Analysis of Bacterial Diversity and Communities Associated With

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J. Microbiol. Biotechnol. (2016), 26(1), 89–98 http://dx.doi.org/10.4014/jmb.1505.05008 Research Article Review jmb

Analysis of Bacterial Diversity and Communities Associated with Tricholoma matsutake Fruiting Bodies by Barcoded Pyrosequencing in Sichuan Province, Southwest China S Qiang Li1,2, Xiaolin Li3, Cheng Chen4, Shuhong Li5, Wenli Huang2, Chuan Xiong1,2, Xing Jin2, and Linyong Zheng1,2*

1College of Life Science, Sichuan University, Chengdu, Sichuan 610065, P.R. China 2Institute of Biological & Nuclear Technology, Sichuan Academy of Agricultural Sciences, Chengdu, Sichuan 610066, P.R. China 3Soil and Fertilizer Institute, Sichuan Academy of Agricultural Sciences, Chengdu, Sichuan 610066, P.R. China 4Institute of plant protection, Sichuan Academy of Agricultural Sciences, Chengdu, Sichuan 610066, P.R. China 5Biotechnology & Germplasm Resources Institute, Yunnan Academy of Agricultural Sciences, Kunming 650221, P.R. China

Received: May 6, 2015 Revised: September 15, 2015 Endophytes play an important role in the growth and development of the host. However, the Accepted: September 28, 2015 study of endophytes is mostly focused on plants, and reports on bacteria associated with fungi First published online are relatively rare. We studied the bacteria associated with fruiting bodies of Tricholoma October 2, 2015 matsutake picked from seven main T. matsutake-producing areas in Sichuan, China, by

*Corresponding author barcoded pyrosequencing. About 8,272 reads were obtained per sample, representing 40 Phone: +86-28-84592187; phyla, 103 classes, and 495 genera of bacteria and archaea, and 361–797 operational taxonomic Fax: +86-28-84592187; E-mail: [email protected] units were observed at a 97% similarity level. The bacterial community was always both more abundant and more diverse than the archaeal community. UniFrac analysis showed there were Present address: Linyong Zheng, some difference of bacterial communities among the samples sites. Three bacterial phyla, Jinjiang, Shizishan Road 106, Sichuan Academy of Agricultural Proteobacteria, Bacteroidetes, and Firmicutes, were dominant in all samples. Correlation Sciences, Chengdu 610066, analysis showed there was a significant correlation between some soil properties and bacterial Sichuan, P.R. China community associated with T. matsutake. This study demonstrated that the bacteria associated S upplementary data for this with T. matsutake fruiting bodies were diversified. Among these bacteria, we may find some paper are available on-line only at http://jmb.or.kr. strains that can promote the growth of T. matsutake.

Introduction environmental conditions, growing in virgin forests without pollution and human intervention [31, 42, 47]. Artificial Tricholoma matsutake (S. Ito et Imai) is an ectomycorrhizal cultivation of T. matsutake has not been successful [21, 46]. basidiomycete associated with Pinaceae and Fagaceae trees It is still unclear how ecological factors such as host in China, Korea, and elsewhere in the Northern Hemisphere plant, soil properties, and the associated microbial [43, 49]. Its fruiting body, the pine mushroom, is commercially communities influence the development of the T. matsutake important as a valuable food because of its medicinal fruiting body [2, 23, 28]. The soil surrounding T. matsutake effects and attractive flavor [14, 23]. Polysaccharides and contains diverse microbial communities that may affect the terpenoids extracted from its fruiting body have antitumor growth of T. matsutake mycelia and the formation of and antioxidant properties [19, 38, 48, 51]. The growth of mycorrhiza [25]. The soil microbial communities may also wild T. matsutake is extremely slow, and it is selective to the play a role in the material exchange between mushroom

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mycelia and plant host. Furthermore, the soil bacteria may live extracellularly inside fungal tissue [15]. Their role in fungal development is still unclear. Therefore, understanding the bacteria affiliated with T. matsutake and the microbial community structure underneath the mushroom has important implications for the artificial domestication and cultivation of T. matsutake. The microbial community in soil-mycelia aggregates and in the T. matsutake fairy ring zone has been studied [26, 41]. Park et al. [32] also found a new species associated with the pine mushroom. This suggests that the surroundings of T. matsutake contains rich microbial resources for us to understand. The microbial communities associated with T. matsutake fruiting bodies have been analyzed using denaturing gradient gel electrophoresis (DGGE) [29]. However, studies on the bacterial diversity associated with T. matsutake fruiting Fig. 1. Location of the sampling sites of Tricholoma matsutake bodies have rarely used barcoded pyrosequencing. fruiting bodies and soil beneath the fruiting bodies. A majority (over 99%) of the microbes living in natural environments have not been cultured. Despite continuing using the pipette method [22]. pH was measured in soil water development of culture techniques for the isolation and extracted by dissolving air-dried soil in distilled water at a ratio of identification of microbes, it is still difficult to assess the 1:5. Organic matter content was estimated using the Tyurin method true diversity in microbial communities using the currently [33]. Total nitrogen was determined by the Kjeldahl method [7]. available culture techniques owing to their limitations [1, Molybdenum antimony ascorbic acid spectrophotography was 34]. Metagenomics is the study of genetic material recovered used to estimate total phosphorus. Total potassium was analyzed directly from environmental samples. Because of its ability by flame photometry. Effective nitrogen was measured by the to reveal the previously hidden diversity of microscopic alkali solution diffusion method. Available phosphorus was life, metagenomics offers a powerful lens for viewing the determined by the baking soda leaching - molybdenum antimony whole microbial world in a sample, including unculturable colorimetric method. Available potassium was determined by microbes [18, 34, 35]. ammonium acetate extraction - flame photometry [30]. Soil Mg, Ca, Cu, Mn, and Zn were determined by inductively coupled In this paper, we studied the bacteria associated with plasma optical emission spectroscopy (Optima 2000 DV; fruiting bodies of T. matsutake picked from seven main PerkinElmer, USA), with yttrium as the internal standard [8]. T. matsutake-producing areas in Sichuan, China. This paper provides knowledge on the fundamental aspects of DNA Extraction and MiSeq Sequencing of 16S rRNA Gene T. matsutake, such as T. matsutake-associated bacterial diversity, Amplicons that may be of particular significance to the artificial DNA extraction was conducted by using the E.Z.N.A. Fungal domestication and cultivation of T. matsutake. DNA Kit (Omega Bio-Tek, USA). The DNA concentration and quality were checked using a NanoDrop Spectrophotometer. Materials and Methods Extracted DNA was diluted to 10 ng/µl and stored at -40°C. Universal primers 515F (5’-GTGCCAGCMGCCGCGGTAA-3’) Mushroom Sampling Strategy and Soil Analyses and 806R (5’-GGACTACHVGGGTWTCTAAT-3’) [45] with 10 nt T. matsutake fruiting bodies were picked from Xiaojin, Yajiang, barcodes were used to amplify the V4 hypervariable regions of Muli, Yanyuan, Yanbian, Huidong, and Mianning county at its 16S rRNA genes for pyrosequencing using the MiSeq sequencer mature stage in Sichuan, China (Fig. 1, Supplemental Fig. S1), and [11, 12]. The PCR mixture (25 µl) contained 1 µl PCR buffer, kept in sterile sealed bags on ice. We collected three fruiting bodies 1.5 mM MgCl2, each deoxynucleoside triphosphate at 0.4 mM, in each producing area, based on altitude difference, and 500 g of each primer at 1.0 mM, 0.5 U of TransStart Fast Pfu DNA soil beneath the fruiting bodies with a soil sampler. DNA was Polymerase (TransGen, China), and 10 ng of soil genomic DNA. extracted from the three fruiting bodies and mixed as one sample The PCR amplification program included initial denaturation at to analyze the bacterial diversity by barcoded pyrosequencing. 94°C for 3 min, followed by 30 cycles of 94°C for 40 sec, 56°C for Soil samples from the same site were mixed separately to analyze 60 sec, and 72°C for 60 sec, and a final extension at 72°C for their properties. Soil particle size distribution was determined 10 min. Two PCRs per sample were combined together after PCR

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amplification. PCR products were subjected to electrophoresis using 1.0% agarose gel. The band with a correct size was excised and purified using the Gel Extraction Kit (Omega Bio-tek, USA) and quantified with Nanodrop. All samples were pooled together, with an equal molar amount from each sample. A TruSeq DNA kit was used to prepare the sequencing samples. The purified library was diluted, denatured, re-diluted, and then mixed with PhiX (about 30% of final DNA amount) as described in the Illumina library preparation protocols, and then the samples were applied to an Illumina MiSeq system for sequencing with the Reagent Kit ver. 2 2×250 bp according to the manufacturer’s manual.

Pyrosequence Data Analysis The sequence data were processed using QIIME Pipeline ver. 1.7.0 (http://qiime.org/tutorials/tutorial.html) [10]. Multiple steps were required to trim the sequences, such as trimming the barcoded fusion primers, and filtering low-quality sequences (read Fig. 2. Rarefaction curves for bacterial operational taxonomic length <150 bp or average quality value <25, ambiguous base calls units (OTUs) in each sampling site (cut-off value at 97% >2) out. Sequences were clustered into operational taxonomic similarity). units (OTUs) at a 97% identity threshold, and the cut-off values In the rarefaction curves, the number of OTUs increased with used for taxonomic assignments were as follows (x = similarity): sequencing reads. X-axis, the number of sequencing reads; Y-axis, the genus (97% > x > 94%), family (94% > x > 90%), order (90% > x > number of OTUs. 85%), class (85% > x > 80%), and phylum (80 > x > 75%). If the similarity was lower than the specific cut-off value, the sequence was assigned as “unclassified” [27]. The aligned 16S rRNA gene different between the sampling sites (Table 1). The sequences were used for chimera check using the Uchime rarefaction curves (Fig. 2) calculated with QIIME pipeline algorithm [17]. All the samples were randomly resampled to 9,700 at 97% similarity also showed a different OTU richness reads. We conducted alpha-diversity (phylogenetic distance whole pattern for all sites. Other OTU richness estimations, such tree, Chao1 estimator of richness, observed species, and Shannon’s as Chao1, indicated that samples picked from Mianning diversity index) and beta-diversity (PCoA, UniFrac) analyses [9], contained the lowest number of bacteria. According to the for which the rarefaction curves were generated from the comprehensive OTU richness results (Table 1 and Fig. 2), observed species. Taxonomy was assigned using the Ribosomal the bacterial community of fruiting bodies from Yanyuan Database Project classifier [44]. was the most diverse (Simpson index, 0.969; Shannon index, 6.38), whereas the lowest bacterial OTU diversity Results (Simpson index, 0.932; Shannon index, 4.90) was in the Diversity Indices samples from Huidong. About 8,272 reads were obtained per sample, representing 40 phyla, 103 classes, and 495 genera of bacteria and archaea, Characteristics of Soil Beneath Tricholoma matsutake and 361–797 OTUs were observed at a 97% similarity level Fruiting Bodies (Table 1). The numbers of observed OTUs were significantly Soil samples, down to a depth of 15 cm, were collected

Table 1. Diversity indices calculated based on a cut-off of 97% similarity of 16S rRNA sequences of 8,272 reads per sample. Sample Chao1 Observed OTUs Shannon index Simpson index Good’s coverage Xiaojin 1,028 717 5.64 0.933 0.959 Yajiang 998 620 5.96 0.959 0.964 Muli 964 658 5.81 0.941 0.965 Yanyuan 1,146 703 6.38 0.969 0.957 Yanbian 1,133 797 6.27 0.956 0.957 Huidong 684 362 4.90 0.932 0.977 Mianning 684 450 5.15 0.923 0.975 Chao1, estimator of richness. Good’s coverage is proportional to the nonsingleton phylotypes in all sequences.

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Table 2. Physical and chemical properties of soil beneath the Tricholoma matsutake fruiting bodies. Sand Silt Clay OM TN TP TK AN AP AK TMn TCu TZn TCa TMg Site pH (%) (%) (%) (g/kg) (g/kg) (g/kg) (g/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) Xiaojin 6.48 66.0 20.5 13.5 84.5 0.48 0.29 32.0 75.2 1.35 232.4 11.9 1.43 0.98 0.85 0.45 Yajiang 6.22 66.9 21.0 12.1 100.1 0.88 0.39 26.8 25.5 2.79 108.7 13.8 1.66 0.70 1.80 0.59 Muli 6.29 74.615.5 9.87 109.1 0.41 0.3621.3 173.7 4.00 300.3 22.3 2.25 0.88 0.94 0.67 Yanyuan 5.55 60.1 17.9 22.1 189.6 4.39 0.42 17.3 134.9 10.12 262.7 32.8 1.02 3.16 12.21 0.54 Yanbian 5.30 50.2 27.5 22.3 185.2 4.24 0.17 26.1 187.2 9.23 226.7 28.2 3.26 2.75 12.31 0.34 Huidong 5.69 60.9 20.8 15.7 123.6 0.97 0.35 28.9 165.9 6.23 289.6 21.9 1.78 1.29 3.97 0.56 Mianning 5.08 88.5 9.73 1.73 106.2 0.36 0.27 29.6 28.6 1.21 183.0 26.6 0.45 0.76 3.18 1.40 OM, organic matter; TN, total nitrogen; TP, total phosphorus; TK, total potassium; AN, effective nitrogen; AP, available phosphorus; AK, available potassium; TMn, total manganese; TCu, total copper; TZn, total zinc; TCa, total calcium; TMg, total magnesium.

from inside the T. matsutake fairy ring at seven different presented 41.0% of the OTUs, and in Yanyuan samples, sites. The physicochemical properties of the soils in the Bacteroidetes presented 44.1% of the OTUs. sampling sites differed slightly (Table 2). The pHs of the A total of 103 classes were identified, and 26 of the 103 soil samples obtained from the fairy rings varied between classes were identified in all seven sites (Fig. 3B). The class 5.08 and 6.48. The soil collected from Yanyuan had the Gammaproteobacteria was dominant in all samples (the highest content of organic matter (189.6 g/kg), total nitrogen average relative abundance was 52.2%). In particular, (4.39 g/kg), total phosphorus (0.42 g/kg), and available Bacilli (39.3%) and Betaproteobacteria (36.9%) were the phosphorus (10.12 mg/kg), and it also contained the lowest dominant classes in Yajiang and Muli samples, respectively. total potassium (17.3 g/kg). Soil samples from Mianning In general, the classes Gammaproteobacteria, Betaproteobacteria, had the most acidic environment (pH 5.08) and the lowest Bacilli, and Sphingobacteria occupied a dominant position available phosphorus (1.21 mg/kg). In terms of soil texture, in T. matsutake fruiting bodies from different sites. the clay composition was lower (1.73%) and the sand A total of 183 orders were observed, and 38 of these were composition was higher (88.5%) in Mianning than in the detected in all samples (Fig. 3C). The most abundant observed other sites. The mineral element contents were slightly orders at all sites were Pseudomonadales (average relative different at different sites (Table 2). abundance 35.8%), Burkholderiales (average relative abundance 15.8%), and Enterobacteriales (average relative Taxonomic Analyses of Bacterial Communities abundance 11.3%). In particular, Burkholderiales (35.4%), Each bacterial 16S rRNA gene sequence was taxonomically Enterobacteriales (28.3%), and Bacillales (37.1%) were the assigned from the phylum level to the species level using dominant classes in the Muli, Yanbian, and Yajiang samples, the Ribosomal Database Project classifier. The proportion respectively. of unclassified bacteria was no more than 0.1%. A total of A total of 310 families were observed and 65 were identified 40 phyla were identified, and 17 of the 40 phyla were in all seven sites (Fig. 3D). The relative abundance of some identified in all seven samples (Fig. 3A). The bacterial families, including Pseudomonadaceae (average of 35.3%), community was always both more abundant and more Enterobacteriaceae (11.3%), Oxalobacteraceae (8.3%), and diverse than the archaeal community. Two archaeal phyla, Sphingobacteriaceae (7.3%), were dominant in all samples, Crenarchaeota and Euryarchaeota, accounted for less than but differed significantly between the sites. Pseudomonadaceae 1% of all bacteria associated with T. matsutake fruiting was the biggest family in most samples except samples bodies. The most abundant archaeal community was from Yanyuan and Yanbian, in which Sphingobacteriaceae Euryarchaeota, about 0.3% of all bacteria detected. More and Enterobacteriaceae occupied a dominant position, than 37 bacterial phyla were detected in the samples. Three respectively. bacterial phyla, Proteobacteria, Bacteroidetes, and Firmicutes, The relative abundance of the genera Pseudomonas (in the were dominant in all samples. The relative abundance of family Pseudomonadaceae), unclassified genus (in the family Proteobacteria was more than 47% of all bacteria detected. Enterobacteriaceae), unclassified genus (in the family Overall, Firmicutes and Bacteroidetes were less abundant. Pseudomonadaceae) and Janthinobacterium (in the family However, in samples picked from Yajiang, Firmicutes Oxalobacteraceae) was dominant in all samples (Table 3).

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Fig. 3. Taxonomic composition analysis of bacterial communities (from phylum to family). This figure shows the relative abundance of bacterial taxa at each taxonomic level: (A) phylum, (B) class, (C), order and (D) family. The results of taxonomic classification using the Ribosomal Database Project classifier server showed a total of 40 phyla, 103 classes, 183 orders, and 310 families and similar bacterial composition patterns between different sites across the higher taxonomic levels (from phylum to family). X-axis, samples from different sites; Y-axis, the relative abundance of bacterial taxa at each taxonomic level (%).

Among them, Pseudomonas was the most abundant (average species (in the family Enterobacteriaceae, Comamonadaceae, abundance 26.3%) and occupied the dominant position in Xanthomonadaceae, Pseudomonadaceae, and so on). The samples from Xiaojin, Yanyuan, Huidong, and Mianning. predominant species were two Pseudomonas sp., belonging Janthinobacterium was the most abundant genus in Muli sample to the family Pseudomonadaceae. (25.4%). Unclassified genus (in the family Enterobacteriaceae) was dominant in Yanbian sample (28.2%). In particular, UniFrac Analysis unclassified genus (in the family Planococcaceae) occupied The differences in bacterial communities between the a dominant position in Yajiang sample (22.1%). samples were estimated using UniFrac analysis (Fig. 4). Several species showed average abundances greater than The bacteria community structure in Yajiang and Yanyuan 1% (Table 4). The communities from the seven different were significantly different from the other sites. The sites shared the same dominant species, Pseudomonas sp., communities from the geographically close and similar Janthinobacterium sp., Janthinobacterium lividum, Pseudomonas environments like Huidong and Mianning were highly viridiflava, Chryseobacterium sp., and other unclassified similar, implying that the environmental conditions can

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Correlation Analysis Between Bacterial Community Associated with the Fruiting Bodies of Tricholoma matsutake and Soil Properties There was a significant correlation between some soil properties and bacterial community associated with T. matsutake fruiting bodies (Table 5). Chao1 and the numbers of observed OTUs were correlated with clay (positively) content and sand (negatively) content. The Simpson index and Shannon index were positively correlated with total nitrogen and available phosphorus. Chao1 was also positively correlated with total nitrogen and total zinc, and negatively correlated with total magnesium. The relative abundance of the phylum Proteobacteria, the predominant phylum in this study, was positively correlated with the Fig. 4. Principal coordinate analysis using weighted UniFrac. presence of total potassium. In contrast, the relative abundance of the phylum Bacteroidetes was negatively affect the T. matsutake-associated bacterial community correlated with total potassium but positively correlated structure. with organic matter. The abundance of Actinobacteria was

Table 3. List of bacterial genera (>1% in at least one experimental site) associated with the Tricholoma matsutake fruiting bodies of different sites. Phylum Family Genus Xiaojin Yajiang Muli Yanyuan Yanbian Huidong Mianning Proteobacteria Pseudomonadaceae Pseudomonas 38.31 18.90 24.13 14.13 14.95 32.06 41.37 Enterobacteriaceae Other 1.01 14.45 2.24 0.64 28.24 24.11 8.04 Pseudomonadaceae Other 5.89 7.75 7.65 4.82 6.23 9.73 21.11 Oxalobacteraceae Janthinobacterium 2.40 1.39 25.42 10.06 3.20 2.92 3.51 Comamonadaceae Other 4.89 0.37 1.25 7.51 10.99 7.42 2.01 Xanthomonadaceae Stenotrophomonas 15.22 0.69 0.14 0.33 0.70 0.34 0.20 Xanthomonadaceae Other 6.99 0.35 0.12 0.43 0.54 0.11 0.13 Oxalobacteraceae Other 0.11 0.69 2.36 1.42 3.36 0.11 0.58 Burkholderiaceae Burkholderia 0.180.44 4.41 0.821.77 0.61 0.34 Other Other 0.16 0.14 1.34 0.15 1.33 0.10 0.53 Alcaligenaceae Achromobacter 0.40 0.05 0.07 0.55 1.10 0.54 0.14 Burkholderiaceae Pandoraea 0.080.181.19 0.27 0.575 0.34 0.16 Nitrospirae [Leptospirillaceae] Leptospirillum 0 1.25 0.97 0 0 0.62 0.87 Firmicutes Planococcaceae Other 0.02 22.11 0.03 0.02 0.02 0.06 0.02 Bacillaceae Bacillus 0.13 8.73 0.17 0.05 0.08 0.12 0.15 Other Other 0.45 3.21 1.02 0.33 0.23 1.06 1.07 Paenibacillaceae Paenibacillus 1.69 1.61 0.02 1.82 0.13 0.02 0.34 Enterococcaceae Other 0.91 0.17 0 0.01 0.081.24 0.39 Bacteroidetes Sphingobacteriaceae Pedobacter 0.17 0.14 5.86 17.73 0.31 1.08 2.65 Sphingobacteriaceae Other 0.07 1.34 1.90 9.683.51 0.04 0.54 Flavobacteriaceae Flavobacterium 0.60 0.11 0.12 8.35 0.24 0.03 1.57 [Weeksellaceae] Chryseobacterium 0.33 0.52 2.15 4.25 0.47 0.79 1.79 Sphingobacteriaceae Other 0 0.09 0.66 1.780.850.01 0.28 Sphingobacteriaceae Sphingobacterium 0.16 0.03 0.05 0.21 0.01 1.34 0.36 Actinobacteria Microbacteriaceae Other 0.39 0.12 0.09 0.25 0.16 6.55 1.12

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Table 4. List of bacterial species (average abundance >1%) associated with the Tricholoma matsutake fruiting bodies of different sites. Phylum Family Species Xiaojin Yajiang Muli Yanyuan Yanbian Huidong Mianning Proteobacteria Pseudomonadaceae Pseudomonas sp. 15.52 8.06 7.64 5.51 5.13 14.61 17.33 Pseudomonadaceae Pseudomonas sp. 13.89 7.57 11.99 5.75 6.04 10.11 16.08 Enterobacteriaceae Other 0.39 6.07 0.90 0.34 11.52 12.483.76 Oxalobacteraceae Janthinobacterium sp. 0.72 0.52 16.31 8.46 1.07 0.06 2.00 Enterobacteriaceae Other 0.22 5.02 0.69 0.14 10.787.14 2.54 Comamonadaceae Other 3.17 0.15 0.484.94 9.00 6.53 1.50 Pseudomonadaceae Other 2.52 2.83 2.89 2.19 1.99 3.46 7.15 Pseudomonadaceae Other 2.41 3.32 2.94 1.582.15 2.25 5.57 Pseudomonadaceae Pseudomonas sp. 6.02 1.53 1.86 1.49 0.80 3.79 3.83 Pseudomonadaceae Other 0.81 1.02 1.35 0.781.56 3.887.79 Oxalobacteraceae Janthinobacterium lividum 1.50 0.66 6.49 0.59 0.33 2.77 1.39 Pseudomonadaceae Pseudomonas viridiflava 1.11 1.182.07 0.91 2.382.72 1.97 Xanthomonadaceae Stenotrophomonas sp. 10.19 0.36 0.00 0.16 0.41 0.14 0.05 Xanthomonadaceae Other 6.60 0.25 0.03 0.10 0.280.04 0.08 Firmicutes Planococcaceae Other 0.00 10.42 0.00 0.01 0.00 0.06 0.02 Bacteroidetes Sphingobacteriaceae Pedobacter cryoconitis 0.00 0.04 5.13 1.480.15 0.70 1.43 Sphingobacteriaceae Pedobacter sp. 0.10 0.03 0.26 6.09 0.06 0.16 0.89 [Weeksellaceae] Chryseobacterium sp. 0.11 0.39 1.65 3.32 0.34 0.15 1.44 Actinobacteria Microbacteriaceae Other 0.32 0.080.05 0.20 0.12 6.43 1.00 negatively correlated with available phosphorus and total Discussion zinc. Total zinc was also negatively correlated with the abundance of the phylum Nitrospirae and Euryarchaeota. Host-associated microbes play an important role in the The abundance of Acidobacteria was positively correlated growth and development of the host [16, 39]. They can with total nitrogen and total copper. There were no clear participate in the metabolic processes of the host, produce correlations between soil pH and phyla abundance. biological macromolecules with growth-promoting or

Table 5. Spearman correlation coefficient (rs) between soil properties and indicators of bacterial community structure. pH Sand Silt Clay OM TN TP TK AN AP AK TMn TCu TZn TCa TMg Simpson index 0.029 -0.600 0.486 0.600 0.543 0.886* 0.657 -0.714 0.143 0.829* 0.0860.429 0.257 0.371 0.429 -0.371

Shannon index -0.086-0.771 0.543 0.771 0.714 0.943** 0.429 -0.771 0.429 0.943** 0.200 0.600 0.371 0.600 0.600 -0.543

Observed OTU 0.257 -0.886* 0.657 0.886* 0.257 0.600 -0.257 -0.143 0.714 0.543 0.371 0.143 0.543 0.771 0.200 -0.943** Chao1 0.086 -0.943** 0.600 0.943** 0.486 0.943** 0.257 -0.429 0.429 0.771 0.257 0.429 0.200 0.829* 0.429 -0.829* Proteobacteria 0.0860.200 -0.086-0.200 -0.600-0.600 -0.771 0.829* -0.029 -0.714 -0.143 -0.486-0.086-0.143 -0.371 -0.086 Bacteroidetes -0.371 0.086-0.543 -0.086 0.829* 0.143 0.371 -0.829* 0.371 0.543 0.543 0.771 -0.200 0.371 0.429 0.371 Firmicutes 0.143 -0.200 0.657 0.200 -0.657 0.143 -0.143 0.657 -0.543 -0.371 -0.771 -0.543 0.086 -0.371 -0.143 -0.371 Actinobacteria -0.0860.771 -0.314 -0.771 -0.771 -0.771 -0.257 0.771 -0.771 -0.943** -0.657 -0.600 -0.371 -0.886* -0.429 0.600 Nitrospirae 0.029 0.600 -0.029 -0.600 -0.371 -0.486 -0.029 0.143 -0.371 -0.371 -0.429 -0.429 0.314 -0.886* -0.257 0.543 Acidobacteria 0.314 -0.600 0.486 0.600 0.314 0.314 -0.314 -0.314 0.886* 0.543 0.543 0.086 0.829* 0.543 0.086-0.657 Euryarchaeota -0.371 0.771 -0.314 -0.771 -0.371 -0.714 -0.314 0.371 -0.486 -0.657 -0.600 -0.257 -0.086 -0.886* -0.0860.714 OM, organic matter; TN, total nitrogen; TP, total phosphorus; TK, total potassium; AN, effective nitrogen; AP, available phosphorus; AK, available potassium; TMn, total manganese; TCu, total copper; TZn, total zinc; TCa, total calcium; TMg, total magnesium. *Significant at p < 0.05; **Significant at p < 0.01. Chao1, estimator of richness.

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antibacterial activities, and affect the yield and quality of evenness. In addition, the relative abundance of the the host [3, 4, 50]. However, the study of host-associated phylum Actinobacteria was similarly positively correlated microbes is mostly focused on plants, and reports on with clay and negatively correlated with sand content [27]. bacteria associated with mushrooms are relatively rare [6, Interestingly, in this study, the clay content and sand 15, 20, 33]. Many large mushrooms have a complex life content were also correlated with the numbers of observed history and demanding environmental requirements. Studying OTUs and Chao1 in the same pattern, yet there was no the community structure of bacteria in mushrooms may be significant correlation with the abundance of the phylum significant for their cultivation. Actinobacteria. In addition, we found that some mineral Most microbes in nature cannot be obtained in pure elements in soil also correlated significantly with some culture because of the difficulty of simulating the conditions bacterial taxa. For example, total zinc was negatively correlated required for their growth and reproduction. Earlier, we with the abundance of Actinobacteria, Acidobacteria, and characterized the T. matsutake fruiting body-associated Euryarchaeota, and total copper positively affected the bacteria using DGGE [29]. For a more comprehensive abundance of Acidobacteria, possibly related to the demand picture of the bacterial diversity, we applied barcoded of these mineral elements by the bacteria. Moreover, there pyrosequencing for culture-independent bacterial community were no clear correlations between phyla abundance and analysis. The results indicated that the bacteria associated soil pH in the fairy ring zone or fruiting bodies. with T. matsutake fruiting bodies were relatively abundant. Altogether, the diverse T. matsutake-associated bacteria In agreement with the earlier results [29], the bacterial showed good prospects for the cultivation of T. matsutake, communities were also varied in different samples, yet the questions to be answered are many. Further study possibly due to the different ecological environments, as of the growth-promoting bacteria should be tested, and the communities from similar environments were similar. their role in the material exchange between host plant and In the fairy ring zone of T. matsutake, Proteobacteria and mycorrhizal fungi and in pest resistance should be Acidobacteria were the dominant phyla, and the relative assessed. In conclusion, this study provides important abundance of the Proteobacteria was approximately twice knowledge about the bacterial community inhabiting the that of the Acidobacteria [27]. The bacterial communities fruiting bodies of T. matsutake and will lay a good associated with the fruiting bodies were different, as foundation for the cultivation of T. matsutake. Proteobacteria, Bacteroidetes, and Firmicutes were distributed in all samples, and Proteobacteria were over 50 times more Acknowledgments abundant than Acidobacteria. This indicated that microbial taxons associated with the T. matsutake fruiting bodies This research was funded by the National Science and were selectively enriched or reduced compared with Technology Pillar Program of Sichuan (2014FZ0004 & microorganisms in the fairy ring zone. It remains to be seen 2013NZ0029), the Foundation for Young Scholars of if this change in the composition of microbial populations Sichuan Province (2014JQ0054), and the Youth Foundation is related to the growth of T. matsutake. Pseudomonas spp. Program of the Financial and Innovational Capacity that were abundant in T. matsutake fruiting bodies have Building Project of Sichuan (2014CXSF-030). shown the ability to promote the growth of plants [13, 40]. Therefore, they might be made into microbial fertilizer applied References in the artificial cultivation of T. matsutake. Janthinobacterium sp. and Pedobacter sp. have shown antimicrobial activity 1. Alvin A, Miller KI, Neilan BA. 2014. Exploring the potential against pathogenic organisms [5, 36]. Whether these bacteria of endophytes from medicinal plants as sources of are active against pathogens inside fruiting bodies needs to antimycobacterial compounds. Microbiol. Res. 169: 483-495. be tested. 2. 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