Plant Pathology Lab: Core Steps and Key Points | 植物病理学实验核心步骤与要点

📚 Plant Pathology Lab: Core Steps and Key Points | 植物病理学实验核心步骤与要点

Plant pathology experiments follow a standardised workflow that combines traditional microbiology, plant physiology and modern molecular biology. The core objective is to prove that a specific pathogen causes the observed disease, and to study the infection process under controlled conditions. These techniques are essential for understanding disease cycles, developing resistant plant varieties and evaluating fungicide efficacy.

植物病理学实验采用标准化流程，将传统微生物学、植物生理学与现代分子生物学相结合。核心目标是证明特定病原体导致了所观察到的病害，并在受控条件下研究侵染过程。这些技术对于理解病害循环、培育抗病品种以及评估杀菌剂药效都至关重要。

1. Aseptic Technique and Workspace Preparation | 无菌操作与工作区准备

All operations must be performed inside a laminar flow cabinet or a still-air box to prevent airborne contamination. Before starting, wipe the work surface with 70% ethanol and expose it to UV light for 15 minutes if available. Wear disposable gloves and a laboratory coat, and flame-sterilise inoculation loops, scalpels and forceps before and after each transfer.

所有操作必须在超净工作台或静止空气箱内进行，以防止空气污染。开始前，用 70% 乙醇擦拭工作台面；如有紫外灯，则开启照射 15 分钟。佩戴一次性手套和实验服，每次转移前后都要对接种环、解剖刀和镊子进行灼烧灭菌。

All culture media and glassware must be sterilised by autoclaving at 121°C for 15–20 minutes. Prepare fresh media such as potato dextrose agar (PDA) for fungi or King’s B medium for bacteria, and pour 20–25 mL per Petri dish. For selective isolation, add antibiotics such as penicillin or streptomycin after autoclaving when the medium has cooled to about 50°C.

所有培养基和玻璃器皿必须在 121°C 高压灭菌 15–20 分钟。配制新鲜培养基，例如真菌用马铃薯葡萄糖琼脂（PDA），细菌用 King’s B 培养基，每个培养皿浇注 20–25 mL。如需选择性分离，可在培养基冷却至约 50°C 时加入青霉素或链霉素等抗生素。

2. Pathogen Isolation from Infected Tissue | 感病组织中的病原体分离

Using a sterile scalpel, cut small pieces of tissue, about 3–5 mm², from the boundary between healthy and diseased tissue, known as the lesion margin. This region usually contains actively growing pathogen structures and is less contaminated by secondary organisms than the decayed centre.

用无菌解剖刀从健康和感病组织的交界处，即病斑边缘，切取约 3–5 mm² 的小块组织。该区域通常含有活跃生长的病原体结构，与腐烂中心相比，受次生杂菌污染较少。

Surface-sterilise the tissue pieces by immersing them in 70% ethanol for 30 seconds, then in 1% sodium hypochlorite solution for 1–2 minutes. Rinse three times in sterile distilled water and blot dry on sterile filter paper. Place the pieces onto agar plates and press gently into the medium surface.

将组织块放入 70% 乙醇中浸泡 30 秒，再放入 1% 次氯酸钠溶液中处理 1–2 分钟进行表面消毒。然后用无菌蒸馏水漂洗三次，在无菌滤纸上吸干水分，将组织块置于琼脂平板上并轻轻压入培养基表面。

Incubate the plates at the optimal temperature for the suspected pathogen, usually 20–25°C. Inspect plates daily for fungal mycelium or bacterial colonies growing from the tissue. Transfer emerging isolates to fresh plates before they contact neighbouring colonies.

将平板置于疑似病原体的最适温度下培养，通常为 20–25°C。每日检查平板，观察组织块周围是否长出真菌菌丝或细菌菌落。一旦出现新分离物，应在与其相邻菌落接触前转移到新鲜平板上。

3. Purification and Pure Culture Maintenance | 纯化与纯培养物保藏

Fungal isolates are purified using the hyphal-tip technique. Under a dissection microscope, excise a small plug containing the leading edge of the mycelium with a sterile needle and transfer it to a fresh PDA plate. Repeat this process two to three times until a uniform culture is obtained.

真菌分离物采用菌丝尖端法进行纯化。在体视显微镜下，用无菌针切取含有菌丝前缘的小块琼脂，将其转移到新鲜 PDA 平板上。重复该过程两到三次，直到获得均一的纯培养物。

Bacterial pathogens are purified by quadrant streaking. Use a sterile loop to spread a small amount of the sample across the first quadrant, then flame-and-strip to the second, third and fourth quadrants. After 24–48 hours, isolated single colonies appear in the later quadrants. Pick a well-isolated colony and re-streak onto a fresh plate.

细菌病原体采用四区划线法纯化。用无菌接种环取少量样品在第一区划线，然后灼烧接种环，依次拖线至第二、第三和第四区。24–48 小时后，后几个区域会出现单个独立菌落。挑取一个分离良好的菌落，再次划线到新鲜平板上。

For long-term storage, prepare glycerol stocks: suspend overnight cultures in a 15–20% glycerol solution and store at −80°C. Media slants are suitable for shorter-term storage at 4°C, but subculturing every few weeks is necessary to maintain viability.

长期保存时，可制备甘油菌种：将过夜培养物悬浮于 15–20% 甘油溶液中，在 −80°C 下保存。短期保存可使用 4°C 培养基斜面，但每数周需转接一次以维持活性。

4. Koch’s Postulates | 柯赫氏法则

Koch’s postulates provide the framework to prove that a microorganism causes a specific disease. The four steps are: (1) the pathogen must be present in all diseased plants, but absent from healthy ones; (2) the pathogen must be isolated and grown in pure culture; (3) when a healthy susceptible host is inoculated with the pure culture, the original symptoms must appear; and (4) the same pathogen must be re-isolated from the experimentally infected plant.

柯赫氏法则是证明某种微生物是否引起特定病害的准则。四步为：(1) 该病原体必须存在于所有感病植株中，而健康植株中没有；(2) 该病原体必须被分离并获得纯培养；(3) 用纯培养物接种健康易感寄主后，必须出现原有症状；(4) 必须从实验感染的植株上再次分离出相同的病原体。

Always include a negative control group in which healthy plants are mock-inoculated with sterile water or sterile agar. Koch’s postulates may fail if the pathogen is a strict biotroph that cannot be cultured, if the host is not genetically uniform, or if the pathogen requires an insect vector. In such cases, molecular methods are used as supportive evidence.

务必设置阴性对照组，即用无菌水或无菌琼脂对健康植株进行模拟接种。若病原体是难以培养的严格活体营养生物、寄主遗传背景不一致，或病原体依赖昆虫介体传播，柯赫氏法则可能无法完成。此时分子方法可用作辅助证据。

5. Pathogen Detection and Identification | 病原体检测与鉴定

Morphological identification begins with direct observation. For fungi, prepare a temporary slide by placing a small piece of mycelium or spores in a drop of lactophenol cotton blue, then observe under a compound microscope. Record the shape, size, colour and septation of conidia, sporangiophores and mycelia. For bacteria, perform Gram staining and observe cell morphology; also test for motility and biochemical reactions such as catalase and oxidase.

形态学鉴定首先从直接观察开始。对于真菌，可取少量菌丝或孢子置于一滴乳酸酚棉蓝染液中制作临时玻片，在复合光学显微镜下观察。记录分生孢子、孢囊梗和菌丝的形状、大小、颜色和分隔情况。对于细菌，进行革兰氏染色并观察细胞形态，同时进行运动性试验以及过氧化氢酶和氧化酶等生化反应测试。

When morphological features are ambiguous, amplify and sequence the internal transcribed spacer (ITS) region of ribosomal DNA for fungi, or the 16S rRNA gene for bacteria. Compare the sequences against databases such as GenBank using BLAST. A 99–100% similarity to a known reference strain supports species-level identification.

当形态特征不明确时，对真菌可扩增并测序核糖体 DNA 的内转录间隔区（ITS），对细菌可测序 16S rRNA 基因。使用 BLAST 将所得序列与 GenBank 等数据库比对。与已知参考

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