用土壤微生物感染植物根系的接种策略
Summary
该协议详细介绍了用土壤微生物接种植物根部的策略。以真菌黄霉菌和大丽黄萎病为例，描述了三种不同的根部感染系统。重点介绍潜在应用和可能的下游分析，并讨论每个系统的优缺点。
Abstract
根际拥有高度复杂的微生物群落，其中植物根系不断受到挑战。根系与各种各样的微生物密切接触，但对土壤相互作用的研究仍然落后于对地上器官进行的研究。虽然文献中描述了一些用模型根病原体感染模型植物的接种策略，但仍然难以获得全面的方法学概述。为了解决这个问题，精确描述了三种不同的根接种系统，可以应用于深入了解根 – 微生物相互作用的生物学。为了说明， 黄萎病 菌种（即 长孢子虫 和 大丽弧菌）被用作根入侵模型病原体。然而，这些方法可以很容易地适应其他根定植微生物 – 包括致病性和有益性。通过在植物木质部定殖，维管束土生真菌（如 黄萎病 属）表现出独特的生活方式。根系侵袭后，它们 通过 木质部血管向肢扩散，到达芽部，并引起疾病症状。选择三种代表性植物物种作为模型宿主： 拟南芥，经济上重要的油菜（Brassica napus）和番茄（Solanum lycopersicum）。给出了分步实验方案。显示了致病性测定的代表性结果，标记基因的转录分析以及报告基因构建的独立确认。此外，还彻底讨论了每种接种系统的优缺点。这些经过验证的协议可以帮助为根 – 微生物相互作用的研究问题提供方法。了解植物如何应对土壤中的微生物对于制定改善农业的新策略至关重要。
Introduction
天然土壤中栖息着数量惊人的微生物，这些微生物可能是中性的，有害的或对植物有益的1。许多植物病原体是土壤传播的，围绕着根部，并攻击地下器官。这些微生物属于各种各样的分支：真菌，卵菌，细菌，线虫，昆虫和一些病毒1，2。一旦环境条件有利于感染，易感植物就会患病，作物产量就会下降。气候变化的影响，如全球变暖和极端天气，将增加土壤传播植物病原体的比例3.因此，研究这些破坏性微生物及其对粮食和饲料生产的影响，以及对自然生态系统的影响将变得越来越重要。此外，土壤中存在与根系紧密相互作用并促进植物生长，发育和免疫力的微生物互助剂。当面对病原体时，植物可以积极地在根际中招募特定的对手，这些对手可以通过抑制病原体来支持宿主的生存4，5，6，7。然而，参与有益根 – 微生物相互作用的机制细节和途径通常仍然是未知的6。
因此，必须扩大对根 – 微生物相互作用的一般理解。用土壤微生物接种根系的可靠方法对于进行模型研究并将研究结果转移到农业应用中是必要的。例如，研究了土壤中的有益相互作用，例如，与Serendipita indica（以前称为Piriformospora indica），固氮根茎属或菌根真菌，而已知的土生植物病原体包括Ralstonia solanacearum，Phytophthora spp.，Fusarium spp.和Verticillium spp.1。后两者是真菌属，分布全球，可引起血管疾病2。黄萎病属（子囊菌）可以感染数百种植物物种 – 主要是双子叶植物，包括草本一年生植物，木本多年生植物和许多作物植物2，8。黄萎病菌丝进入根部，并在细胞间和细胞内向中央圆柱体生长以定植木质部血管2，9。在这些容器中，真菌在其大部分生命周期中保持存在。由于木质部汁液营养贫乏，并且携带植物防御化合物，真菌必须适应这种独特的环境。这是通过分泌与定植相关的蛋白质来实现的，这些蛋白质使病原体能够在其宿主10，11中存活。到达根脉管系统后，真菌可以在木质部血管内向半向扩散到叶子，这导致宿主9，12的全身定植。在这一点上，植物在生长中受到负面影响9，10，13。例如，发育迟缓和黄叶以及过早衰老13，14，15，16。
该属的一个成员是Verticillium longisporum，它高度适应芸苔属宿主，例如农学上重要的油菜，花椰菜和模型植物拟南芥12。几项研究结合了V. longisporum和A. thaliana，以获得对土壤传播的血管疾病和由此产生的根系防御反应13，15，16，17的广泛见解。通过使用长孢弧菌/拟南芥杆菌模型系统可以实现直接的药敏性测试，并且两种生物体都可以获得完善的遗传资源。与长孢子虫密切相关的是病原体大丽黄萎病菌。虽然两种真菌在血管生活方式和侵袭过程中表现相似，但它们从根到叶的繁殖效率和拟南芥引发的疾病症状是不同的：虽然长孢子虫通常诱导早期衰老，但大丽弧菌感染导致枯萎18。最近，一个方法学摘要提出了用长孢子虫或大丽弧菌感染拟南芥的不同根接种策略，有助于规划实验设置19。在田间，长孢子虫偶尔会对油菜生产12造成重大损害，而大丽弧菌的宿主范围非常广泛，包括几种栽培物种，如葡萄藤、马铃薯和番茄8。这使得这两种病原体在经济上都很有趣。
因此，以下方案使用 长孢子虫 和 大丽弧菌 作为根部病原体的模型，以举例说明根部接种的可能方法。拟南芥（拟南芥），油菜（Brassica napus）和番茄（Solanum lycopersicum）被选为模型宿主。有关这些方法的详细说明，请参阅下面的文本和随附的视频。讨论了每种接种系统的优缺点。总而言之，该协议收集可以帮助确定根 – 微生物相互作用背景下特定研究问题的合适方法。
Protocol
Representative Results
Discussion
由于土壤传播的植物病原体1造成的巨大产量损失，需要改进耕作策略或作物品种。对土壤传播疾病的发病机制的有限了解阻碍了更具抗性的植物的发育。需要探索潜在的病理机制，为此需要一个强大的方法学平台。报道的接种程序已经表明，根 – 微生物相互作用中的多因素事件可以通过组合不同的系统很好地解剖19。上述协议旨在为该领域的专家和研究人员提?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
作者感谢Tim Iven和Jaqueline Komorek之前在这些方法上的工作，Wolfgang Dröge-Laser（德国维尔茨堡大学药物生物学系）为这项工作提供了所需的设备和资源，Wolfgang Dröge-Laser以及Philipp Kreisz（都是维尔茨堡大学）对手稿进行了批判性校对。这项研究得到了“Deutsche Forschungsgemeinschaft”（DFG，DR273/15-1，2）的支持。
Materials
| Agar (Gelrite) | Carl Roth | Nr. 0039 | all systems described require Gelrite |
| Arabidopsis thaliana wild-type | NASC stock | Col-0 (N1092) | |
| Autoclave | Systec | VE-100 | |
| BlattFlaeche | Datinf GmbH | BlattFlaeche | software to determine leaf areas |
| Brassica napus wild-type | see Floerl et al., 2008 | rapid-cycling rape | genome ACaacc |
| Cefotaxime sodium | Duchefa | C0111 | |
| Chicanery flask 500 mL | Duran Group / neoLab | E-1090 | Erlenmeyer flask with four baffles |
| Collection tubes 50 mL | Sarstedt | 62.547.254 | 114 x 28 mm |
| Czapek Dextrose Broth medium | Duchefa | C1714 | |
| Digital camera | Nikon | D3100 18-55 VR | |
| Exsiccator (Desiccator ) | Duran Group | 200 DN, 5.8 L | Seal with lid to hold chlorine gas |
| Fluorescence Microscope | Leica | Leica TCS SP5 II | |
| HCl | Carl Roth | P074.3 | |
| KNO3 | Carl Roth | P021.1 | ≥ 99 % |
| KOH | Carl Roth | 6751 | |
| Leukopor | BSN medical GmbH | 2454-00 AP | non-woven tape 2.5 cm x 9.2 m |
| MES (2-(N-morpholino)ethanesulfonic acid) | Carl Roth | 4256.2 | Pufferan ≥ 99 % |
| MgSO4 | Carl Roth | T888.1 | Magnesiumsulfate-Heptahydrate |
| Murashige & Skoog medium (MS) | Duchefa | M0222 | MS including vitamins |
| NaClO | Carl Roth | 9062.1 | |
| Percival growth chambers | CLF Plant Climatics GmbH | AR-66L2 | |
| Petri-dishes | Sarstedt | 82.1473.001 | size ØxH: 92 × 16 mm |
| Plastic cups (500 mL, transparent) | Pro-pac, salad boxx | 5070 | size: 108 × 81 × 102 mm |
| Pleated cellulose filter | Hartenstein | FF12 | particle retention level 8–12 μm |
| poly klima growth chamber | poly klima GmbH | PK 520 WLED | |
| Potato Dextrose Broth medium | SIGMA Aldrich | P6685 | for microbiology |
| Pots | Pöppelmann GmbH | TO 7 D or TO 9,5 D | Ø 7 cm resp. Ø 9.5 cm |
| PromMYB51::YFP | see Poncini et al., 2017 | MYB51 reporter line | YFP (i.e. 3xmVenus with NLS) |
| Reaction tubes 2 mL | Sarstedt | 72.695.400 | PCR Performance tested |
| Rotary (orbital) shaker | Edmund Bühler | SM 30 C control | |
| Sand (bird sand) | Pet Bistro, Müller Holding | 786157 | |
| Soil | Einheitserde spezial | SP Pikier (SP ED 63 P) | |
| Solanum lycopersicum wild-type | see Chavarro-Carrero et al., 2021 | Type: Moneymaker | |
| Thoma cell counting chamber | Marienfeld | 642710 | depth 0.020 mm; 0.0025 mm2 |
| Ultrapure water (Milli-Q purified water) | MERK | IQ 7003/7005 | water obtained after purification |
| Verticillium dahliae | see Reusche et al., 2014 | isolate JR2 | |
| Verticillium longisporum | Zeise and von Tiedemann, 2002 | strain Vl43 |
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