Achieving bio-protection in New Zealand ecosystems mesocosm fungal pathogen OTU table

We established 80 experimental ecosystems (mesocosms), manipulated interactions between plants and soil biota in a fully factorial design. Each mesocosm was grown in a 125 L pot (575 mm diameter), and comprised one of 20 unique, eight-species plant communities varying orthogonally in the proportion of exotic and woody shrub/tree species (0-100% and 0-63%, respectively). These plants were taken from a pool of 20 exotic and 19 native/endemic New Zealand plant species. Soil biota were manipulated using a modified plant-soil feedback approach, where each plant species was grown in monoculture in 10 L pots containing field-collected soil for 9-10 months, allowing the conditioning of typical associated soil biota for each of the plant species. We created ‘home’ soils by taking the conditioned soil from each of the eight representative species in a mesocosm and mixing it together to create a single inoculum. Each ‘home’ soil mixture was also used as an ‘away soil’ in a different mesocosm that did not contain any of the representative plants in that inoculum. These soils were intended to increase the relative biomass in inocula of specialized and preferred interaction partners of the resident (or non-resident) plant species. After approximately one year of growth, we harvested all plants from each mesocosm, took root samples from each individual plant (n=491), extracted DNA and sequenced the fungi in the roots. Fungal sequences were paired and clustered into operational taxonomic units (OTUs) at 97% similarity. We assigned functional attributes to fungal OTUs using the FUNGUILD database and retained only the taxa assigned as “probable” or “highly probable” plant pathogens.

Methods
DNA was extracted from a total of 491 root samples harvested from individual plants from all 80 mesocosms at the end of the experiment, using MoBio PowerSoil (QIAGEN) extraction kits. Root samples were taken from each individual plant using a sterile razor blade, cutting approximately 10-15 fine-root fragments from random places on the washed root ball, then bundling the roots together and slicing a 0.5-1.0 cm cross section piece from the bundle. Root samples were immediately placed into a 96 well plate from the extraction kit and frozen at -80oC as soon as a plate was full. We characterized the fungal communities by amplifying the internal transcribed spacer (ITS) of the ribosomal RNA (rRNA) operon using polymerase chain reaction (PCR) with the barcoded primers fITS7/ITS4. 

PCR was run on each sample and controls using the following thermal cycler conditions: one cycle of initial denaturation at 94°C for 5 mins; 35 cycles of denaturation at 94°C for 30 secs; annealing at 57°C for 30 secs; extension at 72°C for 30 secs; final extension at 72°C for 7 min. PCR products were checked for adequate intensity single bands on a 1% agarose gel with RedSafe. PCR reactions were carried out in duplicates for each sample and pooled prior to library preparation using the 96 well SequalPrep Normalization Kit (Invitrogen). Once PCR products were cleaned and normalized with the SequalPrep Kit, all samples were pooled and sent to Massey Genome Services, (Palmerston North, New Zealand) to be sequenced. Amplicons were sequenced on an Illumina MiSeq analyzer using the 600-cycle Reagent Kit V3, delivering 2 X 300 base pair reads/sequence (Illumina, San Diego, California, USA).

Sequences were paired, putative chimeras removed, and clustered into operational taxonomic units (OTUs) at 97% sequence similarity using Vsearch. Quality and barcode filtering resulted in 6,093,371 reads with a median length of 225 bases.

We assigned functional attributes to OTUs using the FUNGuild database and retained only the taxa assigned as “probable” or “highly probable” plant pathogens for subsequent analyses. We restricted our inclusion of taxa to those that receive most of their nutrients by harming host cells (defined as “pathotrophs” by FUNGuild) and excluded taxa with mixed strategies from our analyses (i.e. “pathotroph-saprotroph”), as many of these taxa are primarily saprotrophs and only occasionally pathogens. We acknowledge that by limiting our pathogen assignment in this way we have likely excluded many taxa that may be pathogens in some environments, so our results represent a conservative analysis of the pathogen communities hosted by our plants. Moreover, the taxa listed here are putative pathogens (hereafter referred to simply as ”pathogens”), as we rely on commonly accepted life history descriptions rather than performing real-time functional assays for each plant-fungal interaction.

我们构建了80个实验生态系统（中宇宙，mesocosms），采用全因子实验设计操控植物与土壤生物群落间的互作关系。每个中宇宙种植于125 L花盆（直径575 mm）中，包含20种独特的8物种植物群落之一，这些群落在外来植物占比和木本灌丛/乔木占比两个维度上采用正交梯度设置，占比范围分别为0-100%和0-63%。供试植物取自由20种外来植物和19种新西兰本土/特有植物组成的种质库。

土壤生物群落的操控采用改良的植物-土壤反馈方法：将每种植物单一种植于装有野外采集土壤的10 L花盆中，培养9-10个月，使各植物物种的典型伴随土壤生物群落得以富集。我们通过将某一中宇宙内8个代表性物种的富集土壤混合，制备得到“本土”土壤接种物。同时，每种“本土”土壤混合物也可作为“异地”土壤，用于不含该接种物对应代表性植物的其他中宇宙。此类土壤接种物旨在提升接种物中本地（或非本地）植物物种的特化偏好互作伙伴的相对生物量占比。

经过约1年的种植后，我们收获每个中宇宙内的所有植物，从每株植物中采集根系样本，总样本量为n=491，随后提取DNA并对根系真菌进行测序。将真菌序列进行配对拼接，并以97%的序列相似度聚类为操作分类单元（operational taxonomic units, OTUs）。我们采用FUNGuild数据库为真菌OTUs赋予功能属性，仅保留被归类为"大概率"或"极大概率"的植物病原菌类群。

### 实验方法
本实验共从80个中宇宙的所有收获植株中采集了491份根系样本，采用MoBio PowerSoil（QIAGEN）提取试剂盒完成DNA提取。具体操作如下：使用无菌剃刀从洗净的根团随机位置切取约10-15条细根片段，将根段捆扎后切取0.5-1.0 cm的横切段作为样本；样本即刻放入提取试剂盒配套的96孔板中，每块板装满后立即置于-80℃冰箱冷冻保存。

我们通过聚合酶链式反应（Polymerase Chain Reaction, PCR）扩增核糖体RNA（ribosomal RNA, rRNA）操纵子的内转录间隔区（internal transcribed spacer, ITS），以此表征真菌群落结构，所用带条形码的引物为fITS7/ITS4。

PCR反应及对照的热循环程序设置如下：94℃初始变性5 min；35个循环：94℃变性30 s，57℃退火30 s，72℃延伸30 s；最终72℃延伸7 min。通过1%琼脂糖凝胶结合RedSafe染色，检测PCR产物是否出现清晰单一目的条带。每个样本设置2次重复PCR，扩增产物混合后使用96孔板SequalPrep归一化试剂盒（Invitrogen）进行文库制备前的归一化处理。

经SequalPrep试剂盒完成纯化与归一化后，将所有样本混合并送至新西兰帕尔默斯顿北的梅西基因组服务中心（Massey Genome Services）进行测序。采用搭载600循环V3型测序试剂试剂盒的Illumina MiSeq测序仪进行双端测序，获得2×300 bp的读长序列（Illumina，美国加利福尼亚州圣地亚哥）。

使用Vsearch工具完成序列拼接、疑似嵌合体去除，并以97%的序列相似度聚类为操作分类单元（OTUs）。经质量过滤与条形码筛选后，共得到6,093,371条读段，中位长度为225 bp。

我们再次通过FUNGuild数据库为OTUs赋予功能属性，仅保留被归类为"pathotrophs"（FUNGuild将其定义为通过损伤宿主细胞获取养分的营养类群）的类群，排除兼具多种营养策略的类群（即"pathotroph-saprotroph"），因此类群多数以腐生营养为主，仅偶尔表现为病原菌。需说明的是，通过此筛选方式，我们可能排除了部分在特定环境中具有致病性的类群，因此本研究对病原菌群落的分析结果属于保守估计。此外，本研究鉴定的类群均为推定病原菌（下文简称为"病原菌"），因我们仅依据通用的生活史特征描述进行功能判定，未针对每株植物与真菌的互作开展实时功能验证。