Large variability of soil microbial diversity and functions in an over 20-year old Eucalyptus grandis plantation

The study was conducted in the south of Brazil, at the experimental station of forest sciences of Itatinga (ESALQ, University of São Paulo), which has a tropical humid climate. The soils are deep Ferralsols (F.A.O., 1998), and their mineralogy and physico-chemical characteristics are detailed in Maquere et al. (2008). The site was planted in 1998 with Eucalyptus grandis at a tree density of 1666 individual trees ha-1. Last fertilization at the site occurred in 2008, with no management for the last 10 years, meaning that the understory vegetation was allowed to develop. A 198 x 276 m stand of rather homogeneous topography was defined and divided in 108 experimental plots of 20 m x 23 m each. Plots were distributed in three blocks separated by a 9 m wide interrow (the blocks were not used in the present study). To account for the natural heterogeneity of this area, a sampling was done in March 2019 (during the dry season). The 10 cm-depth surface soil was collected using an auger in a total of 54 plots spread throughout the stand (data could be processed for 51 plots only). Each sample was a mixture of 9 soil cores taken in the plot (with three cores yielded in three inter-rows in the center of the plot). All samples were sieved at 2 mm and immediately frozen or air-dried for storage before molecular and other analyses, respectively. In order to characterize bacterial and fungal communities, total soil genomic DNA was extracted from 500 mg of soil with the FastDNA SPIN™ kit for soil (MP Biomedicals Santa Ana, CA, USA) as described by Tournier et al. (2015). Extracted DNA was done using PicoGreen fluorescence (Molecular Probes, Paris, France) and kept at -20 °C until amplification. Initial DNA quantity (2.5 ng) was amplified in a total reaction volume of 10 µl containing 1X of Sso advanced SYBR Green supermix (BioRad, Hercules, CA, USA) and 0.5 uM of each primer. qPCR assays were run in duplicate using a Biorad CFX96 Real-time PCR System and the results were analyzed with the Software Bio-Rad CFX Manager 2.0. Bacterial and fungal community diversity were assessed by amplifying the V3-V4 region of the 16S rRNA and the internal transcribed spacer ITS2 of the nuclear ribosomal RNA. To improve recovery and limit PCR bias, three PCR replicates per sample were pooled. All amplicon products were analyzed using paired-end Illumina MiSeq sequencing.

本研究于巴西南部圣保罗大学埃斯塔廷加森林科学实验站（ESALQ）开展，该区域属于热带湿润气候。供试土壤为深厚铁铝土（Ferralsols，F.A.O.，1998），其矿物学组成与理化特征详见Maquere等人2008年的研究成果。该试验地于1998年栽植巨桉（Eucalyptus grandis），种植密度为1666株·公顷⁻¹。末次施肥作业于2008年完成，此后10年未开展任何抚育管理，林下植被得以自然发育。研究划定了一片地形相对均一的林分，面积为198 m × 276 m，并将其划分为108个20 m × 23 m的试验样地；样地被分为3个区组，区组之间以9 m宽的行带间隔（本研究未使用该分区设置）。为考量该区域的自然异质性，研究于2019年3月（旱季）开展采样工作：使用土钻在覆盖整个林分的54个样地中采集10 cm深度的表层土壤样本（仅51个样地的数据可用于后续分析）。每个样本为样地内9个土芯的混合样，具体为在样地中心的3条行间各采集3个土芯。所有样本经2 mm孔径筛筛分后，一部分立即冷冻保存，另一部分风干后储存，分别用于分子生物学及其他相关分析。为表征细菌与真菌群落结构，参照Tournier等人2015年的方法，使用FastDNA SPIN™土壤DNA提取试剂盒（MP Biomedicals，美国加利福尼亚州圣安娜市），从500 mg土壤样品中提取总基因组DNA。提取的DNA采用PicoGreen荧光法进行定量（Molecular Probes，法国巴黎），并于-20 ℃下保存直至扩增环节。以2.5 ng的初始DNA量为模板，在总体积为10 µl的反应体系中进行扩增，体系包含1× Sso advanced SYBR Green预混液（BioRad，美国加利福尼亚州赫拉克勒斯市）及每条引物浓度为0.5 µM。采用Biorad CFX96实时荧光定量PCR系统进行双重复检测，数据分析使用Bio-Rad CFX Manager 2.0软件完成。通过扩增16S rRNA的V3-V4可变区以及核核糖体RNA的内转录间隔区ITS2，分别评估细菌与真菌群落的多样性。为提升扩增回收率并降低PCR偏倚，每个样本设置3个PCR重复并将扩增产物混合。所有扩增子产物均采用Illumina MiSeq双端测序技术进行测序分析。