous untapped source of microbial diversity and prospect of novel bioactive products. The wealth of Rhodococcus reported from North Eastern Asia suggests its importance. Example species include, Rhodococcus kroppenstedtii a novel species from Lahaul-Spiti Valley in Himalayan cold desert, India (Mayilraj et al., 2006), Rhodococcus kunmingensis from Taxus chinensis rhizosphere in Kunming, China (Wang et al., 2008), Rhodococcus kyotonensis from Kyoto, Japan (Li et al., 2007) and Rhodococcus cercidiphylli, an endophyte identified from leaf sample of Cercidiphyllum japonicum from Yunnan province, South-West China (Li et al., 2008). Moreover with increasing altitude, biological processes are posed with the challenge of increased irradiation by ultraviolet rays. Albarracín et al. (2013) reported isolated strains of Rhodococcus displaying an intrinsic and high UV-B resistive capability. These findings might suggest the selective colonization of specific Rhodococcus populations in the rhizosphere at the altitudinal sites of PTSO.

The dominant OTU’s were further explored at the species level for their closest validly published relative available in RDP or Silva by searching against EZtaxon-E One dominant OTU with cultured representative in RDP identified to be B. valentinum (JX514883) was also isolated from the rhizosphere of L. mariae-josephi endemic plant, and was equally abundant in both the lower altitude soil in our present study (Table 2). Navaro et al. (2014) have shown that specific inoculation of Bradyrhizobium symbiont strains in seeds of L. mariae-josephi, can be significant approach toward survival and recovery of an endangered legume species in its natural environment, and suggest it to be a significant approach toward conservation of endangered plant species. Interesting, plant specific root exudates viz; flavonoids, a diverse class of polyphenols secreted by soybean roots attracts the beneficial bacterium B. japonicum thereby serving as signals in plant microbe interaction (Morris et al., 1998). Moreover flavonoids isolated from white lupin roots additionally mobilized inorganic phosphorous and decreased soil microbial respiration, citrate mineralization and soil phosphohydrolase (Tomasi et al., 2008). Similarly, OTU_4 identified to be Rhodoplanes taxonomically was closest to unclassified Rhizobiales (AB240329) and was isolated from the biofilm of Phragmite rhizosphere. Although the structure of Proteobacteria sub-community was explained by TN content (Supplementary Figure S3), negative correlation between relative abundance and NO₃⁺ was observed. High abundance of Bradyrhizobium and Rhodoplanes from sequence reads suggests their important role in N₂ fixation in the rhizosphere (Yarwood et al., 2009). Other OTU’s having closest match to RDP sequences were uncultured clones or DGGE fragment isolated from diverse environments such as bamboo forest at high elevation, boreal forest soil with pH and Nitrogen gradient, un-vegetated soil at Singy Island, Antarctic habitat, snow melt soil at Alps.

The Indian Himalayan mountain ecosystem is one the most fragile in the world and the biodiversity of the region is vulnerable due to unprecedented stress owing to human perturbations. Our study concerns one of the medicinal plants endemic to the region and finding of this study will aid in maneuvering in situ as well as ex situ conservation strategies. We conclude by stating that, bacterial community differences in the rhizosphere of R. arboretum Sm. subsp. delavayi in the EH high altitude can be mostly predicted by pH, TN, OM. We believe our study is the first of its kind from this region summarizing the rhizospheric bacterial population of medicinally important endemic R. arboretum Sm. subsp. delavayi growing at high altitudes in cold climatic conditions. Although pH influence on bacterial community responses is a documented phenomenon, our study provides additional support on soil edaphic factors influence on different dominant taxas thriving at high altitudes experiencing extremes of weather conditions. The shift of acidobacterial relative abundance with pH changes provides evidence of niche specialization of acidobacterial subpopulations in high altitude cold soil. With rapid changes in biogeography and selection over time, the bacterial community is also undergoing a gradual replacement; hence findings of the current study will be invaluable assessors in understanding community ecology shifts and in long term sustainability, conservation of R. arboreum.

Conceived and designed the experiments: RD and RS. Performed the experiments: RD, AY, and RS. Bioinformatics and statistical data analysis: RD. Contributed reagents/materials/Analysis tools: RS and RD. Manuscript preparation: RD. Contributed in critically reviewing/correction: VG, BS, and PH.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.