Artificial Inoculation of Epichloë festucae into Lolium perenne, and Visualisation of Endophytic and Epiphyllous Fungal Growth
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Author:
Date:
2018-09-05
[Abstract] Natural hosts for the fungal endophyte Epichloë festucae include Festuca rubra (fine fescue) and Festuca trachyphylla (hard fescue). Some strains also form stable associations with Lolium perenne (perennial ryegrass). L. perenne is a suitable host to study fungal endophyte–grass interactions, such as endophytic fungal growth within the plant and epiphyllous growth on the plant surface. Here we provide a detailed protocol based on work by, for artificial inoculation of E. festucae into L. perenne, and newly developed staining and visualization techniques for observing endophytic and epiphyllous hyphae and the expressorium, an appressorium-like structure used by the fungus to exit the plant. The staining method uses a combination of ...
[摘要] 真菌内生菌Epichloëfestucae的天然宿主包括 Festuca rubra (细羊茅)和 Festuca trachyphylla (硬羊茅)。一些菌株也与黑麦草(多年生黑麦草)形成稳定的关联。 升。 perenne 是研究真菌内生菌 - 草相互作用的合适宿主,例如植物内生真菌生长和植物表面的叶生生长。在这里,我们提供了一个基于工作的详细协议,用于 E的人工接种。 festucae 进入 L. perenne ,以及新开发的染色和可视化技术,用于观察内生和附生的菌丝和表达菌,这是一种类似真菌的结构,用于离开植物。染色方法使用葡聚糖结合苯胺蓝二铵盐(AB)和几丁质结合小麦胚凝集素缀合的Alexa Fluor ® 488-(WGA-AF488)的组合。该协议将是研究Epichloë -grass相互作用的有用工具,特别是不同Epichloë - 草关联,各种内生菌 - 宿主发育阶段的比较,以及分析突变Epichloë菌株。
【背景】Latch和Christensen(1985)开发了一种用Epichloë内生菌人工感染草的方案。这项工作一直是广泛的 E的基础。 festucae Fl1- L. perenne 共生研究(Scott et al。,2012年综述)。在我们最近的工作中,我们描述了一种新发现的真菌结构,即能够实现 E的表达式。 ...
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Visualization of Growth and Morphology of Fungal Hyphae in planta Using WGA-AF488 and Propidium Iodide Co-staining
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Author:
Date:
2018-07-20
[Abstract] Fungal pathogens colonizing plants show a varying degree of symptoms. Microscopy techniques have been used to study the infection and proliferation of fungal hyphae inside the host. One of the best optimized and commonly used method is the co-staining with Wheat Germ Agglutinin- Alexa Fluor 488 conjugate (WGA-AF488) and propidium iodide (PI), which stains fungal hyphae and the plant cell wall in contrasting shades. This technique is widely used to characterize the various behaviors of fungal hyphae, e.g., in fungal knockout mutants being attenuated during differential stages of host colonization. We describe the protocol for sample preparation of WGA-AF488– PI staining of infected plant tissue. Here, we have used an infected sample with the basidiomycetous smut fungus Ustilago ...
[摘要] 定植植物的真菌病原体表现出不同程度的症状。显微镜技术已被用于研究宿主内真菌菌丝的感染和增殖。最优化和常用的方法之一是与小麦胚芽凝集素-Alexa Fluor 488缀合物(WGA-AF488)和碘化丙啶(PI)共染色,其以对比色调染色真菌菌丝和植物细胞壁。该技术广泛用于表征在宿主定植的不同阶段减毒的真菌敲除突变体中真菌菌丝的各种行为,例如。。我们描述了感染植物组织的WGA-AF488-PI染色的样品制备方案。在这里,我们使用受感染的样本与担子菌黑色真菌 Ustilago maydis 感染其宿主植物玉米( Zea mays L.)和 Ustilago hordei 感染大麦( Hordeum vulgare L.)。该协议通过共聚焦激光扫描显微镜有助于了解植物中真菌的生长,生物量和形态(Doehlemann 等。,2011; Redkar 等。,2015)。
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