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Bacterial community structure and co-occurrence networks in the rhizosphere and root endosphere of the grafted apple
嫁接苹果根际及根内圈细菌群落结构及共生网络

Hui Cao 1 , 2 ∗ + 1 , 2 ∗ + ^(1,2^(**+)){ }^{1,2^{*+}}, Longxiao Xu² , Jianfei Song 2 2 ^(2){ }^{2}, Mi Xun², Weiwei Zhang 2 2 ^(2){ }^{2} and Hongqiang Yang 2 ∗ 2 ∗ ^(2^(**)){ }^{2^{*}}
曹慧 1 , 2 ∗ + 1 , 2 ∗ + ^(1,2^(**+)){ }^{1,2^{*+}} 、徐龙晓²、宋剑飞 2 2 ^(2){ }^{2} , 米寻², 张薇薇 2 2 ^(2){ }^{2} 和杨红强 2 ∗ 2 ∗ ^(2^(**)){ }^{2^{*}}

Abstract  抽象的

Background Compared with aerial plant tissues (such as leaf, stem, and flower), root-associated microbiomes play an indisputable role in promoting plant health and productivity. We thus explored the similarities and differences between rhizosphere and root endosphere bacterial community in the grafted apple system.
背景与气生植物组织（如叶、茎和花）相比，根相关微生物组在促进植物健康和生产力方面发挥着无可争议的作用。因此，我们探讨了嫁接苹果系统中根际和根内圈细菌群落的异同。

Results Using pot experiments, three microhabitats (bulk soil, rhizosphere and root endosphere) samples were obtained from two-year-old apple trees grafted on the four different rootstocks. We then investigated the bacterial community composition, diversity, and co-occurrence network in three microhabitats using the Illumina sequencing methods. Only 63 amplicon sequence variants (ASVs) out of a total of 24,485 were shared in the rhizosphere and root endosphere of apple grafted on the four different rootstocks (M9T337, Malus hupehensis Rehd., Malus robusta Rehd., and Malus baccata Borkh.). The core microbiome contained 8 phyla and 25 families. From the bulk soil to the rhizosphere to the root endosphere, the members of the phylum and class levels demonstrated a significant enrichment and depletion pattern. Co-occurrence network analysis showed the network complexity of the rhizosphere was higher than the root endosphere. Most of the keystone nodes in both networks were classified as Proteobacteria, Actinobacteriota and Bacteroidetes and were low abundance species.
结果通过盆栽实验，从嫁接在四种不同砧木上的两年生苹果树获得了三个微生境（土壤、根际和根内圈）样品。然后，我们使用 Illumina 测序方法研究了三个微生境中的细菌群落组成、多样性和共现网络。在四种不同砧木（M9T337、Malus hupehensis Rehd.、MalusRobusta Rehd. 和 Malus baccata Borkh.）上嫁接的苹果的根际和根内圈中，总共 24,485 个扩增子序列变异（ASV）中只有 63 个是共享的。核心微生物组包含 8 个门和 25 个科。从大块土壤到根际再到根内圈，门和类水平的成员表现出显着的富集和消耗模式。共现网络分析表明根际网络复杂度高于根内圈。两个网络中的大多数关键节点都被归类为变形菌门、放线菌门和拟杆菌门，并且是低丰度物种。

Conclusion The hierarchical filtration pattern existed not only in the assembly of root endosphere bacteria, but also in the core microbiome. Moreover, most of the core ASVs were high-abundance species, while the keystone ASVs of the network were low-abundance species.
结论 分层过滤模式不仅存在于根内球细菌的组装中，而且也存在于核心微生物组中。此外，大多数核心ASV是高丰度物种，而网络的关键ASV是低丰度物种。

Keywords Rhizosphere, Root endosphere, Bacterial community diversity, Co-occurrence network
关键词 根际, 根内圈, 细菌群落多样性, 共现网络
† † ^(†){ }^{\dagger} Hui Cao and Longxiao Xu contributed equally to this work.
† † ^(†){ }^{\dagger} 曹辉和徐龙晓对这项工作做出了同等贡献。
*Correspondence:  *一致：
Hui Cao  曹慧
caohui716@163.com
Hongqiang Yang  杨红强
hqyang@sdau.edu.cn
1 1 ^(1){ }^{1} College of Life Sciences, Zaozhuang University, Zaozhuang 277000, Shandong Province, China
1 1 ^(1){ }^{1} 枣庄学院生命科学学院, 山东省枣庄市 277000
2 2 ^(2){ }^{2} College of Horticulture Science and Engineering, Shandong Agricultural University, State Key Laboratory of Crop Biology, Tai’an 271018, Shandong Province, China
2 2 ^(2){ }^{2} 山东农业大学园艺科学与工程学院，作物生物学国家重点实验室，山东省泰安市 271018

Background  背景

Apple is one of the fruits with the most widely cultivated acreage in the world [1], and its cultivated soil types and rootstock types are varied, which leads to the diversity of apple root morphology and root-zone soil microbiomes. These differences are crucial significance for apple to absorb and utilize nutrients from the soil and promote the improvement of fruit yield and quality.
苹果是世界上种植面积最广的水果之一[1]，其栽培土壤类型和砧木类型多样，导致苹果根系形态和根区土壤微生物组的多样性。这些差异对于苹果吸收利用土壤养分、促进果实产量和品质的提高具有至关重要的意义。
The root, which plays a crucial role in the interaction between soil microbiomes and plants, is the basis of the plant. Root tissues provide colonization site and secrete essential organic compounds to maintains the plant-specific microbiota of root-zone soils [2,3], which includes
根是植物的基础，在土壤微生物组和植物之间的相互作用中起着至关重要的作用。根组织提供定殖位点并分泌必需的有机化合物，以维持根区土壤的植物特异性微生物群 [2,3]，其中包括
not only soil attached microorganisms adhering to the roots and inhabit the root surface, but also the colonized microorganisms in the root interior environment [4]. Depending on the chemical signals and nutrients released by the root, microorganisms are enriched and grown in the rhizosphere soil, and then they pass through the regulation and selection by plant own metabolism to stably colonize the root tissues [5-7].
不仅是附着在根部并栖息在根部表面的土壤附着微生物，还有根部内部环境中定植的微生物[4]。依靠根部释放的化学信号和养分，微生物在根际土壤中富集和生长，然后通过植物自身代谢的调节和选择，稳定定植于根部组织[5-7]。
Root-associated microbiomes mainly include viruses, bacteria, archaea, protozoa, and fungi [8]. Among them, the diversity and abundance of bacteria are enormous, and they play a more important role in promoting plant health and improving crop productivity [9]. For instance, the rhizosphere bacterial community can promote the decomposition of mineral nutrients, defend against soilborne diseases and improve plant resilience to adverse growth conditions [10-12]. It is reported that the inoculated plant-growth-promoting rhizobacteria strains contributed to the increase in young apple tree growth and fruit yield [13]. Often the beneficial effects of root endophytes, without causing any evident damage to the host plants, are greater than many rhizosphere bacteria [5, 14]. Plant-growth-promoting bacterial endophytes facilitate plant growth by producing phytohormones, antimicrobial metabolites, and increasing supply of nutrients [15, 16].
根相关微生物群主要包括病毒、细菌、古细菌、原生动物和真菌[8]。其中，细菌的多样性和丰度巨大，在促进植物健康、提高作物生产力方面发挥着更为重要的作用[9]。例如，根际细菌群落可以促进矿质养分的分解，防御土传疾病并提高植物对不利生长条件的抵抗力[10-12]。据报道，接种促进植物生长的根际细菌菌株有助于苹果幼树生长和果实产量的增加[13]。通常，根内生菌的有益作用在不对宿主植物造成任何明显损害的情况下比许多根际细菌还要大 [5, 14]。促进植物生长的细菌内生菌通过产生植物激素、抗菌代谢物和增加营养供应来促进植物生长[15, 16]。
In apple orchards, the roots of the rootstocks uptake nutrients from soil for the plant and are the primary site of rhizosphere microorganisms [2]. Chai et al. (2020) used Illumina MiSeq sequencing to determine the bacterial community of the rhizosphere from different rootstocks, they found apple rootstocks with different phosphorus efficiency showed alteration of the microbes in rhizosphere [17]. They also found the rhizosphere bacterial community structure significantly differed among the apple rootstocks of different nitrogen tolerance, for example, the bacterial phyla Proteobacteria and Actinobacteria were the dominant groups in the rhizosphere and presented higher abundance in the low nitrogen-tolerant rhizosphere [18]. Liu et al. (2022) also demonstrated a clear impact of root genotype on microbial composition and diversity [19]. Previous studies have indicated that the apple rootstock also has an important effect on the endophytic microbiota of different rootstock/scion combinations, interestingly, “M.M.111” rootstock with weak growth control properties had more beneficial and growth promoting fungal and bacterial taxa than “M.9” rootstock with strong growth control properties [20]. As reviewed by previous studies, rootstock genotypes can influence the taxonomy, structure,composition and network properties of the rhizosphere bacterial community in grapes [21, 22]. However, studies unveiling the bacterial community structure and network in the rhizosphere
在苹果园中，砧木的根部从土壤中为植物吸收养分，是根际微生物的主要场所[2]。柴等人。 (2020)利用Illumina MiSeq测序来确定不同砧木根际细菌群落，他们发现不同磷效率的苹果砧木显示出根际微生物的变化[17]。他们还发现，不同耐氮性的苹果砧木根际细菌群落结构存在显着差异，例如，细菌门变形菌门和放线菌门是根际的优势类群，在低耐氮根际丰度较高[18]。刘等人。 (2022)还证明了根基因型对微生物组成和多样性的明显影响[19]。前期研究表明，苹果砧木对不同砧木/接穗组合的内生微生物群也有重要影响，有趣的是，生长控制能力较弱的“MM111”砧木比“M.9”具有更多有益和促进生长的真菌和细菌类群。 ”具有很强的生长控制特性的砧木[20]。根据以往的研究综述，砧木基因型可以影响葡萄根际细菌群落的分类、结构、组成和网络特性[21, 22]。然而，研究揭示了根际细菌群落结构和网络
and root endosphere of grafted apple are lacking. In this study, we use a grafted apple system with four different rootstocks to study root-associated bacterial communities by 16 S rRNA gene high-throughput sequencing. We compared the changes of the bacterial community diversities and co-occurrence network in the bulk soil, rhizosphere, and root endosphere. Our results will lay the groundwork for regulating the rhizosphere and root endosphere microbiomes to promote apple healthy growth.
嫁接苹果缺乏根内膜。在本研究中，我们使用具有四种不同砧木的嫁接苹果系统，通过 16 S rRNA 基因高通量测序来研究根相关细菌群落。我们比较了大块土壤、根际和根内圈的细菌群落多样性和共现网络的变化。我们的研究结果将为调节根际和根内圈微生物组以促进苹果健康生长奠定基础。

Materials and methods  材料和方法

Experimental materials and design
实验材料与设计

The 2 -year-old apple scion variety (Malus domestica Borkh.cv.Red Fuji), grafted on four rootstocks (M9T337, Malus hupehensis Rehd., Malus robusta Rehd., and Malus baccata Borkh.) were used in the study. The native soil from an arable site in Taian city ( 36 ∘ 10 ′ N , 117 ∘ 07 ′ E ) 36 ∘ 10 ′ N , 117 ∘ 07 ′ E (36^(@)10^(')N,117^(@)07^(')E)\left(36^{\circ} 10^{\prime} \mathrm{N}, 117^{\circ} 07^{\prime} \mathrm{E}\right), Shandong Province, China, was collected at 0 − 20 − cm 0 − 20 − cm 0-20-cm0-20-\mathrm{cm} depth. The soil is loam ( 21 % 21 % 21%21 \% clay, 29 % 29 % 29%29 \% powder and 50 % 50 % 50%50 \% sand) with a pH of 6.7 , bulk density of 1.37 g ⋅ cm − 3 1.37 g ⋅ cm − 3 1.37g*cm^(-3)1.37 \mathrm{~g} \cdot \mathrm{~cm}^{-3}, available nitrogen of 80.50 mg ⋅ kg − 1 80.50 mg ⋅ kg − 1 80.50mg*kg^(-1)80.50 \mathrm{mg} \cdot \mathrm{kg}^{-1}, available phosphorus of 66.46 mg ⋅ kg − 1 66.46 mg ⋅ kg − 1 66.46mg*kg^(-1)66.46 \mathrm{mg} \cdot \mathrm{kg}^{-1}, available potassium of 129.84 mg ⋅ kg − 1 129.84 mg ⋅ kg − 1 129.84mg*kg^(-1)129.84 \mathrm{mg} \cdot \mathrm{kg}^{-1}, organic matter of 10.05 g ⋅ kg − 1 10.05 g ⋅ kg − 1 10.05g*kg^(-1)10.05 \mathrm{~g} \cdot \mathrm{~kg}^{-1} and it is classified as a Cinnamon soil. The experiment used a potted method, four different grafted seedlings were planted in pots with three replicates established in a completely randomized block design.
研究中使用了嫁接在四种砧木（M9T337、Malus hupehensis Rehd.、MalusRobusta Rehd. 和 Malus baccata Borkh.）上的 2 年生苹果接穗品种（Malus Domestica Borkh.cv.Red Fuji）。泰安市耕地原生土 ( 36 ∘ 10 ′ N , 117 ∘ 07 ′ E ) 36 ∘ 10 ′ N , 117 ∘ 07 ′ E (36^(@)10^(')N,117^(@)07^(')E)\left(36^{\circ} 10^{\prime} \mathrm{N}, 117^{\circ} 07^{\prime} \mathrm{E}\right) ，中国山东省，收集于 0 − 20 − cm 0 − 20 − cm 0-20-cm0-20-\mathrm{cm} 深度。土壤为壤土（ 21 % 21 % 21%21 \% 黏土， 29 % 29 % 29%29 \% 粉末和 50 % 50 % 50%50 \% 沙），pH值为6.7，堆积密度为 1.37 g ⋅ cm − 3 1.37 g ⋅ cm − 3 1.37g*cm^(-3)1.37 \mathrm{~g} \cdot \mathrm{~cm}^{-3} , 有效氮 80.50 mg ⋅ kg − 1 80.50 mg ⋅ kg − 1 80.50mg*kg^(-1)80.50 \mathrm{mg} \cdot \mathrm{kg}^{-1} , 有效磷 66.46 mg ⋅ kg − 1 66.46 mg ⋅ kg − 1 66.46mg*kg^(-1)66.46 \mathrm{mg} \cdot \mathrm{kg}^{-1} , 有效钾 129.84 mg ⋅ kg − 1 129.84 mg ⋅ kg − 1 129.84mg*kg^(-1)129.84 \mathrm{mg} \cdot \mathrm{kg}^{-1} , 有机质 10.05 g ⋅ kg − 1 10.05 g ⋅ kg − 1 10.05g*kg^(-1)10.05 \mathrm{~g} \cdot \mathrm{~kg}^{-1} 它被归类为肉桂土。实验采用盆栽方法，将四种不同的嫁接苗种植在盆中，并以完全随机区组设计设置三个重复。

Sample collection of the rhizosphere, root endosphere, and bulk soil
根际、根内圈和土体的样品采集

We separated the rhizosphere soil from the root endosphere according to the methods described previously [23]. Briefly, roots were manually removed from the pot using sterile gloves and gently shaken to remove loose soil. Root segments with adhering soil of 8 cm starting 2 cm below the root base were dissected with a sterile scalpel and placed into sterile tubes containing PBS-S buffer ( 130 mM NaCl , 7 mM Na 2 HPO 4 , 3 mM NaH PO 4 130 mM NaCl , 7 mM Na 2 HPO 4 , 3 mM NaH PO 4 130mMNaCl,7mMNa_(2)HPO_(4),3mMNaHPO_(4)130 \mathrm{mM} \mathrm{NaCl}, 7 \mathrm{mM} \mathrm{Na}{ }_{2} \mathrm{HPO}_{4}, 3 \mathrm{mM} \mathrm{NaH} \mathrm{PO}_{4}, pH7.0, 0.02% Silwet L-77). The root segments were washed twice with shaking at 180 rpm for 20 min each time, and the two washing buffers were combined. The pellet resulting from the centrifugation of the washing buffer for 20 min at 4000 g was defined as the rhizosphere samples and frozen for storage at − 80 ∘ C − 80 ∘ C -80^(@)C-80^{\circ} \mathrm{C}.
我们根据之前描述的方法将根际土壤与根内圈分离[23]。简而言之，使用无菌手套手动将根从盆中取出，并轻轻摇动以除去松散的土壤。用无菌手术刀从根基下方 2 厘米处切下带有 8 厘米附着土壤的根段，并将其放入含有 PBS-S 缓冲液的无菌管中（ 130 mM NaCl , 7 mM Na 2 HPO 4 , 3 mM NaH PO 4 130 mM NaCl , 7 mM Na 2 HPO 4 , 3 mM NaH PO 4 130mMNaCl,7mMNa_(2)HPO_(4),3mMNaHPO_(4)130 \mathrm{mM} \mathrm{NaCl}, 7 \mathrm{mM} \mathrm{Na}{ }_{2} \mathrm{HPO}_{4}, 3 \mathrm{mM} \mathrm{NaH} \mathrm{PO}_{4} ，pH7.0，0.02% Silwet L-77)。将根段洗涤两次，每次 180 rpm 振荡 20 分钟，并将两种洗涤缓冲液合并。将洗涤缓冲液在 4000 g 下离心 20 分钟所得的沉淀物定义为根际样品，并冷冻保存于 − 80 ∘ C − 80 ∘ C -80^(@)C-80^{\circ} \mathrm{C} 。
The treated root segments were washed with water and moved to a new sterile tube. Next, the root segments were sterilized with 70 % 70 % 70%70 \% alcohol and a sodium hypochlorite solution containing 2.5 % 2.5 % 2.5%2.5 \% active Cl − Cl − Cl^(-)\mathrm{Cl}^{-}, as described in Sun et al. [24]. Finally, the root segments were rinsed in sterile, distilled water several times. The sterile root segments were defined as the root endosphere samples and frozen for storage at − 80 ∘ C − 80 ∘ C -80^(@)C-80^{\circ} \mathrm{C}.
处理过的根段用水洗涤并移至新的无菌管中。接下来，将根段消毒 70 % 70 % 70%70 \% 酒精和次氯酸钠溶液含有 2.5 % 2.5 % 2.5%2.5 \% 积极的 Cl − Cl − Cl^(-)\mathrm{Cl}^{-} ，如 Sun 等人所述。 [24]。最后，用无菌蒸馏水冲洗根段数次。将无菌根段定义为根内球样品并冷冻保存于 − 80 ∘ C − 80 ∘ C -80^(@)C-80^{\circ} \mathrm{C} 。
The bulk soil samples were collected from unplanted apple tree pots and the soil depth from 2 to 10 cm from the surface corresponding to 8 cm root length, then stored at − 80 ∘ C − 80 ∘ C -80^(@)C-80^{\circ} \mathrm{C} until further processing.
大量土壤样品从未种植的苹果树盆中采集，土壤深度为距表面 2 至 10 cm，相当于 8 cm 根长，然后储存在 − 80 ∘ C − 80 ∘ C -80^(@)C-80^{\circ} \mathrm{C} 直至进一步处理。

DNA extraction  DNA提取

Microbial community genomic DNA from the bulk soil, rhizosphere, and root endosphere samples were extracted using the E.Z.N.A. ® ® ^(®){ }^{\circledR} Soil DNA Kit (Omega, USA) according to the manufacturer’s instructions. Then, total DNA was detected on 0.8 % 0.8 % 0.8%0.8 \% agarose gel electrophoresis and a Nanodrop 2000 UV-vis Spectrophotometer (Thermo Scientific, Wilmington, USA) was used to determined DNA concentration and quality.
使用 EZNA 从大量土壤、根际和根内圈样品中提取微生物群落基因组 DNA ® ® ^(®){ }^{\circledR} 土壤 DNA 试剂盒（Omega，美国）根据制造商的说明。然后，检测总DNA 0.8 % 0.8 % 0.8%0.8 \% 使用琼脂糖凝胶电泳和 Nanodrop 2000 紫外可见分光光度计（Thermo Scientific，威尔明顿，美国）测定 DNA 浓度和质量。

PCR amplification and sequencing
PCR 扩增和测序

The V3-V4 hypervariable region of the bacterial 16S rRNA gene was amplified with primer pairs 338F (forward primer 5 ′ 5 ′ 5^(')5^{\prime}-ACTCCTACGGGAGGCAGCA-3’) and 806R (reverse primer 5 ′ 5 ′ 5^(')5^{\prime}-GGACTACHVGGGTWT CTAAT-3’). The PCR mixtures contain 5 × 5 × 5xx5 \times reaction buffer 5 μ L , 5 × GC 5 μ L , 5 × GC 5muL,5xxGC5 \mu \mathrm{~L}, 5 \times \mathrm{GC} buffer 5 μ L 5 μ L 5muL5 \mu \mathrm{~L}, dNTP ( 2.5 mM ) 2 μ L 2 μ L 2muL2 \mu \mathrm{~L}, Q5 DNA Polymerase 0.25 μ L 0.25 μ L 0.25 muL0.25 \mu \mathrm{~L} from Q5 ® ® ^(®){ }^{\circledR} High-Fidelity DNA Polymerase (New England Biolabs [NEB], MA, USA), forward primer ( 10 μ M ) 1 μ L ( 10 μ M ) 1 μ L (10 muM)1muL(10 \mu \mathrm{M}) 1 \mu \mathrm{~L}, reverse primer ( 10 μ M ) ( 10 μ M ) (10 muM)(10 \mu \mathrm{M}) 1 μ L 1 μ L 1muL1 \mu \mathrm{~L}, template DNA 2 μ l 2 μ l 2mul2 \mu \mathrm{l}, and finally ddH 2 O ddH 2 O ddH_(2)O\mathrm{ddH}_{2} \mathrm{O} up to 25 μ L 25 μ L 25 muL25 \mu \mathrm{~L}. The PCR conditions 98 ∘ C 98 ∘ C 98^(@)C98^{\circ} \mathrm{C} for 2 min , followed by 30 cycles of 98 ∘ C 98 ∘ C 98^(@)C98^{\circ} \mathrm{C} for 15 s , 55 ∘ C 15 s , 55 ∘ C 15s,55^(@)C15 \mathrm{~s}, 55^{\circ} \mathrm{C} for 30 s , 72 ∘ C 30 s , 72 ∘ C 30s,72^(@)C30 \mathrm{~s}, 72^{\circ} \mathrm{C} for 30 s , and a final extension at 72 ∘ C 72 ∘ C 72^(@)C72^{\circ} \mathrm{C} for 5 min . PCR reactions were performed in triplicate. The amplified PCR products were separated on 0.8 % 0.8 % 0.8%0.8 \% agarose gels, purified using an AxyPrep DNA Gel Extraction Kit (AXYGEN, USA) and quantified using a Quant-iT PicoGreen dsDNA Assay Kit and Microplate reader (BioTek, FLx800). Briefly, After obtaining the pure purified amplicons, we used the TruSeq ® ® ^(®){ }^{\circledR} DNA PCR-Free Sample Preparation Kit (Illumina, USA) for library construction according to the manufacturer’s protocol. Nuclease free water (QIAGEN, Valencia, CA, USA) replaced template DNA in negative controls. The library quality was preliminary determined by Qubit ® ® ^(®){ }^{\circledR} 2.0 Fluorometer (Thermo Scientific, USA), and Q-PCR according to Wang et al. (2022) was used for accurate and quantitative library detection [25]. After the library was qualified, the bacterial communities of all samples including negative controls were sequenced using the Illumina Miseq System by Personal Biotechnology Co., Ltd. (Shanghai, China). All sequence data have been deposited into the NCBI Sequence Read Archive database under accession number SRP280070.
使用引物对 338F（正向引物）扩增细菌 16S rRNA 基因的 V3-V4 高变区 5 ′ 5 ′ 5^(')5^{\prime} -ACTCCTACGGGAGGCAGCA-3') 和 806R（反向引物 5 ′ 5 ′ 5^(')5^{\prime} -GGACTACHVGGGTWT CTAAT-3')。 PCR 混合物包含 5 × 5 × 5xx5 \times 反应缓冲液 5 μ L , 5 × GC 5 μ L , 5 × GC 5muL,5xxGC5 \mu \mathrm{~L}, 5 \times \mathrm{GC} 缓冲 5 μ L 5 μ L 5muL5 \mu \mathrm{~L} , dNTP (2.5 mM) 2 μ L 2 μ L 2muL2 \mu \mathrm{~L} , Q5 DNA 聚合酶 0.25 μ L 0.25 μ L 0.25 muL0.25 \mu \mathrm{~L} 从Q5开始 ® ® ^(®){ }^{\circledR} 高保真 DNA 聚合酶（New England Biolabs [NEB]，MA，美国），正向引物 ( 10 μ M ) 1 μ L ( 10 μ M ) 1 μ L (10 muM)1muL(10 \mu \mathrm{M}) 1 \mu \mathrm{~L} , 反向引物 ( 10 μ M ) ( 10 μ M ) (10 muM)(10 \mu \mathrm{M}) 1 μ L 1 μ L 1muL1 \mu \mathrm{~L} , 模板 DNA 2 μ l 2 μ l 2mul2 \mu \mathrm{l} ，最后 ddH 2 O ddH 2 O ddH_(2)O\mathrm{ddH}_{2} \mathrm{O} 最多 25 μ L 25 μ L 25 muL25 \mu \mathrm{~L} 。 PCR条件 98 ∘ C 98 ∘ C 98^(@)C98^{\circ} \mathrm{C} 2分钟，然后进行30个循环 98 ∘ C 98 ∘ C 98^(@)C98^{\circ} \mathrm{C} 为了 15 s , 55 ∘ C 15 s , 55 ∘ C 15s,55^(@)C15 \mathrm{~s}, 55^{\circ} \mathrm{C} 为了 30 s , 72 ∘ C 30 s , 72 ∘ C 30s,72^(@)C30 \mathrm{~s}, 72^{\circ} \mathrm{C} 持续 30 秒，最后延长至 72 ∘ C 72 ∘ C 72^(@)C72^{\circ} \mathrm{C} 5分钟。 PCR 反应一式三份进行。将扩增的 PCR 产物分离 0.8 % 0.8 % 0.8%0.8 \% 琼脂糖凝胶，使用 AxyPrep DNA 凝胶提取试剂盒（AXYGEN，美国）纯化，并使用 Quant-iT PicoGreen dsDNA 检测试剂盒和酶标仪（BioTek，FLx800）定量。简而言之，在获得纯的纯化扩增子后，我们使用 TruSeq ® ® ^(®){ }^{\circledR} DNA PCR-Free 样品制备试剂盒（Illumina，美国），用于根据制造商的方案构建文库。无核酸酶水（QIAGEN，巴伦西亚，加利福尼亚州，美国）取代了阴性对照中的模板 DNA。文库质量由Qubit初步判定 ® ® ^(®){ }^{\circledR} 2.0 荧光计（Thermo Scientific，美国），以及根据 Wang 等人的 Q-PCR。 (2022) 用于准确定量的文库检测 [25]。文库合格后，使用上海个人生物科技有限公司（中国上海）的 Illumina Miseq 系统对包括阴性对照在内的所有样品的细菌群落进行测序。 所有序列数据均已存入 NCBI 序列读取存档数据库，登录号为 SRP280070。

Processing and analysis of sequencing data
测序数据的处理和分析

The raw data were performed using QIIME 2 version 2023.2. Briefly, raw sequence data were demultiplexed
原始数据使用 QIIME 2 版本 2023.2 进行。简而言之，原始序列数据被解复用
using the demux plugin, and primers were cut with cutadapt plugin. Sequences were then quality filtered, denoised, chimera removed and merged amplicon sequence variants (ASVs) using the DADA 2 plugin [26]. Finally, singletons ASVs were removed, and the sequencing depth of per sample was rarefied to counts up to 92,636 reads (the lowest sequencing depth of all samples). The taxonomy annotation of each ASVs representative sequence was analyzed using the Greengenes2 database (http://greengenes.secondgeno me.com/ )[27].
使用 demux 插件，并使用 cutadapt 插件切割引物。然后使用 DADA 2 插件对序列进行质量过滤、去噪、嵌合体去除和合并扩增子序列变体 (ASV) [26]。最后，单例 ASV 被去除，每个样本的测序深度被稀疏化至 92,636 个读数（所有样本中最低的测序深度）。使用 Greengenes2 数据库（ http://greengenes.secondgeno me.com/）[27 ] 分析每个 ASV 代表序列的分类注释。

Statistical analysis  统计分析

The α α alpha\alpha-diversity indexes (Chao1, Observed ASVs, Shannon, and Simpson) and rarefaction curves were evaluated by QIIME 2. The relationship between bacterial community structures of different samples was visualized using a principal coordinate analysis ( PCoA ) and clustering analysis based on Bray-Curtis distances. The Venn-diagram analysis was performed to calculate the shared ASVs among the rhizosphere and root endosphere (http:// bioinformatics.psb.ugent.be/webtools/Venn/). The Sankey plots were performed in R version 3.6 .1 using the network D3 package. According to the described previously [28], the significant differential abundance of bacteria at phylum and genus levels were performed using the STAMP software by Welch’s test. Statistically significant difference differences in α α alpha\alpha-diversity indexes between rhizophere, endosphere and microhabitat were assessed using Kruskal-Wallis test. The Stats package (R version 3.6.1) was used to perform the Mann-Whitney-Wilcoxon test.
这 α α alpha\alpha -通过QIIME 2评估多样性指数（Chao1、Observed ASVs、Shannon和Simpson）和稀疏曲线。使用主坐标分析（PCoA）和基于Bray-Curtis的聚类分析可视化不同样本的细菌群落结构之间的关系距离。进行维恩图分析以计算根际和根内圈之间共享的 ASV ( http://bioinformatics.psb.ugent.be/webtools/Venn/ )。桑基图是使用网络 D3 包在 R 版本 3.6 .1 中执行的。根据前面的描述[28]，利用STAMP软件通过Welch检验对门和属水平上细菌丰度的显着差异进行了检验。统计上显着的差异 α α alpha\alpha -使用Kruskal-Wallis检验评估根际、内圈和微生境之间的多样性指数。 Stats 包（R 版本 3.6.1）用于执行 Mann-Whitney-Wilcoxon 测试。
To explore the interaction between the root-associated bacteria of grafted apple, the ASVs that had average relative abundance > 0.05 % > 0.05 % > 0.05%>0.05 \% and presented in 50 % 50 % 50%50 \% of the samples were selected for Co-occurrence network analyses. Based on Random Matrix Theory (RMT) approach, the Co-occurrence networks of the rhizosphere and root endosphere bacteria were constructed using the Molecular Ecological Network Analyses Pipeline (MENA) (http://ieg4.rccc.ou.edu/mena) at the ASV level [29]. We screened for significant congruent pairs of rhizosphere and root endosphere bacteria based on the statistical significance ( P < 0.05 ) ( P < 0.05 ) (P < 0.05)(P<0.05) and strength ( ρ > 0.9 ) ( ρ > 0.9 ) (rho > 0.9)(\rho>0.9) of the correlation. The visualization of the Co-occurrence networks was performed by Cytoscape version 3.7.2. According to the described previously [ 29 , 30 ] [ 29 , 30 ] [29,30][29,30], the topological roles of each node can be defined by its within-module connectivity ( Zi ) and among-module connectivity ( Pi ). The keystone nodes (species) contain three types: network hubs ( Zi > 2.5 Zi > 2.5 Zi > 2.5\mathrm{Zi}>2.5 and Pi > 0.62 Pi > 0.62 Pi > 0.62\mathrm{Pi}>0.62 ) and module hubs ( Zi > 2.5 ( Zi > 2.5 (Zi > 2.5(\mathrm{Zi}>2.5 and Pi ≤ 0.62 ≤ 0.62 <= 0.62\leq 0.62 ).
探讨嫁接苹果根部相关细菌与平均相对丰度 ASV 之间的相互作用 > 0.05 % > 0.05 % > 0.05%>0.05 \% 并提出于 50 % 50 % 50%50 \% 选择样本进行共现网络分析。基于随机矩阵理论（RMT）方法，使用分子生态网络分析管道（MENA）（ http://ieg4.rccc.ou.edu/mena ）构建了根际和根内圈细菌的共现网络。 ASV 水平[29]。我们根据统计显着性筛选了显着一致的根际和根内圈细菌对 ( P < 0.05 ) ( P < 0.05 ) (P < 0.05)(P<0.05) 和力量 ( ρ > 0.9 ) ( ρ > 0.9 ) (rho > 0.9)(\rho>0.9) 的相关性。共现网络的可视化由 Cytoscape 版本 3.7.2 执行。根据前面的描述 [ 29 , 30 ] [ 29 , 30 ] [29,30][29,30] ，每个节点的拓扑角色可以通过其模块内连接性（ Zi ）和模块间连接性（ Pi ）来定义。基石节点（种类）包含三种类型：网络集线器（ Zi > 2.5 Zi > 2.5 Zi > 2.5\mathrm{Zi}>2.5 和 Pi > 0.62 Pi > 0.62 Pi > 0.62\mathrm{Pi}>0.62 ) 和模块集线器 ( Zi > 2.5 ( Zi > 2.5 (Zi > 2.5(\mathrm{Zi}>2.5 和 Pi ≤ 0.62 ≤ 0.62 <= 0.62\leq 0.62 ）。
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