ECTOMYC: Impact of Beech transplantation experiment (BTE) on root associated Fungi

The study was conducted in mono-specific European beech (Fagus sylvatica) forests in Central Europe (Germany). Three regions, one in southern Germany (Schwaebische Alb = ALB), one in the middle (Hainich = HAI) and one in the north-east (Schorfheide = SCH) were used to collect beech nuts. The nuts were collected in each region on four plots (ALB plots: were A05s, A07s, A08s, A09s; HAI plots: H10s, H11s, H12s, H06s; and SCH plots: S46s, S05s, S06s, S09s). The collection was conducted in autumn 2011 (28th Sept to 20th Oct) with 30 beechnuts per tree using 100 trees per plot, i.e. resulting in 3000 beech nuts per plot. The beech nuts were germinated after sterilization and grown in sterilized soil. The plants originating from different plots were termed plot-progeny. Ten plants of each plot-progeny were planted into each of nine forest plots per region in fall (September 2012). The plants were grown for 2 years and harvested in summer 2014 (July to August). Since many plants did not survive after out-planting, we selected plots with sufficient plants for harvesting, resulting in 5 plots in ALB (A05, A06, A29, A39, A42), 5 plots in SCH, (S34, S35, S37, S38, S49), and 4 plots in HAI (H05, H12, H16, H21). In addition, roots of on old tree per plot were harvested. This resulted in a total of 182 root samples for analyses of root-associated fungi. The roots were briefly washed and kept frozen at -80 Celsius. The frozen roots with their adhering fungal communities were milled and used for total DNA extraction. Some sample did not yield intact DNA and therefore omitted, resulting in about 170 samples in total. The DNA was purified and amplied (Phusion-Polymerase (Phusion High-Fidelity DNA Polymerase, New England Biolabs GmbH, Frankfurt a. M., Deutschland) using ITS3 KYO2 (TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGATGAAGAACGYAGYRAA, Microsynth AG, Balgach, Schweiz) as forward and ITS4 (GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTCCTCCGCTTATTGATATGC, Microsynth AG, Balgach, Schweiz) as reverse primer. The amplicons were purified with a DNA-Bead-Kit (MagAttract PowerClean DNA Kit, Qiagen, Hilden, Germany) and adjusted to 2 ng microl-1. The samples were sequenced at the Goettingen Genomics Laboratory (Department of Genomic and Applied Microbiology, Griesebachstrasse 8, 37077 Goettingen, Deutschland) on an Illumina MiSeq Instrument.

本研究于欧洲中部（德国）的单优欧洲山毛榉（Fagus sylvatica）林分中开展。共选取3个采样区域：德国南部的施瓦本阿尔卑斯（Schwaebische Alb，简称ALB）、中部的海尼希（Hainich，简称HAI）以及东北部的肖尔夫海德（Schorfheide，简称SCH），用于采集山毛榉坚果。每个区域内设置4个样地：ALB区域样地为A05s、A07s、A08s、A09s；HAI区域样地为H10s、H11s、H12s、H06s；SCH区域样地为S46s、S05s、S06s、S09s。坚果采集工作于2011年秋季（9月28日至10月20日）完成，每棵采种母树采集30粒坚果，每个样地选取100棵母树，最终每个样地可获得3000粒山毛榉坚果。山毛榉坚果经灭菌处理后萌发，并种植于灭菌土壤中。源自不同样地的植株被称为样地子代（plot-progeny）。2012年秋季（9月），每个样地子代的10株植株被移栽至对应区域的9个森林样地中。植株培育2年后，于2014年夏季（7月至8月）收获。由于多数植株定植后未能存活，我们仅选取植株数量满足收获要求的样地，最终ALB区域保留5个样地（A05、A06、A29、A39、A42），SCH区域保留5个样地（S34、S35、S37、S38、S49），HAI区域保留4个样地（H05、H12、H16、H21）。此外，我们采集了每个样地内1棵成年树的根系，最终共获得182份根系样本用于根系相关真菌分析。根系经短暂清洗后于-80℃冷冻保存。带有附着真菌群落的冷冻根系被研磨后用于总DNA提取。部分样本未能获得完整DNA，因此被剔除，最终有效样本总量约为170份。DNA经纯化后，使用Phusion高保真DNA聚合酶（Phusion High-Fidelity DNA Polymerase，纽英伦生物实验室有限公司，美因河畔法兰克福，德国）进行扩增，正向引物为ITS3 KYO2（序列：TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGATGAAGAACGYAGYRAA，Microsynth AG，巴尔加赫，瑞士），反向引物为ITS4（序列：GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTCCTCCGCTTATTGATATGC，Microsynth AG，巴尔加赫，瑞士）。扩增产物使用DNA磁珠纯化试剂盒（MagAttract PowerClean DNA Kit，凯杰公司，希尔德，德国）纯化，并调整至2 ng·μL⁻¹。样本在哥廷根基因组学实验室（基因组与应用微生物学系，格里塞巴赫街8号，37077哥廷根，德国）的Illumina MiSeq测序仪上完成测序。