Use of confocal laser scanning microscopy to locate Stenocarpella maydis in corn stalk (Zea mays)

Abstract

Abstract Stenocarpella maydis causes high production losses in almost all countries where corn ( Zea mays ) is cultivated. The rot caused by S. maydis may occur on the stalks and ears of corn plants. S. maydis in corn poses a significant threat to human and animal nutrition, mainly due to mycotoxins such as diplodiatoxin. This study aimed to validate an efficient methodology for visualizing S. maydis colonization in corn using clarification protocol, fluorochromes, and Confocal Laser Scanning Microscopy (CLSM). Conidial suspensions were inoculated into the corn stalk at the V6 stage using a syringe. Corn stalk fragments of 1 cm 2 were collected 21 days after inoculation (dai) for CLSM analysis. The samples were fixed in Karnovsky’s solution and clarified in KOH and chloral hydrate. The fungal structures were labeled with Alexa488-WGA at 1.0 mg mL − 1 for 30 min under vacuum (excitation at 488 and emission at 510–540 ƞm). Thereafter, the corn plant tissues were labeled with Calcofluor White at 0.1 mg mL − 1 (excitation at 405 and emission at 440–490 ƞm) for 30 min. The Laser Confocal LSM780 Zeiss Observer Z.1 microscope, LCI Plan-Neofluar 25×/0.8, and C-Apochromat 63×/1.20 objectives were used to acquire fluorescent images. At 21 dai, it was possible to observe the colonization and formation of pycnidia with bicellular conidia of the fungus S. maydis in corn stalk fragments. The fungus colonized parenchymatic tissues and vascular bundles of the corn stalk. In contrast, at 21 dai, colonization of the fungus S. maydis was not observed in the parenchymatic tissues and vascular bundles of the corn stalk from uninoculated control plants. Our study made it possible to validate a new methodology for studying the infectious process of S. maydis in corn stalk using clarification protocols, fluorochromes, and CLSM.

Plant phenotyping relevance

トウモロコシ茎内の病原菌定着という植物病態を可視化するため、組織透明化・蛍光染色・共焦点顕微鏡法を開発・検証しており、植物表現型取得が中心である。

Code and data availability

This is a short microscopy methods paper (CLSM imaging of Stenocarpella maydis in corn stalks). No public dataset, image repository, or analysis code is deposited. The only paper-specific assets (CLSM fluorescence images and microscopy acquisition data) are stated to be available only upon request from the authors.

No evidence-backed public reproduction asset is currently recorded.