Summary

松树线虫 Bursaphelenchus xylophilus 感染松树体内和体外及分离诱导挥发物

Published: September 27, 2024
doi:

Summary

该方案描述了松树的 体内体外 松木线虫 感染及其通过 气相色谱 (GC) 和 GC 与质谱联用 (GC-MS) 进行的挥发性分析。

Abstract

松木线虫 (PWN) 是一种植物寄生虫,可在针叶树物种中引起松树枯萎病 (PWD)。这种植物寄生线虫严重导致了亚洲国家(例如日本、中国和韩国)的松树森林砍伐。在过去的二十年里,在欧洲,葡萄牙和西班牙受到了很大影响。对易感宿主物种 PWN 感染和/或 PWD 进展机制的研究依赖于温室条件下松树幼苗的受控感染。这种技术很费力,并且需要调动大量的经济和人力资源。此外,由于与某些松树物种相关的遗传多样性以及外部因素的干扰,它可能容易产生变异性。作为替代方案,松树与 PWN 的体外共培养为研究生化变化提供了更有利的系统,因为它们 a) 允许控制单一环境或营养变量,b) 占用更少的空间,c) 需要更少的时间来获得,以及 d) 不受污染或宿主遗传变异。以下方案详细介绍了海洋松树 Pinus pinaster 的标准体内 PWN 感染,以及使用 PWN 建立松芽的新型体外共培养,作为研究这种植物寄生虫对松树挥发物影响的改进方法。通过加氢蒸馏和蒸馏-萃取从体内体外感染的松树中提取 PWN 诱导的挥发物,并使用纤维或填充柱技术通过固相微萃取 (SPME) 捕获排放的挥发物。

Introduction

松木线虫(PWN), Bursaphelenchus xylophilus (Steiner & Bührer 1934) Nickle 1970,是一种主要寄生在 松树 物种上的植物寄生线虫。这种植物寄生虫在昆虫成熟取食期间由 Monochamus 属的昆虫传播到易感松树种的树木中。PWN 通过攻击树木的树脂管并减少树脂流动,并破坏其维管组织,导致水柱中断来杀死树木。树冠缺水会引起松树萎病 (PWD) 的第一个可见症状,即松针在光合作用停止后变得绿化,并因干燥而下垂。松树通常通过产生树脂和挥发性化合物来响应生物和非生物胁迫1。因此,了解松树防御的机制对于确定 PWN 攻击的具体效果和寻找害虫防治的替代方法非常重要2

目前,在田间条件下的实验取决于受感染松树的可用性、PWN 感染的确认和可变的环境条件。在温室条件下,这些参数可以更容易地控制;然而,宿主的遗传多样性成为变异性的重要来源3。例如,在一项关于 Pinus pinaster 抗性反应的研究中,萜烯柠檬烯和树脂酸的产生与 PWN 感染有关4。然而,由于样品数量少且自然条件多变,仅记录到一半样品的可检测变化。在另一项使用温室种植的松树幼苗的研究中,尽管环境条件更容易控制,但天然松树遗传多样性导致提取的挥发物具有很大的变异性5。由于疾病诱导的松树挥发物会受到环境和遗传变异的极大影响,因此求助于体外嫩芽培养是研究松树组织对 PWN 感染的化学和生化反应的良好替代方案 5,6。通过在体外繁殖植物基因型,其基因组成可以无限期地维持和克隆,从而在比体内条件下更小的空间和更短的时间内建立更多基因相同的个体。这些培养物在易于操纵的营养和环境条件下是一种简单的工作系统,因此在评估挥发物的产生和排放方面,它们比传统系统具有额外的优势 7,8。这些系统特别有利于木本物种的研究,因为大多数时候需要大量资源,即目标树木有时位于难以到达的地点,需要昂贵的设备、专门的劳动力和更长的分析时间8在体外,松树与 PWN 的共培养能够评估线虫和植物在不同阶段的代谢相互作用9。对于挥发物的分析,这一点非常重要,因为分析技术已经变得非常准确,采样的微小变化会导致挥发性分布的重大变化。气相色谱-质谱联用 (GC-MS) 是一种用于分析挥发物的强大技术,可快速、简化挥发物的分析10。这里介绍的方案描述了在温室条件下感染体内松树幼苗的技术,以及为诱导挥发物的提取和分析而优化的基因相同松树的体外芽培养物的技术。

Protocol

1. 体外生长 松 木线虫 注意:松木线虫是通过以灰葡萄孢 (de Bary) Whetzel11 的非孢子菌株的真菌菌丝体为食而生长的。 对于常规传代培养,将培养塞(直径 0.5 cm)从真菌菌落的最外缘转移到无菌马铃薯葡萄糖琼脂 (PDA) 板上,并在 25 ± 1°C 下保持 7 至 10 天,或直到真菌菌落到达板的边缘?…

Representative Results

PWN 在最佳条件下繁殖迅速,世代时间可低至 4 天,每只雌性一生中产卵约80 个 28 个。使用上述方法,可以根据真菌的生长获得大量的 PWN。在 8 天的生长期内,PWN 的种群数量可以增加 100 倍(图 1)。为了提高 PWN 数量的一致性,请使用灭菌的 PWN,因为未知细菌或真菌的污染会对 PWN 种群产生负面影响。 成功感…

Discussion

此处介绍的方案概述了一种增强的方法来分析受 PWN 感染的海松中的挥发性化合物,其中环境和遗传变异性减少,并且不会影响结果。使用 体外 海洋松树基因型的纯品系,提取和排放的挥发物可以作为宿主对松树林最具破坏性的生物威胁之一的反应进行分析。

通过在体外培养灰葡萄孢芽孢杆菌(非孢子菌株)并将其用作线虫的食…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项研究部分由欧盟通过 101060634 的赠款协议在 PurPest 项目下资助,并由 Fundação para a Ciência e a Tecnologia (FCT) 通过 NemACT 项目资助,DOI 10.54499/2022.00359.CEECIND/CP1737/CT0002;NemaWAARS,DOI:10.54499/PTDC/ASP-PLA/1108/2021;CESAM UIDP/50017/2020+UIDB/50017/2020+ LA/P/0094/2020;CE3C,DOI 10.54499/UIDB/00329/2020;绿色信息技术,DOI 10.54499/UIDB/04551/2020 和 10.54499/UIDP/04551/2020。

Materials

38 mesh test sieve Retsch 60.131.000038
6-Benzylaminopurine (6-BAP) Duchefa Biochemie B0904
Charcoal activated Duchefa Biochemie C1302
Clevenger apparatus WINZER Laborglastechnik 25-000-02
Hydrogen peroxide solution Sigma-Aldrich H1009-500ML
Indole-3-butyric acid (IBA) Duchefa Biochemie I0902
Likens-Nickerson apparatus VitriLab LDA. c/IN29/32
Microbox round containers Sac O2 O118/80+OD118
n-Pentane Sigma-Aldrich 1.00882
PARAFILM M sealing film BRAND HS234526B-1EA
Phyto agar Duchefa Biochemie P1003
Potato Dextrose Agar BD DIFCO 213400
Scalpel blade no. 24 Romed Holland BLADE24
Schenk & Hildebrandt Basal salt medium Duchefa Biochemie S0225
Schenk & Hildebrandt vitamin mixture Duchefa Biochemie S0411
SPME fiber assembly Polydimethylsiloxane (PDMS) Supelco 57300-U
SPME Fiber Holder Supelco 57330-U
Sucrose Duchefa Biochemie S0809
Tenax TA- stainless steel tubes- conditioned + capped Markes International C1-AAXX-5003

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Faria, J. M. S., Figueiredo, A. C., Teixeira, D. M., Inácio, M. L. Infection of In Vivo and In Vitro Pines with the Pinewood Nematode Bursaphelenchus xylophilus and Isolation of Induced Volatiles. J. Vis. Exp. (211), e67149, doi:10.3791/67149 (2024).

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