Endophytic Bacterial Communities Associated with Roots and Leaves of Plants Growing in Chilean Extreme Environments

Several studies have demonstrated the relevance of endophytic bacteria on the growth and fitness of agriculturally-relevant plants. To our knowledge, however, little information is available on the composition, diversity, and interaction of endophytic bacterial communities in plants struggling for existence in the extreme environments of Chile, such as the Atacama Desert (AD) and Patagonia (PAT). The main objective of the present study was to analyze and compare the composition of endophytic bacterial communities associated with roots and leaves of representative plants growing in Chilean extreme environments. The plants sampled were: Distichlis spicate and Pluchea absinthioides from the AD, and Gaultheria mucronata and Hieracium pilosella from PAT. The abundance and composition of their endophytic bacterial communities was determined by quantitative PCR and high–throughput sequencing of 16S rRNA, respectively. Results indicated that there was a greater abundance of 16S rRNA genes in plants from PAT (1013 to 1014 copies g−1 DNA), compared with those from AD (1010 to 1012 copies g−1 DNA). In the AD, a greater bacterial diversity, as estimated by Shannon index, was found in P. absinthioides, compared with D. spicata. In both ecosystems, the greater relative abundances of endophytes were mainly attributed to members of the phyla Proteobacteria (14% to 68%), Firmicutes (26% to 41%), Actinobacteria (6 to 23%) and Bacteroidetes (1% to 21%). Our observations revealed that most of operational taxonomic units (OTUs) were not shared between tissue samples of different plant species in both locations, suggesting the effect of the plant genotype (species) on the bacterial endophyte communities in Chilean extreme environments, where Bacillaceae and Enterobacteriacea could serve as keystone taxa as revealed our linear discriminant analysis.

plant (e.g., Arabidopsis thaliana), commercially relevant plants for agriculture (e.g., wheat, soybean, rice, maize, etc.) and wild plant species (e.g., weeds and trees) grown under laboratory, greenhouse and fields conditions 1,2,7,8 . Consequently, we only have limited knowledge on the composition and interactions of microbiota and plants, especially endophytic bacterial communities, on native plant vegetation growing in extreme environments, such as hot and/or cold deserts. Thus, our understanding on microbial interactions in plant holobiont will be key in the develop of efficient strategies for native plant conservation and/or exploit the full yield potential of crop plants under climate change scenario 9 .
The country of Chile is long (4,270 km) and narrow (mean width 177 km) and harbors a great variety of pristine ecosystems. The Atacama Desert (AD) is located in the northern region of Chile (from 18°24′S to 29°55′S) and is considered among the driest places on earth. In contrast, the Chilean Patagonia (from 41°08S to 56°30′S) is located in the far south of the country and is a sub Antarctic region. Both regions have extreme environments and their plant-associated bacterial communities have been barely studied thus far. In this context, we have reported that members of the orders Enterobacteriales, Actinomycetales, and Rhizobiales comprise dominant groups of the bacterial communities in the rhizosphere (the soil influenced by plant roots) of shrubs grown in AD and Patagonia (PAT), namely Atriplex sp. and Chuquiraga sp., respectively 10 . Results of this study also suggested that some isolates, belonging to the genera Enterobacteria, Pseudomonas, and Bacillus, were putative PGPB. The ability of the native isolates from AD to act as PGPB was confirmed by formulation and inoculation of a bacterial consortium onto plants. These studies revealed that wheat plants inoculated with the consortium produced greater biomass under water shortage and field conditions, compared with uninoculated seedlings 11 . A recent study also showed a greater protection against salt stress in wheat plants inoculated with rhizosphere bacteria isolated from Andean Altiplano native plant (Parastrephia quadrangularis) in AD 12 . However, these studies did not take into account the composition and interaction of native endophytic bacteria in Chilean extreme environments, as well as their potential use as PGPB.
During the last several years, advances in high-throughput DNA sequencing (HTS) technologies (e.g., Illumina ® , PacBio ® and Oxford Nanopore ® ) have opened new windows into the microbial ecology of a variety of environments, allowing the detailed study of complex bacterial communities in nature as never seen before. Thus, HTS platforms have widely been used to decipher the structure and function of microbiota in different compartments of plants, including as the rhizosphere, endosphere (inner tissues of plants), and phyllosphere (the aerial part of plant leaves) 1,13 . Results of 454-pyrosequencing studies showed that the Proteobacteria (mainly Gammaproteobacteria) were the dominant taxa in the rhizospheres of Atriplex sp. and Stipa sp. (shrubs) grown in the AD 14 . These authors also postulated that native plants from Chilean extreme environments may attract, select, and conserve specific bacterial groups in order to sustain plant growth and tolerance to local harsh conditions. Based on this supposition, the main goal of the present study was to describe and compare the relative abundances and composition of bacterial communities associated with roots and leaves of plants grown in the AD and PAT regions of Chile by using HTS of 16S rRNA genes.
DNA extraction. Roots and leaves samples were separated and surface sterilized by repeated immersion in 70% (v/v) ethanol for 3 min, followed by 2.5% (v/v) sodium hypochlorite (NaOCl) for 5 min as described by Barra et al. 15 . Roots were exhaustively rinsed with sterile distilled water. Triplicate portions of roots and leaves were aseptically cut, frozen in liquid nitrogen, macerated and homogenized with a mortar and pestle, and stored at −80 °C until DNA extraction. Samples of the homogenized tissues (0.25 g) were used for DNA extraction with Quick-DNA TM Plant/seed Miniprep kits according to manufacturer instructions (Zymo Research, CA, USA). The quantity and purity of DNA extracts were determined by measuring absorbance at 260 nm and 280 nm by using a microplate spectrophotometer (Multiskan GO, Thermo Fisher Scientific, Inc., MA, USA).
Quantitative pCR. The abundance of endophytic bacteria in each tissue sample was determined by quantitative PCR (qPCR) by using a universal primer set for the bacterial 16S rRNA gene (Bac1369F 5′-CGG TGA ATA CGT TCY CGG-3′) and Prok1492R (5′-GGW TAC CTT GTT ACG ACT-3′) as previously described 16,17 . Briefly, PCR conditions were run with an enzyme activation step at 95 °C for 10 min, followed by 40 cycles of 15 s at 95 °C, and 1 min of annealing plus extension at 60 °C. PCR reactions were performed in triplicate per plant species (including technical triplicates) with 20 µg L −1 of total DNA in a StepOnePlus TM Real-Time PCR System (Applied Biosystems, Inc., CA, USA) using PowerUp TM SYBR TM Green Master Mix (Applied Biosystems, Inc.), by following the manufacturer instructions. The numbers obtained were normalized and analyzed by using one-way ANOVA, and comparisons were done by using Tukey's post-hoc test. Differences were considered to be significant when the P value was ≤0.05.
High-throughput DNA sequencing. The distribution and relative abundances of endophytic bacteria in root and leaf tissues was assessed by HTS using triplicate samples of each plant species as follow. The V4 hypervariable region of the 16S rRNA genes were amplified, for bacteria and archaea, by using primer set 515F (5′-GTG CCA GCM GCC GCG GTA A-3′) and 806R (5′-GGA CTA CHV GGG TWT CTA AT-3′). Sequencing was done www.nature.com/scientificreports www.nature.com/scientificreports/ by the University of Minnesota Genomics Center (UMGC, Minneapolis, MN, USA) 18 using barcode primers and the dual indexing method. Amplicons were gel purified, pooled, and paired-end sequenced at a read length of 300 nt on the Illumina MiSeq platform (Illumina, Inc., San Diego, CA, USA) from UMGC.
Bioinformatics and statistical Analysis. Sequences were analyzed by using mothur program ver. 1.34.0 (https://www.mothur.org) 19 . The first 150 nt were trimmed from sequences to remove low-quality regions at the ends of reads. Fastq-join software was used to join paried-end sequencing reads 20 , the joined sequencing reads were trimmed to maintain an average quality score >35, a homopolyer length >8 nt. Sequences with >2 mismatches in primer sequences and ambiguous bases were removed. High quality sequencing reads were aligned on the basis of the SILVA database ver. 123 21 , and subjected to a 2% pre-clustering step to remove possible sequence errors 22 . The UCHIME software was used to identify and remove probable chimeric sequences 23 . To avoid the influence of non-microbiota (e.g., Chloroplast and mitochondria), the sequences were futher filtered by Qiime to remove non-microbiota taxa before a subsequent analysis. Sequence data was rarefied to 700 and 4,500 sequence reads per data set prior to statistical analysis for AD and PAT, respectively. Raw sequencing data were deposited in the Sequence Read Archive (SRA) of NCBI under Accession Number SRP156290.
For statistical analysis, Alpha diversity indices, as well as Good's coverage, were calculated using the Shannon index and the abundance-based coverage estimate (ACE) through the mothur program. Visualization of the taxonomic distribution of microbial communities was performed using the "ggplot2" package in R 24 . Differences in beta diversity was evaluated by using analysis of similarity (ANOSIM) and permutational multivariate analysis of variance (PERMANOVA). Principal coordinate analysis (PCoA) was performed based on unweighted unifrac distance for the ordination 25 . The VennDiagram package in R was used to identify shared OTUs of endophytic bacterial communities between root and leave tissues 26 . Variations in taxa associating with root and leaf tissues were evaluated using linear discriminant analysis (LDA) of effect sizes 27 , which employs Kruskal-Wallis and Wilconxon rank-abundance tests and then utilizes linear discriminant analysis (LDA) to estimate effect sizes of the features. www.nature.com/scientificreports www.nature.com/scientificreports/

Results
Abundances of Bacteria. In general terms, plant tissues from Patagonia (G. mucronata and H. pilosella) had greater abundances of bacteria (from 10 13 to 10 14 16S rRNA gene copies g −1 template DNA), compared with those from AD (D. spicata and P. absinthioides) (from 10 10 to 10 12 16S rRNA genes copies g −1 template DNA), except roots from G. mucronata (Table 1). In AD plants, a significantly (P ≤ 0.05) greater abundance of bacteria in both tissues was found in P. absinthioides (2.6 × 10 11 and 2.7 × 10 12 16S rRNA gene copies g −1 template DNA in roots and leaves, respectively) compared with those from D. spicata (4.4 × 10 10 and 5.4 × 10 11 16S rRNA gene copies g −1 template DNA in roots and leaves, respectively). In PAT plants, a significantly (P ≤ 0.05) greater abundance of bacteria in both tissues was found in H. pilosella (8.1 × 10 13 and 5.4 × 10 14 16S rRNA gene copies g −1 template DNA in roots and leaves, respectively) compared with those from G. mucronata (1.3 × 10 13 and 3.0 × 10 10 16S rRNA gene copies g −1 template DNA in roots and leaves, respectively).

Composition of endophytic Bacterial Community in extreme environments. Sequence analyses
showed a lower estimated coverage in AD (from 91 to 95%) compared with PAT (from 98 to 99%) ( Table 2). The values of observed OTUs (S obs ) were lower in plant tissues from AD compared with those from PAT, ranging in values from 68 to 113 and 208 to 220 in roots, and 103 to 152 and 151 to 160 in leaves, respectively. Similarly, lower ACE values were also observed in AD compared to PAT plants, ranging in values of 148 to 188 and 263 to 314 in roots, and 139 to 211 and 190 to 245 in leaves, respectively. However, and as revealed by the Shannon index, there was significant differences (Tukey's post-hoc test, P < 0.05) in bacterial diversity in both tissues of P. absinthioides compared with tissues from D. spicata in AD (Table 2). In contrast, no significant differences in bacterial diversity were found in plant tissues from PAT plants.
In both ecosystems, the assignment of taxonomic affiliation to endophytic bacterial communities at the phylum level indicated that there were high relative abundances of Proteobacteria (14.88% to 68.53%), Firmicutes (26.03% to 41.59%), Actinobacteria (6.45% to 23.69%), and Bacteroidetes (1.09% to 21.21%) in both tissues ( Fig. 2A). It is noteworthy that the lowest relative abundance of Bacteroidetes was found in roots from D. spicata. This tissue, however, presented a relative abundance of 31.01% of members belonging to Euryarchaeota phylum ( Fig. 2A).
With respect to minor taxa, broad taxonomic diversity among samples was found. The tissues of AD plants (D. spicata and P. absinthioides) showed large relative abundances of Cyanobacteria, Lentisphaerae and Chloroflexi in roots, and Verrucomicrobia and Fusobacteria and Cyanobacteria in leaves (Fig. 2B). The tissues from PAT plants   mucronata and H. pilosella) showed a large relative abundance of Elusimicrobia, Fusobacteria, Spirochaetes and Acidobacteria in roots, and Elusimicrobia, Fusobacteria, TM7 and Verrucomicrobia in leaves (Fig. 2B). At family level, a wide taxonomic diversity among samples was also found. In AD plants, higher relative abundances of Halobacteriaceae (31.01%), Bacillaceae (24.67%) and Nocardiopsaceae (17.78%) are highlight in roots of D. spicata whereas a higher relative abundance of Halomonadaceae (25.81%) is highlight in leaves of P. absinthioides (Fig. 3). In PAT plants, a higher relative abundance of members belonging to Pseudomonaceae was found in roots (21%) and leaves (57.93%) from G. mucronata.
Differences between tissues and plant species were also confirmed by PCoA. In AD plants, a clear grouping between roots and leaves from D. spicata, and roots from P. absinthioides was observed (Fig. 4, AD). Similarly, a clear grouping between roots and leaves from H. pilosella, and roots from G. mucronata was also observed in PAT plants (Fig. 4, PAT).
shared and Unique operational taxonomic Units and Keytone taxa in extreme ecosystems. In relation to the distribution of shared and unique OTUs among endophytic bacterial communities in ecosystems, 53 out of 1075 OTUs were shared in AD, while 115 out of 1713 OTUs shared in PAT ecosystem ( Fig. 6 and Table 3). In AD plants, among these 53 shared OTUs, most belonged to the Firmicutes (20), followed by Proteobacteria (15) and Actinobacteria (13). In PAT plants, among these 115 shared OTUs, most belonged to the Firmicutes (37), followed by the Actinobacteria (29), Bacteroidetes (25) and Proteobacteria (24). In the PAT, there were a greater number of unique OTUs, relative to those seen at AD, where 305 and 341 and 306 and 195 plant specific OTUs were found in root and leaves from G. mucronata and H. pilosella, respectively   ( Table 3 and Fig. 5). In the AD, 234 and 224, and 218 and 93 plant specific OTUs were found in root and leaves from D. spicata and P. absinthioides, respectively (Table 3 and Fig. 5). In AD plants, most of unique OTUs belonged to the Firmicutes (353), followed by the Proteobacteria (213), Actinobacteria (90) and Bacteroidetes (87). Similarly, in PAT plants, most of unique OTUs belonged to the Firmicutes (512), followed by Proteobacteria (397), Actinobacteria (159) and Bacteroidetes (119) ( Table 3).
Linear discriminant analysis (LDA) of effect size was performed to determine which taxa varied among plant components at two ecosystems. Several bacterial taxa, which belong to the Bacillacea, Nocardiopsaceae, Ectothiorhodospiraceae, and Moraxellaceae (at family level), elucidative served as the keystone taxa in roots of D. spicata, while only the Propionivacteriaceae and Corynebacteriaceae could be used to indicate leaves of D. spicata and P. absinthioides, respectively (Fig. 6). Moreover, the Bacillaceae had the greatest effect size among the plant components in AD. Contrastingly, taxa belonging to the family level of the Enterobacteriaceae, Pseudomonadaeceae, Dermabacteraceae, Coriobacteriaceae, and Bacteroidaceae were the keystones presenting endophytic microbiota among plant compartments in PAT ecosystem (Fig. 6). Additionally, the Enterobacteriaceae had the highest effect size in PAT.

Discussion
Microbial endophytes play a central role in the ecology, evolution, and growth promotion of plants 2,4,28 . However, despite their importance, there is scant knowledge concerning endophytic microbial populations in plants living in extreme environments, including Chile. Our study showed that the abundances of bacteria from the endosphere of root and leaf tissues ranged from 10 10 to 10 12 and from 10 10 to 10 14 16S rRNA gene copies g −1 template DNA in AD and PAT, respectively; corresponding from 10 3 to 10 7 copies of 16S rRNA genes copies g −1 fresh tissue.
A wide range of endophytic prokaryote densities have been reported in tissues of different plants thus far. Similar to our finding, 10 14 16S rRNA gene copies g −1 template DNA of total endophytic prokaryotes were reported in endospheres of olive tree (Olea europaea L.) leaves collected from diverse Mediterranean ecosystems 29 . Higher abundances than those observed in this current study were reported to be present in the endosphere of rice (10 7 to 10 8 16S rRNA gene copies g −1 root) and crops (10 10 to 10 13 copies of 16S rRNA genes g −1 root) by Ruppel et al. 30 and Breidenbach et al. 31 , respectively. Under stress conditions, Blain et al. 32 recently reported abundances of endophytic bacteria from 10 3 to 10 5 16S rRNA gene copies g −1 from fresh roots of natural vegetation growing in a hydrocarbon-contaminated site. In addition, and to our knowledge, there are no studies www.nature.com/scientificreports www.nature.com/scientificreports/ reporting the abundances of endophytic bacterial populations in plants from Chilean extreme environments. It should be noted, however that a previous study of the rhizosphere of plants from AD and PAT revealed values of 10 9 and 10 11 16S rRNA genes copies g −1 of soil, respectively 33 .
With respect to analyses of alpha diversity of endophytic bacterial communities in root and leave tissues, the number of OTUs observed (97% similarity) range from 68 to 152 and from 151 to 220 in the endospheres of plants from AD and PAT, respectively. These values are in accordance with other studies reporting OTUs values from plant endospheres ranging from 100 to 300 2,28,34,35 . However, a significantly greater number of OTUs (from 450 to 3700) have also been reported in plant endospheres by other authors 29,36,37 .
The Shannon index values ranged from 2.28 to 4.41. and from 2.76 to 4.41 in the endospheres of plants from AD and PAT, respectively. The diversity of bacteria in root and leaf tissues of tree peony (Paeonia Sect. Moutan) had greater Shannon index values (7 to 9) compared to our study 37 . In contrast, a recent study reported Shannon index values that were similar to those we found (3 to 4) in root and leaf endospheres of groundsel (Senecio vulgaris L., Asteraceae), also by using the Illumina platform 34 . Similarly, but by using 454-pyrosequencing, Correa-Galeote et al. 35 reported Shannon index values of 3 to 4 in the root endosphere of maize cultivated at 3,537 meters above sea level in Perú. Interestingly, the Shannon index values found in endosphere tissues from AD plants were generally lower than those reported in other AD habitats, such as soil, lakes and sediments of flat mats, with Shannon index values ranging from 3 to 9 [38][39][40][41] . This suggests that endospheres of AD plants may harbor less bacterial diversity compared to other niches in the AD, which is considered oldest and driest place on the Earth 42 .
Interestingly, compared with G. mucronata, H. pilosella had high bacterial abundances and diversity at the PAT ecosystem, as determined by qPCR and Shannon index, respectively. H. pilosella is an exotic weed at the  www.nature.com/scientificreports www.nature.com/scientificreports/ Chilean Patagonia, which recognized by its explosive expansion in Patagonian grasslands, often replacing forage plants with the concomitant economic loss for livestock and soil degradation by overgrazing 43 . Therefore, the higher abundance and diversity of endophytic bacteria in H. pilosella might give this plant species a competitive advantage against other Patagonian plants. Thus, alteration of the composition and activity of endophytic bacteria may be a useful strategy for biological control of invasive plants. However, our study is limited to few sampled plants and major efforts are required to validate this statement and evaluate the potential biocontrol of H. pilosella expansion in Chilean Patagonia grasslands.
Our Illumina-based analyses revealed the dominance of members of the phyla Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes in the endosphere. It was previously reported that Proteobacteria and Firmicutes are common inhabitants of plant endospheres with relative abundances ranging from 39% to 97% and 14% to 44%, respectively 4,28,36 . Studies done to analyze root and leave endosphere tissues have also shown a great dominance (over 86%) by the Proteobacteria, Firmicute and other phyla (such as Bacteroidetes, Acidobacteria and Actinobacteria) in olive trees, peony and groundsel 29,34,37 . Interestingly, a high relative abundance (31%) of Euryarchaeota was found in roots of D. spicata. Member of Euryarchaeota have also been found colonizing the endospheres of Mediterranean olive trees 29 and compartments (rhizosphere, endosphere and phyllosphere) of a halophyte plant (Salsola stocksii) from Pakistan 44 .
Relative to what was seen in tissues from the AD and PAT as minor taxa, both ecosystems had a great diversity, represented by members of the Lentisphaerae, Chloroflexi, Verrucomicrobia, Elusimicrobia, Fusobacteria, Spirochaetes and Acidobacteria. Low numbers of sequences of these phyla were observed by Hardoim et al. 4 , when endophytic data sets from all peer-reviewed publications were revised in the ISI Web of Science and PubMed databases. Similarly, our analyses interestingly revealed the presence of members of the phylum Cyanobacteria in roots and leaves of AD plants. High abundances (24% to 47%) of members of this phyla have also been reported in the endospheres of grasses (Spartina alterniflora) and mangrove (Kandelia obovata) 45 . Lower abundances (1.7%) of Cyanobacteria have also been reported in the endosphere and other compartments (rhizosphere and rhizoplane) of the medicinal perennial plant Stellera chamaejasme L. 46 . It is noteworthy that Cyanobacteria are