DOI: 10.1515/biolog-2015-0048. Bacterial diversity and community structure of Western Indian. Himalayan red kidney bean (Phaseolus vulgaris) rhizosphere.
Biologia 70/3: 1—, 2015 Section Cellular and Molecular Biology DOI: 10.1515/biolog-2015-0048
Bacterial diversity and community structure of Western Indian Himalayan red kidney bean (Phaseolus vulgaris) rhizosphere as revealed by 16S rRNA gene sequences* Deep Chandra Suyal1**, Amit Yadav2**, Yogesh Shouche2 & Reeta Goel1*** 1
Department of Microbiology, College of Basic Sciences and Humanities; G.B.P.U.A&T, Pantnagar – 263145, Uttarakhand, India; e-mail: rg55@rediffmail.com 2 Microbial Culture Collection, National Centre for Cell Science, Pune University Campus, Ganeshkhind, Pune – 411 007, Maharashtra,India
Abstract: Agriculture is an important livelihood activity in the Himalayan regions. Our previous studies revealed the presence of diverse diazotrophic assemblage in indigenous red kidney bean (RKB) rhizospheric soil from two different locations of Western Indian Himalaya, namely S1 (Chhiplakot, 30.70◦ N/80.30◦ E) and S2 (Munsyari, 30.60◦ N/80.20◦ E), selected on the basis of real-time PCR analysis. In this study, two 16S rRNA gene clone libraries (SB1 and SB2, respectively) were constructed using the same rhizospheric soil samples for assessing the total bacterial diversity and their community structure. A total of 760 clones were obtained, with ∼54–59% of these sequences belonging to the phylum Proteobacteria. While sequences belonging to Bacteroidetes, Chloroflexi, Acidobactria, Planctomycetes, Firmicutes, Nitrospira, Gemmatimonadetes, Cyanobacteria, Verrucomicrobia, OD1, OP11 and Actinobacteria were encountered in both the soils, sequences belonging to bacteria from the classes Chlorobi and BRC1 were only detected in the S1 soil. Both the libraries showed similar bacterial community compositions, with Pseudomonas (∼33–34%) as predominant genus. Phylogenetic analysis revealed that all the clone sequences were clustered in different bacterial groups as per their resemblance with their respective phylogenetic neighbours. Major clusters were formed by Gammapreoteobacteria followed by Bacteroidetes and Alphaproteobacteria. A good fraction of the clone sequences has no resemblance with existing groups, thereby suggesting the need of culture-dependent studies from Himalayan regions. To the best of our knowledge, this study is the first major metagenomic effort on Himalayan RKBs rhizobacteria revealing fundamental information that needs to be explored for functional studies. Key words: Western Indian Himalayan; Phaseolus vulgaris; bacterial diversity; rhizosphere; 16S rRNA gene. Abbreviations: DOTUR, distance-based OTU and richness; OTU, operational taxonomic units; PAST, paleontological statistics; qPCR, real-time PCR; R, recombination; RKB, red kidney bean; rRNA, ribosomal RNA; TOC, total organic carbon; WIH, Western Indian Himalaya.
Introduction Himalayan ecosystems contain a series of climatically very different zones within short distances and elevations, displaying a range of micro-habitats with great biodiversity. They are fragile and vulnerable to changes due to their particular and extreme climatic and bio-geographic conditions. Moreover, changes in land use practices, infrastructure development, unsustainable tourism, overexploitation of natural resources, fragmentation of habitats, and climate change create an additional pressure on Himalayan ecosystems. A huge range of traditional crops including red kidney bean (RKB) are grown in the Himalayan agro-ecosystems,
which have been managed by local farming communities since time immemorial. These crops are adapted to the local environmental conditions and possess the inherent qualities to withstand the environmental risks and other natural hazards. Further, rhizosphere microbiome also contributes to the ability of some plant species to survive under extreme conditions (Jorquera et al. 2012). Hence, microbial diversity associated with these plants must be unique and novel. The diversity of rhizobia nodulating RKB has been studied in detail by Diaz-Alcantara et al. (2013). Recently, Sanchez et al. (2014) characterized twenty different plant growth promoting bacteria from the rhizosphere of Phaseolus vulgaris. However, all the reports
* Electronic supplementary material. The online version of this article (DOI: 10.1515/biolog-2015-0048) contains supplementary material, which is available to authorized users. ** These authors contributed equally to this work *** Corresponding author
c 2015 Institute of Molecular Biology, Slovak Academy of Sciences
2 were confined only to nodulating rhizobial populations and scarce information is available about its rhizospheric diversity, especially from the Himalayan ranges. Previous studies confirmed that Himalayan soils have bacterial community with a tremendous potential of biodegradation (Soni et al. 2008) and plant growth promotion (Kumar et al. 2014; Suyal et al. 2014a). Moreover, Prema et al. (2009) and Suyal et al. (2014b) highlighted the prevalence of csp and nif from the high altitude soils of Indian Himalayas. Our previous study revealed the presence of diverse nitrogen-fixing microbial assemblages in the same RKB rhizospheric soil sample (Suyal et al. 2014b). Recently, seven cold adapted bacterial diazotrophs were isolated from the same rhizosphere and proteome of the psychrophilic nitrogenfixing Pseudomonas migulae S10724 for low temperature diazotrophy was documented (Suyal et al. 2014c) confirming its ability to fix atmospheric N2 at the fluctuating temperatures. Therefore, the present study aimed to investigate the bacterial diversity in the rhizosphere of RKB and speculate the bacterial community structure. The obtained data will improve our understanding of the legume rhizosphere and will help in developing appropriate sustainable agricultural development strategies under high altitude agro-ecosystems. Moreover, unravelling the rhizosphere microbiome holds potential to uncover numerous yet unknown microorganisms, genes and functions for various applications. Material and methods Sampling sites and sample collection The sampling sites were located on the upper reaches of Kumaun Himalaya (Suyal et al. 2014b). Rhizospheric soil samples (not deeper than 15 cm) were collected from the rhizosphere of RKB (P. vulgaris L.) using sterile spatula in sterile polythene bags and transported to laboratory under sterile and cold conditions. Each soil sample was collected in triplicates, which were later mixed to make a single composed sample per site. Samples for chemical analysis were stored at 4 ◦C, and samples for clone library analysis were stored at –20 ◦C till further use. Total soil DNA extraction Total soil DNA was extracted from each 0.5 g (fresh weight) soil sample by using the PowersoilTM DNA isolation kit (Mobio Lab. Inc., USA) according to the manufacturer’s instructions. After extraction, DNA samples were quantified spectrophotometrically at 260 nm and used immediately for nifH library construction; remaining DNA was stored in TE buffer (10 mM Tris, 1 mM ethylenediaminetetraacetic acid, pH 8.0) at –80 ◦C till further use. Real-time PCR (qPCR) analysis Copy number of 16S rDNA genes from collected soil samples was quantified using iCycleriQTM Multicolor (BioRad Lab, Hercules, USA) qPCR machine as described previously (Soni & Goel 2010). In brief, genomic DNA of the bacterium Pseudomonas putida was isolated, quantified and used as a standard for qPCR analysis in serial dilutions (0.1 ng to 100 ng). PCR reactions were carried out in volume of 25 µL containing 1X iQTM SYBR
D.C. Suyal et al. Green Supermix (Bio-Rad), 100 pmol of each primers, primer 1 (5’-CCTACGGGAGGCAGCAG-3’) and primer 2 (5’-ATTACCGCGGCTGCTGG-3’) and 50 ng of template DNA. Cycling conditions included 3 min of the denaturation step at 94 ◦C, followed by 35 cycles of 1 min at 94 ◦C, 1 min at 58 ◦C, and 2 min at 72 ◦C (Soni & Goel 2010). 16S rDNA library construction and sequence analysis The 16S rRNA genes were amplified from the extracted community DNA using universal bacterial primers 27F (5’AGAGTTTGATCMTGGCTCAG-3’) and 907R (5’-CCGT CAATTCCTTTRAGTTT-3’) as described earlier (Surakasi et al. 2010). The amplicons were excised from 1.2% (w/v) agarose gel and purified using the HipuraTM Quick gel purification kit (Himedia). The PCR products were analyzed on 1.2% agarose gel and quantified spectrophotometrically at 260 nm. Purified PCR products were cloned into the pCR4-TOPO Escherichia coli vector (Invitrogen) by using the ‘TOPO TA cloning ’ kit (Invitrogen) for sequencing with ‘One Shot TOP10 Electro-Competent cells ’ according to the manufacturer’s instructions (Invitrogen) on the same day. Randomly selected colonies were picked and subjected to colony PCR in 25 µL of PCR mixture as described earlier (Surakasi et al. 2010). PCR products were polyethylene glycol purified and sequenced in both directions using the AB dye terminator (Applied Biosystems) as described previously (Surakasi et al. 2010). Statistical and phylogenetic analysis of the 16S rDNA clone sequences Phylogenetic analysis of the clone sequences was carried out as reported by Surakasi et al. (2010). All sequences were assigned to an operational taxonomic unit using distance-based operational taxonomic units (OTU) and richness (DOTUR) at 5% sequence distance cutoff. The diversity of OTU was further examined using rarefaction analysis. Coverage of 16S rRNA gene clone libraries and various other diversity indices were determined with paleontological statistics (PAST) version 1.77 (http://palaeo-electronica.org/2001 1/past/) (Cetecioglu et al. 2009). Shannon-Wiener diversity index (http://www. changbioscience.com/genetics/shannon.html/) was used to calculate Shannon index (H0), evenness and the Simpson’s index (D). The sequences were then used to construct the phylogenetic tree using the MEGA software, version 5 (Tamura et al. 2011) in accordance with the interior test of phylogeny using a neighbour-joining algorithm. Nucleotide diversity analyses In order to obtain information about the population structure and the nucleotide diversity of RKB rhizospheric bacteria based on 16S rDNA gene sequences, statistical analyses were performed using the software DnaSP 5.10 (www.ub.edu/dnasp/) (Singh et al. 2013). Nucleotide sequence accession numbers All the 16S rRNA gene clone libraries sequences reported in this paper have been deposited in the GenBank database (Benson et al. 2013) under accession numbers KF145207KF145966.
Results Two soil samples S1 and S2 were selected among twenty different RKB rhizospheric samples collected from various agro-climatic zones of Western Indian Himalaya
Bacterial community structure of Himalayan red kidney bean rhizosphere
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Fig. 1. Relative abundance of bacterial phyla across the 16S rDNA clone libraries SB1 and SB2, constructed from the soils S1 and S2, respectively. The percentage of sequences is plotted on X-axis. Proteobacteria was the predominant phylum in both the soils.
(WIH), on the basis of 16S rRNA gene abundance using qPCR technique (Table S1). The collection sites S1 and S2 were from Chhiplakot and Munsyari, respectively. Physico-chemical analysis of S1 and S2 revealed the significant difference between the total nitrogen and total organic carbon (TOC) content of both the soils (Suyal et al. 2014b). Mountainous cold-temperate areas have high TOC content but large spatial variability due to variable climate and vegetation (Li et al. 2010). Various studies have reported the influence of topography (Yoo et al. 2006), climatic conditions (Davidson & Janssens 2006), soil composition (Jobbagy & Jackson 2000), litter quality and its decomposition rate (Yang et al. 2005) and species composition or vegetation type (Schulp et al. 2008) on the spatial distribution of TOC. Cloning and analysis of 16S rDNA sequences A total of 760 positive clones were obtained from two clone libraries SB1 (416 sequences) and SB2 (344 se-
quences); constructed using the soils S1 and S2, respectively. Proteobacteria was dominant phylum in SB1 and SB2 (Fig. 1), with maximum occurrence of Pseudomonas (Gammaproteobacteria), averaging ∼34% of total bacterial clones in both (Fig. S1). Bacteroidetes, Acidobacteria, Choloflexi, Nitrospira, Acidobacteria and Actinobacteria were also observed in both the libraries, but at a lower percentage of the clones (Fig. 2). A good fraction of the clone sequences belonged to unclassified bacteria viz. unclassified Actinobacteria (>1%), Proteobacteria (>1%), Chloroflexi (>3%), etc (Fig 2a). In addition, Verrucomicrobia, Firmicutes, Cyanobacteria, Gemmatimonadetes, Planctomycetes, OP1 and OD1 made up a fraction of the bacterial clones in both the libraries (Fig 2b). Furthermore, 5-8% of the clone sequences were not assigned to any bacterial phylum, thereby suggesting the need of culture-dependent studies from these Himalayan regions (Fig. 2a).
Fig. 2a. Comparison of bacterial communities among the 16S rDNA clone libraries SB1 and SB2, at genus level. Only the members which were above 1% are shown here.
4 D.C. Suyal et al.
Fig. 2b. Comparison of bacterial communities among the 16S rDNA clone libraries SB1 and SB2, at genus level. Only the members which were less than 1% are shown here.
Bacterial community structure of Himalayan red kidney bean rhizosphere 5
6 The overall frequency distribution of the clones indicated that Gammaproteobacteria were most predominant (>36%) in both the libraries represented by genera Pseudomonas, Steroidobacter and Cellvibrio in SB1, and by three additional genera Aspromonas, Lysobacter and Stenotrophomonas in SB2 (Fig. S1). Furthermore, 3% of SB1 and 9% of SB2 Gammaproteobacteria clone sequences remained unclassified. Among Alphaproteobacteria, genera Dongia, Altererythrobacter, Novosphingobium, Hyphomicrobium, Bradyrhizobium, Mesorhizobium and Rhizobium were shared by both the libraries. In addition to this, genera Defluviicoccus, Devosia, Microvirga, Phenylobacterium and Sphingosinicella were exclusively present in SB1, while genera Croceicoccus, Sphingomonas, Elioraea and Ensifer were observed in SB2 only. In case of Betaproteobacteria, most of the clone sequences remained unclassified. Genera Chitinilyticum, Chitinimonas and Chitinimonas were observed exclusively in SB1, while Massilia, Diaphorobacter and Ramlibacter were present in SB2 only. Among Deltaproteobacteria, SB1 contained only unclassified Deltaproteobacterial members, while SB2 represented three genera Syntrophobacter, Anaeromyxobacter and Geobacter along with a few unclassified members. Members of Bacteroidetes were also prevalent in both the libraries (Figs 1 and 2). Genera Ohtaekwangia, Flavobacterium, Terrimonas, Haliscomenobacter, Adhaeribacter and Solitalea were some major genera shared by both SB1 and SB2. Firmicutes were represented by genera Bacillus, Clostridium, Anaerobacillus, Pasteuria and Paenisporosarcina in SB1 and genera Bacillus and Paenisporosarcina in SB2. In case of Actinobacteria, genera Arthrobacter and Marmoricola were common in both the libraries. In addition to this, genera Ilumatobacter, Tetrasphaera and Nocardioides were present in SB1, while genera Solirubrobacter and Rhodococcus were detected in SB2. The Acidobacteria observed in this study were related to six different groups viz. Gp3, Gp4, Gp5, Gp5, Gp11 and Gp16 along with very few unclassified members (Figs 2a and 2b). Moreover, a few members of Planctomycetes were also detected in both the libraries with the genera Planctomyces and Phycisphaera in SB1 and genus Phycisphaera in SB2. In a minor fraction of bacterial communities, members of OP1, OD1, Verrucomicrobia, Cyanobacteria, Chloroflexi, Gemmatimonadetes and Nitrospira were also observed in both the libraries. In case of SB1, members of BRC1, Armatimonadetes and Chlorobi were also detected. Statistical and phylogenetic analyses of 16SrDNA clones Determination of OTU is one of the preferred methods that are currently available for comparing diversity from different clone libraries. Based on similarity criteria of ≥97%, 16S rRNA gene clone sequences of SB1 and SB2 were grouped into 244 and 186 OTUs, respectively. Both the libraries had similar richness and diversity with slight differences (Table 1). All the clone
D.C. Suyal et al. Table 1. Comparative diversity analysis among the 16S rDNA libraries (using PAST version 1.77). 16S rDNA libraries Diversity indices
Individuals Dominance D Simpson 1-D Shannon H Evenness eˆH/S Menhinick Margalef Berger-Parker
SB1
SB2
416 0.1322 0.8678 3.006 0.3063 3.558 11.28 0.3413
344 0.137 0.863 2.978 0.2846 3.383 11.13 0.3372
sequences of SB1 and SB2 were clustered in 19 and 17 different bacterial groups, respectively, as per their resemblance with their respective phylogenetic neighbours (Fig. S2). Nucleotide diversity analyses The nucleotide diversity analysis using the software DnaSP 5.10 (www.ub.edu/dnasp/) showed polymorphic sites were more in the soil 1, indicating more diversity in S1; hence, it might have undergone more evolution than S2 (Table 2). When the Parsimony informative sites were calculated, they did not mark much difference (264 for S1 and 263 for S2). Additionally, a greater nucleotide diversity per site also suggests a larger extent of DNA polymorphism in S1 species as compared to S2 species. Theta per site was also larger in S1 species suggesting a larger extent of evolution indicating towards more prominent role S1 species in evolution. Moreover, a lower value of average nucleotide distance between the most distant sites clearly displays that S1 species might be evolved more than the S2 species. Apart from that, a higher value of R (recombination) per gene and estimate of R between adjacent sites in S1 species implies larger extent of recombination in this class than the S2 species (Singh et al. 2013). This clearly implies that S1 was more responsible for driving evolution. Discussion The Western Himalayas, which comprise different subtropical, temperate and subalpine zones, are wellknown for their rich biodiversity. Soil biological activity in these high-altitude agricultural lands is reportedly low due to suboptimal or freezing soil temperatures, as seasonal freeze-thaw cycles change various physicochemical properties of the soil providing both challenges and opportunities for the survival of indigenous microflora (Schmidt et al. 2009). These high-altitude ecosystems are potentially under threat of biodiversity loss from global warming – a consequence of both geographical range contraction and mountain-top ecosystem extinction risk (La Sorte & Jetz 2010). Therefore, there is an immense need to document and preserve
Bacterial community structure of Himalayan red kidney bean rhizosphere
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Table 2. Molecular diversity analysis of 16S rDNA sequences of clone libraries and bacterial isolates from S1 and S2. Clone libraries Parameters
Polymorphic sites (segregating sites, S) Parsimony informative sites Nucleotide diversity per site (Pi) Theta per site from total (number of mutations, Eta) Average number of nucleotide (differences, k)
SB1
SB2
302 264 0.17453 0.23526 73.826
308 263 0.17426 0.23512 75.281
987.179 26.898 0.010 0 1344.09
654.007 23.876 0.092 0 1567.87
Recombinational analyses Variance of the sample (distribution of kij, Sk2) Theta per gene R per gene Estimate of R between adjacent sites Average nucleotide distance between the most distant sites DNA divergence between the groups Average number of nucleotide differences between groups Number of net nucleotide subpopulations per site between the groups, Da Mutations polymorphic in group 1, but monomorphic in group 2 Mutations polymorphic in group 2, but monomorphic in group 1
the microbial diversity from such fragile ecosystems, which could further be utilized in sustainable development. The high copy number of 16S rRNA gene in the rhizospheric soils is in accordance with our previous study (Soni et al. 2010). Suyal et al. (2014b) reported the highest nifH load in the same rhizospheric samples and established a good insight into correlation between nitrogen-fixing microorganisms and fertility of Himalayan agricultural soils of high altitude. Bacterial diversity and community structure Remarkably diverse 16S rDNA sequences were extracted from the RKB rhizosphere, and most of them were found to be novel and/or distantly related to those of cultivated organisms. High abundance of Proteobacteria in both the samples could be supported by the fact that Proteobacteria, Bacteroides and high G+C gram-positive bacteria are common in most cold habitats. The existence of wide range of bacterial phyla in WIH rhizospheric samples is in agreement with earlier studies, which had also indicated that clones affiliated to Verrucomicrobia, Chlorobi (Pradhan et al. 2010), Chloroflexi (Wagner et al. 2009; Pradhan et al. 2010), Gemmatimonadetes (Pradhan et al. 2010; Shivaji et al. 2011), Nitrospira and Planctomycetes (Pradhan et al. 2010) are present in other cold habitats at a low frequency. Results are also in agreement with the earlier studies from Himalayan regions (Shivaji et al. 2011; Suyal et al. 2014a) which had also indicated that gramnegative bacteria predominated over gram-positive bacteria. Many independent studies have depicted Proteobacteria viz. bacteria from the Pseudomonadaceae family as dominant members of the rhizosphere microbiota. This is in line with Proteobacteria being generally fast-growing with the ability to utilize a broad range of root-derived carbon substrates (Philippot et al. 2013).
70.263 –0.00048 13 13
Among the two libraries, bacterial diversity was higher in SB1 (Chhiplakot), which could be attributed to the pristine environment of the high-altitude regions, to the climatic conditions and to lower anthropogenic activities. Microbial community assembly in the rhizosphere is governed by abiotic and biotic factors. The complex physico-chemical characteristics of soils affect plant physiology and root exudation patterns, which in turn influence the composition of the rhizosphere microbiota (Philippot et al. 2013). Comparison of the bacterial diversity observed in the present study with that of other Himalayan cold habitats, such as Tibetan plateau glacier (Liu et al. 2009), soil samples from the western Himalayas (Shivaji et al. 2011), Puruogangri ice (Zhang et al. 2008) and soil from Roopkund lake and glacier (Pradhan et al. 2010), indicated that the diversity of bacteria in SB1 and SB2 are comparable with that reported from the above cold habitats with respect to the presence of similar bacterial groups. The occurrence of identical phyla in geographically diverse cold environments is indicative of the ability of microorganisms to adapt to similar strategies to survive at low temperatures. The values of diversity indices also pointed out the existence of more bacterial diversity in S1 than that of S2. Shannon index was higher in SB1 library (3.006) than the SB2 (2.978). This index is increased either by having additional unique species, or by having greater species evenness (Shanon 1948). These results are well supported by the values of Margalef and Menhinick indices (higher in SB1), which estimate the species richness independently of the sample size (Magurran 2004). Moreover, the value of Berger-Parker index was also higher in SB1. This index expresses the proportional importance of the most abundant species. Increase in the value of this index accompanies an increase in diversity and a reduction in dominance (Magurran 2004).
8 For the Simpson index, values near zero correspond to highly diverse or heterogeneous ecosystems and values near one correspond to more homogeneous ecosystems (Simpson 1949). As the observed values of this index were 0.8678 (SB1) and 0.863 (SB2), tending towards 1, it indicates the saturation risk of western Himalayan diazotrophic diversity. These results are well supported by the outcomes of nucleotide diversity analysis using the software DnaSP 5.10 revealing that the nucleotide diversity and recombination frequency is much greater in S1 than that in S2. Furthermore, DNA divergence studies reveal the presence of major evolutionary hotspots in Chhiplakot soil metagenome. In both the libraries, major clusters were of Gammapreoteobacteria followed by Bacteroidetes and Alphaproteobacteria. Both the phylogenetic trees were similar with slight variation in the positioning of a few bacterial groups viz. in SB1, Cyanobacteria were found to cluster with Proteobacterial members, while in SB2 they were grouped with Bacteroidetes, which was clustered with Chlorobi in earlier case. Similarly, Acidobacteria were grouped with Firmicutes and Nitrospira with Verrucomicrobia in SB1, while in SB2 both were clustered separately. It could be justified by the fact that phylogenetic structure is a net result of forces that shape communities. Significant clustering may be evidence of positive interactions, such as phenotypic attraction and spatial isolation; over-dispersion can result from negative interactions, such as competition, predation, minimal niche overlap, or the presence of many niches (DeAngelis & Firestone 2012). Interestingly, all the unclassified bacteria were clustered into a separate major cluster, which indicates the existence of novel bacterial communities in Himalayan regions. Although considerable progress has been made in our understanding of the microbial ecology of the rhizosphere, rhizosphere ecologists face several major challenges. These include the development of new crops and cropping systems to produce sufficient biomass for food, feed, fibre and bio-energy at low environmental costs. Production methods need to focus on efficient recycling of nutrients and effective control of pests and pathogens. In these systems, associated microbial communities play a major part in plant adaptation to biotic and abiotic stresses. Therefore, ‘going back to the roots’ of native plant communities holds a great promise to further improve the sustainability of crop production for food, feed, fibre and fuel. Conclusion In conclusion, this study provides the qualitative as well as quantitative assessment of RKB-associated bacterial diversity and identifies the two diazotrophic hotspots, namely Chhiplakot and Munsyari from WIH. Our results expand the knowledge of WIH bacterial wealth, which will be useful in making of microorganisms-based sustainable development strategies. Further, a good fraction of the clone sequences have no resemblance with existing groups, which may play a significant yet unknown ecological role; thereby suggesting the need of
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