Impact of phage therapy on Pseudomonas syringae pv. syringae and plant microbiome dynamics through coevolution and field experiment - Field trial data set

This entry contains raw data tables, metadata and R scripts used in the analysis of field trial data. Method excerpts: A field trial with phages was conducted between 19th June 2023 and 19th July 2023 and then three months after in September, at a cherry orchard research plot in NIAB, East Malling (N 51° 17' 31.7’’, E 0° 26' 52.1’’). The trees used were 15-year old Prunus avium cultivar Sweetheart on Gisela 5 rootstock. The distance between trees was 1.5 m, with 3 m distance between each row. There were two untreated guard trees between every experimental tree. A fully randomised block design with two factors (each with two levels), over five blocks with four plots per block (one tree per plot) was used. The two factors were ‘phages’ (cocktail 5C yes/no) and ‘Pss’ (Pss bacteria yes/no) for four total treatment groups: ‘Control’ consisting of PBS buffer; ‘cocktail 5C’ (108 PFU ml-1); ‘Pss’ (2 x 108 CFU ml-1); and a combination of ‘Pss+ cocktail 5C’, where Pss was applied first followed by cocktail 5C within 12 h. Subsequent sampling was done in the morning of day 1-, 2-, 3-, 4- and 30-day post treatment (DPT) to quantify Pss and phage populations. Three (out of four treated) random leaves from two shoots per tree were collected and combined into a single sample. Similarly, two random shoot sections (0.5 - 0.8 cm thick and 4 cm long) were collected from each tree at each time point and processed as described below to assess both Pss and phage populations sizes and collect representative strains. Using samples obtained from the field experiment, DNA extraction was performed on the remaining 5 ml of leaf washes, from all samples at 3 DPT and 30 DPT, for a total of 40 samples. 3 DPT was chosen as the changes in bacterial CFU and phage PFU were detectable from this time point onward. Leaf washes were centrifuged at 5000 g, for 20 min, at 4°C, resuspended in 500 µl of sterile PBS and stored in sterile 2 ml tubes at -20°C. All samples were centrifuged (16,000 g, 10 min) and the supernatant were carefully discarded. Each pellet (ca. 5 mg) was resuspended in 400 µl of lysis buffer and the DNA extracted according to the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) manufacturer’s protocol, including the optional RNase A digestion step after lysis. Yields were analysed using a Nanodrop spectrophotometer (Thermo Scientific, Waltham, MA, US). The total bacterial (16S) and fungal (ITS) microbiome abundance was quantified using quantitative PCR (qPCR) using the same primer pairs as used in amplicon sequencing below (Papp-Rupar et al., 2022). The effect of phages on the total 16S and ITS microbiome sizes (as log 10 copy number) was analysed using the same statistical approach as in analysis of CFU and PFU data. The samples were sent to Novogene UK (Cambridge, UK) for PCR, library prep and amplicon sequencing of fungal ITS1 amplicon using ITS1-1F (5’-CTTGGTCATTTAGAGGAAGTAA-3’ (Gardes & Bruns, 1993) and ITS2 primer (5’-GCTGCGTTCTTCATCGATGC-‘3 (White et al., 1990); and bacterial 16S V5-V7 amplicon using 799F (5’-AACMGGATTAGATACCCKG-3’ (Chelius & Triplett, 2001) and 1193R (5’-ACGTCATCCCCACCTTCC-3’ (Bodenhausen et al., 2013)). Samples were sequenced on an Illumina NovaSeq platform in paired end mode with read length of 250 nt. Amplicon Sequence Variants (ASVs) were generated from a combined set of leaf and shoot samples (across both 3 DPT and 30 DPT) but analysed separately, using a previously published pipeline (Papp-Rupar et al., 2022). The following raw sequence reads were discarded in quality control step: reads with incorrect bases in the barcode or primer regions; and reads containing adapter contamination. Forward and reverse reads used in the ASV generation step were merged using the UPARSE pipeline V. 11.0 (Edgar, 2013) with stringent criteria: minimum read length of 250 nt, zero differences in read overlap region, maximum expected error threshold of 0.2 (16S) and 0.1 (ITS) per sequence (Edgar and Flyvbjerg, 2015) and minimal merged read length of 400 (16S) or 185 (ITS). Reads were then dereplicated, chimeric sequences removed and sequences with less than eight replicates discarded before generation of denoised ASVs. For frequency table generation, reads were merged using “differences in read overlap region” set to 100 to ensure effectively all reads were merged. These unfiltered merged reads were aligned to the ASV representative sequences at the level of 97% similarity to produce an ASV frequency table. Finally, the SINTAX algorithm (https://www.drive5.com/usearch/manual/sintax_algo.html) was used to assign taxonomic ranks to each ASV with the Unite V8.3 (2021-05-10) fungal database (Kõljalg et al., 2013) and “the RDP training set V18” database for the 16S rRNA gene (Cole et al., 2014). The SINTAX algorithm only resolves bacterial ASVs to the genus level, but may resolve fungal ASVs to the species level. Taxonomy assignment confidence was at the 80% level.本数据集包含用于田间试验数据分析的原始数据表、元数据（metadata）与R脚本。 方法节选： 本研究于2023年6月19日至2023年7月19日开展噬菌体（phages）田间试验，并于三个月后的同年9月进行后续采样，试验地点位于东梅林（East Malling）NIAB的樱桃园试验地块，地理坐标为北纬51°17'31.7''，东经0°26'52.1''。试验所用果树为种植15年的欧洲甜樱桃（Prunus avium）品种'Sweetheart'，嫁接于Gisela 5砧木。株间距为1.5米，行间距为3米；每株试验果树两侧各设置2株未作处理的保护行果树。 本试验采用完全随机区组设计，设置2个因素（每个因素均设2个水平），共包含5个区组，每个区组设4个样地（每个样地对应1株果树）。两个因素分别为“噬菌体（phages）”（是否施加5C噬菌体鸡尾酒）与“Pss”（是否接种Pss细菌），共形成4个处理组：①对照组（Control）：仅施加磷酸盐缓冲液（PBS）；②5C噬菌体鸡尾酒组（cocktail 5C）：施加浓度为10^8 噬菌斑形成单位/毫升（plaque forming unit, PFU ml⁻¹）的5C噬菌体鸡尾酒；③Pss细菌组（Pss）：施加浓度为2×10^8 菌落形成单位/毫升（colony forming unit, CFU ml⁻¹）的Pss菌液；④Pss+5C噬菌体鸡尾酒联合组（Pss+ cocktail 5C）：先接种Pss菌液，12小时内再施加5C噬菌体鸡尾酒。 后续采样分别于处理后第1、2、3、4及30天（day post treatment, DPT）的清晨进行，用于定量检测Pss细菌与噬菌体的种群数量。每株果树采集2个枝条上的3片随机叶片，混合为单一样本。同理，在每个采样时间点从每株果树采集2段随机枝条段（直径0.5~0.8 cm，长度4 cm），按照下述流程处理，用于检测Pss细菌与噬菌体的种群密度，并收集代表性菌株。 基于田间试验获取的样本，本研究对3 DPT与30 DPT两个时间点的所有剩余5 ml叶片洗脱液样本进行DNA提取，共计40个样本。选择3 DPT作为采样时间点，是因为自此时间点起可检测到细菌CFU与噬菌体PFU的数量变化。将叶片洗脱液以5000 g离心20分钟（4℃），重悬于500 μl无菌PBS中，随后转移至无菌2 ml离心管内，于-20℃保存。所有样本再次以16000 g离心10分钟，小心弃去上清液。将获得的菌体沉淀（约5 mg）重悬于400 μl裂解缓冲液中，按照DNeasy植物微量试剂盒（Qiagen，德国希尔德）的制造商说明书进行DNA提取，包含裂解后可选的RNase A消化步骤。使用Nanodrop分光光度计（赛默飞世尔科技，美国马萨诸塞州沃尔瑟姆）对DNA提取产量进行检测。采用与下述扩增子测序相同的引物对，通过实时定量PCR（quantitative PCR, qPCR）定量检测总细菌（16S rRNA基因）与真菌（内转录间隔区，ITS）的微生物组丰度（Papp-Rupar等，2022）。采用与CFU、PFU数据分析相同的统计学方法，分析噬菌体对总细菌16S与真菌ITS微生物组丰度（以log10拷贝数计）的影响。 将样本送至英国诺禾致源（Novogene UK，英国剑桥）进行PCR、文库制备以及真菌ITS1扩增子测序，所用引物为ITS1-1F（5’-CTTGGTCATTTAGAGGAAGTAA-3’，Gardes & Bruns, 1993）与ITS2引物（5’-GCTGCGTTCTTCATCGATGC-3’，White等，1990）；同时进行细菌16S V5-V7区扩增子测序，所用引物为799F（5’-AACMGGATTAGATACCCKG-3’，Chelius & Triplett, 2001）与1193R（5’-ACGTCATCCCCACCTTCC-3’，Bodenhausen等，2013）。测序在Illumina NovaSeq平台上进行，采用双端测序模式，读长为250 nt（核苷酸）。 基于3 DPT与30 DPT两个时间点的叶片及枝条样本的合并数据集，生成扩增子序列变异体（Amplicon Sequence Variants, ASVs），并按照已发表的分析流程分别进行分析（Papp-Rupar等，2022）。在质量控制步骤中，以下原始序列读段将被剔除：条形码或引物区域存在错误碱基的读段，以及含有接头污染的读段。在生成ASV的步骤中，使用UPARSE分析流程V11.0（Edgar, 2013）对正向与反向读段进行拼接，采用严格的筛选标准：最小读长250 nt，读段重叠区域无碱基错配，每条序列的最大预期错误阈值分别为0.2（16S）与0.1（ITS）（Edgar & Flyvbjerg, 2015），拼接后的最小读长分别为400 nt（16S）与185 nt（ITS）。在生成去噪ASV之前，需先对读段进行去重复、剔除嵌合序列，并丢弃复制数少于8的序列。在生成频率表时，将读段重叠区域的碱基错配数设置为100以确保几乎所有读段均可完成拼接。将未经过滤的拼接读段与ASV代表序列以97%的相似度进行比对，从而生成ASV频率表。最后，使用SINTAX算法（https://www.drive5.com/usearch/manual/sintax_algo.html）为每个ASV分配分类学等级：针对真菌ITS序列采用Unite V8.3（2021-05-10）数据库（Kõljalg等，2013），针对细菌16S rRNA基因采用“RDP训练集V18”数据库（Cole等，2014）。SINTAX算法仅能将细菌ASV鉴定至属水平，却可将真菌ASV鉴定至种水平，分类学鉴定的置信度阈值为80%。