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A Reliable Method for Phytophthora cajani Isolation, Sporangia, Zoospore Production and in Planta Infection of Pigeonpea
一种可靠的分离疫霉属水霉、产生孢子囊和游动孢子以及感染木豆植株的实验方法

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Abstract

Pigeonpea (Cajanus cajan L.) is an important legume crop of rainfed agriculture. High levels of protein in pigeonpea make it a valuable protein source for developing countries. Phytophthora blight caused by Phytophthora cajani (P. cajani) is a potential threat to pigeonpea (Cajanus cajan L.) production, affecting the crop irrespective of cropping system, cultivar grown and soil types (Pande et al., 2011; Sharma et al., 2006). The primary mode of infection of P. cajani is sporangium and zoospore. Therefore, sensitive and reliable methods for zoospore production and estimating infection severity are desirable in case of Phytophthora blight of pigeonpea (Sharma et al., 2015). Here we present a protocol for isolation of P. cajani from infected plants, sporangia and zoospore production and in planta infection technique of pigeonpea seedlings. These methods will be important tool to devise a platform for rapid and reliable screening against Phytophthora blight disease of pigeonpea as well as for host x pathogen x environment interaction studies.

Keywords: Phyophthora cajani(phyophthora cajani), Pigeonpea(木豆), Phytophthora stem blight(大豆茎枯病), Inoculation technique(接种技术)

Materials and Reagents

  1. Cotton (Jaycot Industries)
  2. Glass slide (75 x 25 x 1.35 mm) (Blue star)
  3. Parafilm (Sigma-Aldrich, catalog number: P7793 )
  4. Petridish (100 x 19.5 mm) (Borocil, catalog number: 5550300 )
  5. Phytophthora cajani isolate ICPC 1 (NCBI, GenBank Acc, catalog number: 10534 )
  6. Pigeonpea seedlings, cultivar ICP 7119
  7. Agar (HiMedia Laboratories, catalog number: RM201 )
  8. Ampicillin (Srlchem, catalog number: 61314 )
  9. Calcium carbonate (CaCO3) (HiMedia Laboratories, catalog number: GMR 397 )
  10. Dextrose (HiMedia Laboratories, catalog number: GRM077 )
  11. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: 472301 )
  12. Ethanol
  13. Pentachloronitrobenzene (PCNB) (Purity: ≥ 94%) (Sigma-Aldrich, catalog number: P2205 )
  14. Pimaricin (Sigma-Aldrich, catalog number: P9703 )
  15. Rifampicin (Sigma-Aldrich, catalog number: R7382 )
  16. Sodium hypochlorite (Scribd Inc., Qualigens, catalog number: 27905 )
  17. Sterilized pond water
  18. Sterilized water
  19. Sterilized river sand
  20. V8 Agar media (HiMedia Laboratories, catalog number: M638 )
  21. Vermiculite (locally available)
  22. Tomato juice
  23. Tomato juice agar (see Recipes)
  24. V8 juice agar (see Recipes)
  25. PARP solutions (see Recipes)
  26. Tomato broth (see Recipes)

Equipment

  1. Autoclave (Tomy Seiko)
  2. Beaker (100 and 400 ml) (Borocil, catalog number: 1000D16 and 1000D23 )
  3. Conical flask (50 ml) (Borocil, catalog number: 4060012 )
  4. Cork borer (approximately 5 mm diameter)
  5. Greenhouse (25-28 °C)
  6. Haemocytometer (1/400 mm2 and 1/10 mm deep) (Sigma-Aldrich)
  7. Hot air oven (Thermo Fisher Scientific)
  8. Incubator (Temperature range: 5-60 °C, humidity: 0-100%) (Tomy Percival)
  9. Inoculating needle
  10. Horizontal laminar flow clean benches (Esco Micro Pte, Labculture, model: LHC-4C )
  11. Light microscope (OLYMPUS)
  12. Measuring cylinder (100 ml) (Borocil, catalog number: 3024016 )
  13. Plastic tray (30 x 48 x 10 cm)
  14. Test tube (18 x 150 mm) (Borocil, catalog number: 9820U06 )

Procedure

  1. Pathogen isolation
    1. Collect pigeonpea plants exhibiting Phytophthora blight symptoms (brown to dark brown lesions distinctly different from healthy green portions on main stem, branches and petioles) from infected field (Figure 1).
    2. Place symptomatic plant materials in labelled plastic bags for transportation in cooler box (4 °C) and store in 4 °C until fungal culture were isolated and purified from the samples in the laboratory.
    3. Select stem tissues with typical Phytophthora blight lesions for isolation of the pathogen Phytophthora (Figure 1).


      Figure 1. Phytophthora blight lesions on pigeonpea stem

    4. Cut symptomatic tissues in small pieces and surface sterilise with 1% sodium hypochlorite for 1 min followed by 2-3 times washing in sterile distilled water.
    5. Place the surface cleaned tissues onto sterilized V8 juice agar media in a petri-plate (Figure 2) (V8 juice supplemented with L-Asparagine, CaCO3, glucose, yeast extract and agar) amended with PARP antibiotics (pimarcin 400 μl; ampicillin 250 mg; rifampicin 1,000 μl; and pentachloronitrobenzene 5 ml.L-1 media) (see Recipes for detail).


      Figure 2. P. cajani (indicated with red circle) colony isolated from infected stem tissue of pigeonpea on V8 juice agar media

    6. Incubate plates at 25 °C in the 12 h/12 h day-night photoperiod for 3-4 days.
    7. Transfer putative Phytophthora colonies (indicated with red circle in Figure 2) to 20% tomato extract agar (tomato extract 200 ml, CaCO3 2 g and agar 20 g.L-1 , see Recipes for detail) and again incubate at 25 °C in the 12 h/12 h day-night for 7 d (Figure 3).
    8. Further subculture is done after every 15-20 days.


      Figure 3. Purified P. cajani isolate on tomato extract agar media

    9. Identify the culture based on morphological characters like mycelial type and sporangia shape as well as molecular level using ITS region sequencing.
    10. Maintain cultures under in vitro at 18 ± 1 °C in darkness.
    11. To maintain the pathogen virulence, prepare new cultures every 2-3 months by passing the pathogen through the host and reisolating from infected plants.
      Note: All the steps have to be performed under aseptic conditions in horizontal laminar air flow system.

  2. Sporangia and zoospore production
    1. Choose virulent isolate of P. cajani for sporangia and zoospore production.
    2. Place one piece (5 mm) of mycelial bit from actively growing pure culture (5-7 day old culture) in 100 ml conical flask containing 25 ml of 20% tomato extract broth and incubate at 25 °C under dark condition for 72 h for mycelial growth (Figure 4A-B).
    3. After 72 h of incubation, decant tomato extract broth from flask and wash the mycelial mat with sterilized water and replace with 25 ml of sterilized pond water. Incubate again at 25 °C for 4 h in the darkness (Figure 4C).
    4. Decant the sterilized pond water and replace again with sterilized pond water and incubate for 3 h in the darkness.
    5. Repeat step 4 for one more time and incubate flask containing the mycelia again at 25 °C for approximately 20 h in the darkness (Figure 4D).


      Figure 4. P. cajani sporangia and zoospore production. A. Inoculated tomato extracts broth with P. cajani mycelial bit. B. Mycelial mat after 72 h post incubation. C. Replacement of tomato extracts broth with sterilized pond water. D. Mycelial mat in sterilized pond water.

    6. Observe sporangial initiation after 6 h during an incubation period and abundant sporangial production (mature sporangia) after 15-16 h under the light microscope (Figure 5A).
    7. Numerous swimming zoospores were noted within 16-20 h (Figure 5B-C). Zoospore concentration was determined using a haemocytometer and adjusted to desired concentration (1.5 x 105 ml-1) via dilution with sterilized deionized water and used for in planta infection.


      Figure 5. Sporangia and zoospore of P. cajani isolate ICPC 1. A. Mature sporangia after 15-16 h incubation. B. Zoospore release from mature sporangia. C. Swimming zoospores after 16-20 h incubation.

  3. In planta infection with zoospore
    1. Take apparently healthy seeds of any susceptible variety to P. cajani, in our case ICP 7119 (HY3C).
    2. Surface sterilize the seeds in sodium hypochlorite (1% v/v) for 2-3 min and then wash in sterile distilled water 2-3 times.
    3. Sow the sterilized seeds in plastic trays (35 x 25 x 8 cm) filled with a mixture of sterilised river sand and vermiculite (10:1 v/v) in a greenhouse maintained at 25-28 °C under natural light conditions for 7 day. Ten rows were sown in each tray and each row consisted of eight seeds (Figure 6A).
    4. Saturate the 7 day old seedlings grown in trays containing mixture of sand and vermiculite with sterilized water.
    5. Inoculate seedlings with diluted zoospore suspension usually containing 1.5 x 105 zoospores/ml (approximately 2 ml of zoospore suspension per plant) (Figure 6B).
    6. Trays with inoculated seedlings were kept in greenhouse at 28 ± 2 °C under ambient light conditions. Similar number of seedlings inoculated with only sterilized water served as un-inoculated control.
    7. After zoospore inoculation, flood the trays with sterilized water for 48 h and maintain saturation thereafter till completion of experiments (Figure 6C).
    8. After 5-7 day of infection, count the infected plants and calculate the disease incidence by estimating the percentage of plants infected with Phytophthora blight (Figure 6D).



      Figure 6. In planta infection of pigeonpea with P. cajani zoospore. A. Seven days old seedlings on sterilized sand and vermiculite mix. B. Inoculation of pigeonpea seedlings with P. cajani zoospore. C. Inoculated seedlings tray flooded with sterilized water. D. Phytophthora blight infected seedlings with lesions (indicated with circle).

Recipes

  1. Tomato juice agar (1 L)
    200 ml tomato juice
    2 g CaCO3
    20 g agar
    Combine tomato juice, CaCO3 and agar; bring to 1 L with water, mix on low heat
    Autoclave for 20 min at 121 °C temperature and 15 lbs pressure
  2. V8 juice agar (1 L)
    Suspend 44.3 g V8 juice agar media in 1 L distilled water on low heat
    Autoclave for 20 min at 121 °C temperature and 15 lbs pressure
  3. PARP solutions (1 L)
    0.4 ml pimaricin (prepare 2.5 % aqueous solution of pimaricin)
    0.25 g ampicillin
    0.01 g rifampicin [suspend 10 mg Rifampicin in 1 ml of dimethyl sulfoxide (DMSO)]
    5 ml pentachloronitrobenzene (PCNB) (dissolve 1 g PCNB to 200 ml ethanol at 70 °C in water bath)
    Preserve these solutions at 4 °C in refrigerator
    Add the required quantity of PARP in media before pouring in to the petri dish
  4. Tomato broth (1 L, 20%)
    200 ml tomato juice
    800 ml water
    50 ml of tomato juice and 0.5 g CaCO3 in 950 ml of distilled water
    Mix 200 ml tomato juice and 800 ml water
    Autoclave for 20 min at 121 °C temperature and 15 lbs pressure

Acknowledgements

The funding support from Department of Science and Technology-Climate Change Division and National Food Security Mission (NFSM), Department of Agriculture & Cooperation, Ministry of Agriculture, Govt. of India is gratefully acknowledged. The authors are thankful for technical assistance from Mr. Bal Krishna and K Ramulu from Legumes Pathology group.

References

  1. Pande, S., Sharma, M., Mangla, U. N., Ghosh, R. and Sundaresan, G. (2011). Phytophthora blight of Pigeonpea [Cajanus cajan (L.) Millsp.]: an updating review of biology, pathogenicity and disease management. Crop Prot 30: 951-7.
  2. Sharma, M., Ghosh, R., Tarafdar, A. and Telangre, R. (2015). An efficient method for zoospore production, infection and real-time quantification of Phytophthora cajani causing Phytophthora blight disease in pigeonpea under elevated atmospheric CO(2). BMC Plant Biol 15: 90.
  3. Sharma, M., Pande, S., Rao, J. N., Kumar, P. A., Reddy, D. M., Benagi, V. I., Mahalinga, D., Zhote, K. K., Karanjkar, P. N. and Eksinghe, B. S. (2006). Prevalence of Phytophthora blight of pigeonpea in the Deccan Plateau of India. Plant Pathol J 22: 309-13.

简介

番茄(Cajanus cajan)是雨养农业的重要豆科作物。高水平的蛋白质使其成为发展中国家有价值的蛋白质来源。由 Phytophthora cajani (p.cajani )引起的疫霉病是对木豆(Cahanus cajan 种植系统,栽培品种和土壤类型(Pande等人,2011; Sharma等人,2006)。感染的主要模式。 cajani 是孢子囊和游动孢子。因此,对于动物疫霉生产和估计感染严重性的灵敏和可靠的方法在鸽种疫霉病的情况下是期望的(Sharma等人,2015)。在这里,我们提出了隔离 P的协议。 cajani 从感染的植物,孢子囊和游动孢子的生产和在木豆幼苗的植物的感染技术。这些方法将是一个重要的工具,设计一个平台,以快速和可靠的筛选疫霉病疫霉病以及宿主x病原体x环境相互作用研究。

关键字:phyophthora cajani, 木豆, 大豆茎枯病, 接种技术

材料和试剂

  1. 棉花(Jaycot Industries)
  2. 玻璃载玻片(75 x 25 x 1.35 mm)(蓝色星)
  3. 石蜡膜(Sigma-Aldrich,目录号:P7793)
  4. 培养皿(100×19.5mm)(Borocil,目录号:5550300)
  5. Phytophthora cajani 分离ICPC 1(NCBI,GenBank Acc,目录号:10534)
  6. 鸽子幼苗,品种ICP 7119
  7. 琼脂(HiMedia Laboratories,目录号:RM201)
  8. 氨苄青霉素(Srlchem,目录号:61314)
  9. 碳酸钙(CaCO 3)(HiMedia Laboratories,目录号:GMR 397)
  10. 葡萄糖(HiMedia Laboratories,目录号:GRM077)
  11. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:472301)
  12. 乙醇
  13. 五氯硝基苯(PCNB)(纯度:≥94%)(Sigma-Aldrich,目录号:P2205)
  14. 匹马霉素(Sigma-Aldrich,目录号:P9703)
  15. 利福平(Sigma-Aldrich,目录号:R7382)
  16. 次氯酸钠(Scribd Inc.,Qualigens,目录号:27905)
  17. 灭菌池水
  18. 灭菌水
  19. 灭菌河砂
  20. V8琼脂培养基(HiMedia Laboratories,目录号:M638)
  21. 蛭石(本地可用)
  22. 番茄汁
  23. 番茄汁琼脂(见配方)
  24. V8果汁琼脂(见配方)
  25. PARP解决方案(参见配方)
  26. 番茄汤(见食谱)

设备

  1. 高压釜(Tomy Seiko)
  2. 烧杯(100和400ml)(Borocil,目录号:1000D16和1000D23)
  3. 锥形烧瓶(50ml)(Borocil,目录号:4060012)
  4. 软木钻孔器(直径约5mm)
  5. 温室(25-28°C)
  6. 血细胞计数器(1/400mm 2和1/10mm深)(Sigma-Aldrich)
  7. 热风炉(Thermo Fisher Scientific)
  8. 孵育器(温度范围:5-60℃,湿度:0-100%)(Tomy Percival)
  9. 接种针
  10. 水平层流清洁台(Esco Micro Pte,Labculture,型号:LHC-4C)
  11. 光学显微镜(OLYMPUS)
  12. 量筒(100ml)(Borocil,目录号:3024016)
  13. 塑料托盘(30 x 48 x 10厘米)
  14. 试管(18×150mm)(Borocil,目录号:9820U06)

程序

  1. 病原体隔离
    1. 收集表现出疫霉病病斑的木本植物(棕色 到明显不同于健康绿色部分的深棕色病变 在主干,分枝和叶柄上)(图1)。
    2. 将有症状的植物材料放在贴有标签的塑料袋中 运输在冷藏箱(4°C),并存储在4°C直到真菌 培养物从实验室的样品中分离和纯化。
    3. 选择具有典型疫霉病病原体的茎组织以分离病原体疫霉(图1)。


      图1.木豆茎上的疫霉病病毒

    4. 切断小块的症状组织和表面消毒 1%次氯酸钠洗涤1分钟,然后洗涤2-3次 无菌蒸馏水。
    5. 将表面清洁的组织放在上面 灭菌的V8汁琼脂培养基(图2)(V8汁 补充有L-天冬酰胺,CaCO 3,葡萄糖,酵母提取物和琼脂) 用PARP抗生素(pimarcin400μl;氨苄青霉素250mg; 利福平1,000μl;和五氯硝基苯(5mL)。 详细信息)。


      图2. P。 cajani (用红色圆圈表示)菌落从V8汁琼脂培养基上的鸽子感染的茎组织分离

    6. 孵育平板在25°C在12 h/12 h昼夜光周期3-4天。
    7. 转移推定的疫霉菌落(用红色圆圈表示 在图2中)至20%番茄提取物琼脂(番茄提取物200ml,CaCO 3 3g) ?和琼脂20g L <-1-1 ,详见配方),并再次在25℃孵育 在12小时/12小时昼夜7天(图3)
    8. 每15-20天进行进一步传代培养。


      图3.已净化 P。 cajani 隔离番茄提取物琼脂培养基

    9. 基于形态特征如菌丝体鉴定培养物 ?类型和孢子囊形状以及使用ITS区的分子水平 测序。
    10. 在黑暗中在18±1℃下在体外维持培养物。
    11. 为了保持病原体的毒力,准备新的文化每2-3 通过使病原体通过宿主并重新分离 感染植物 注意:所有步骤必须在无菌条件下在水平层流气流系统中进行。

  2. 孢子囊和游动孢子生产
    1. 选择 P的有害分离物。 cajani 用于孢子囊和游动孢子生产。
    2. 放置一片(5毫米)的菌丝体从积极生长纯 培养物(5-7天龄培养物)的100ml锥形瓶中 的20%番茄提取物肉汤,并在25℃下在黑暗条件下孵育 72小时用于菌丝体生长(图4A-B)。
    3. 72小时后 孵育,倾析从烧瓶的番茄提取物肉汤并洗涤菌丝体 ?垫用无菌水,并更换为25毫升灭菌池 水。在黑暗中在25℃再次孵育4小时(图4C)。
    4. 倾倒灭菌的池塘水,并再次更换无菌池塘水,并在黑暗中孵育3小时
    5. 重复步骤4再一次,孵育含有的烧瓶 菌丝体在25℃下在黑暗中再次培养约20小时 4D)。


      图4. P。 cajani 孢子囊和游动孢子生产。 用番茄接种番茄提取物肉汤。 cajani 菌丝体。乙。 菌丝垫72小时后孵育。 C.替代番茄 用无菌池塘水提取肉汤。灭菌的菌丝垫 ?池塘水
    6. 观察6小时后的孢子引发 孵化期和丰富的孢子生成(成熟 孢子囊)在光学显微镜下15-16小时后(图5A)。
    7. 在16-20小时内观察到许多游泳游动孢子(图5B-C)。 使用血细胞计数器测定游动孢子浓度 通过稀释调节至所需浓度(1.5×10 5个/mL -1 ) 灭菌的去离子水,用于在植物中感染。

      图 5.P的孢子囊和游动孢子。 cajani 隔离ICPC 1. A.成人 孢子囊。 B.游动孢子从成熟释放 孢子囊。 C.孵育16-20小时后游泳游动孢子。

  3. 在动植物感染的 感染
    1. 将任何易感品种的明显健康的种子用于P. cajani ,在我们的情况下为ICP 7119(HY3C)。
    2. 在次氯酸钠(1%v/v)中对种子进行表面消毒2-3分钟,然后用无菌蒸馏水洗2-3次。
    3. 播种灭菌的种子在塑料托盘(35×25×8厘米)填充 与无菌河砂和蛭石(10:1v/v)的混合物 温室在自然光照条件下保持在25-28℃7 天。在每个托盘中播种十排,每排由八个 种子(图6A)
    4. 饱和生长在含有沙子和蛭石的混合物的盘中的7天大的幼苗与无菌水。
    5. 通常用稀释的游动孢子悬浮液接种幼苗 含有1.5×10 5个游动孢子/ml(约2ml的游动孢子 悬浮液/植物)(图6B)
    6. 托盘与接种 将幼苗在环境光下在28±2℃的温室中保持 条件。相似数目的幼苗仅接种灭菌 水作为未接种的对照
    7. 游动孢子后 接种,用灭菌水冲洗托盘48小时并保持 ?饱和,直到实验完成(图6C)。
    8. 在感染5-7天后,计数感染的植物并计算 通过估计感染的植物的百分比来确定疾病发病率 (Phytophthora blight)(图6D)。



      图6. 在植物中感染 ?的鸽子。 cajani zoospore。 A。七天龄的幼苗 无菌砂和蛭石混合物。 B.鸽种的接种 幼苗与p。 cajani zoospore。 C.接种的秧苗盘淹没 用无菌水。 D.疫霉感染幼苗 病变(用圆圈表示)。

食谱

  1. 番茄汁琼脂(1L)
    200毫升番茄汁
    2g CaCO 3
    20克琼脂
    组合番茄汁,CaCO 3和琼脂;用水将1升,低热量混合
    在121°C温度和15磅压力下高压灭菌20分钟
  2. V8果汁琼脂(1L)
    在低热量下将44.3g V8汁琼脂培养基悬浮在1升蒸馏水中
    在121°C温度和15磅压力下高压灭菌20分钟
  3. PARP溶液(1L)
    0.4毫升匹马菌素(制备2.5%的匹马霉素水溶液) 0.25克氨苄青霉素 0.01 g利福平[悬浮10mg利福平在1 ml二甲亚砜(DMSO)中]
    5ml五氯硝基苯(PCNB)(在70℃下在水浴中将1g PCNB溶解于200ml乙醇中)
    在4℃下,在冰箱中保存这些溶液
    在加入培养皿
    之前,在培养基中加入所需数量的PARP
  4. 番茄肉汤(1L,20%)
    200毫升番茄汁
    800毫升水
    50ml番茄汁和0.5g CaCO 3在950ml蒸馏水中的溶液 混合200毫升番茄汁和800毫升水
    在121°C温度和15磅压力下高压灭菌20分钟

致谢

科学技术部气候变化司和国家粮食安全特派团(NFSM),农业和农业部,合作,农业部,政府。的印度感谢。作者感谢来自豆科植物病理学小组的Bal Krishna和K Ramulu先生的技术援助。

参考文献

  1. Pande,S.,Sharma,M.,Mangla,U.N.,Ghosh,R。和Sundaresan,G。(2011)。 鸽子的疫霉病[ Cajanus cajan (L.)Millsp。] :a biological review of biology,pathogenicity and disease management。 Crop Prot 30:951-7。
  2. Sharma,M.,Ghosh,R.,Tarafdar,A.和Telangre,R。(2015)。 一种用于动物疫霉生产,感染和实时定量测定 Phytophthora cajani的有效方法在高架大气CO(2)下在鸽种中引起疫霉病。 BMC Plant Biol 15:90。
  3. Sharma,M.,Pande,S.,Rao,J.N.,Kumar,P.A.,Reddy,D.M.,Benagi,V.I.,Mahalinga,D.,Zhote,K.K.,Karanjkar,P.N.and Eksinghe, 印度Deccan高原的木豆疫病流行率 植物病理学> 22:309-13。
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引用:Sharma, M. and Ghosh, R. (2016). A Reliable Method for Phytophthora cajani Isolation, Sporangia, Zoospore Production and in Planta Infection of Pigeonpea. Bio-protocol 6(2): e1706. DOI: 10.21769/BioProtoc.1706.
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