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. pISSN 1017-7825, eISSN 1738-8872 Keywords: Tricholoma matsutake, barcoded pyrosequencing, community structure, soil Copyright© 2016 by properties The Korean Society for Microbiology and Biotechnology 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 January 2016 ⎪ Vol. 26⎪ No. 1 90 Li et al. 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 J. Microbiol. Biotechnol. Bacteria Associated with T. matsutake Fruiting Bodies 91 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