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Infection Assays of Tomato and Apple Fruit by the Fungal Pathogen Botrytis cinerea
真菌病原体灰葡萄孢菌对番茄和苹果果实的感染分析   

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Abstract

Botrytis cinerea (B. cinerea) is an aggressive fungal pathogen that infects more than 200 plant species. Furthermore, the pathogen can attack fruits of some plants, such as tomato and apple. B. cinerea has become one of the model systems in molecular phytopathology because of its economic importance and sophisticated genetic operation methods. Virulence assays are very important in the study of fungal pathogenesis. This protocol details the artificial inoculation procedure of B. cinerea on tomato and apple fruits. It also can be used to analyse the virulence of postharvest fungal pathogens on other fruits, such as pear, peach, jujube and so on.

Materials and Reagents

  1. Freshly harvested tomatoes and apples (fully ripe)
  2. Botrytis cinerea (B05.10): Supplied by Prof. Paul Tudzynski Westfaelische Wilhelms-Universitaet Muenster, Germany
  3. KH2PO4 (Beijing Chemical Works, catalog number: HG/T 1274-1993 )
  4. Glucose (Beijing Chemical Works, catalog number: HG/T 3475-1999 )
  5. 2% sodium hypochlorite (Xilong Chemical, catalog number: 7681-52-9 )
  6. Potato
  7. Dextrose
  8. PDA medium (see Recipes)
  9. 2% sodium hypochlorite (see Recipes)
  10. KH2PO4-glucose solution (see Recipes)

Equipment

  1. Glass stick
  2. Hemacytometer (QIUJING, model: XB-K-25 )
  3. Cheesecloth
  4. Vortexer (Select BioProducts, model: SBS100-2 )
  5. Optical microscope (Chongqing Optec Instrument, model: B203LED )
  6. Clean bench (Donglian Electronic & Technology Development, model: SCB-1520 )
  7. Pipette
  8. Plastic basket
  9. Sterile nail (approximately 3 mm in diameter)

Procedure

  1. Fruit disinfection
    1. The fruits with uniformity and without physical injuries were used as experimental materials.
    2. These fruits were immersed in 2% sodium hypochlorite solution for 2 min, rinsed with sterile tap water, and air-dried in a clean bench (approximately 2 h).
    3. After being air dried, the fruits were wounded (approximately 4 mm in depth) at the equator with sterile nail (sterilized with alcohol lamp) prior to inoculation with pathogen (Figure 1).


      Figure 1. Wounding of apple fruit A and tomato fruit B with sterile nail

  2. Pathogen inoculum preparation
    1. Five microliter spore suspension (5 x 106/ml in 15% glycerol, stored at -80 °C) of B. cinerea was inoculated on PDA plates and cultured for 2 weeks at 22 °C.
    2. Fungal spores were harvested by flooding the surface of the culture with KH2PO4-glucose solution containing 0.05% (v/v) Tween-80, then filtered through four layers of sterile cheesecloth. The concentration of spore suspension was determined by observing under optical microscope using hemacytometer. Then, the concentration of spore suspension was adjusted to 5 x 104 per milliliter by KH2PO4-Glucose solution.

  3. Inoculation
    Tomato and apple fruits were inoculated with 10 μl spore suspension (the suspension was mixed by Vortexer before inoculation) in each wounded site using a pipette, respectively, KH2PO4-Glucose solution was used as control. Ten inoculated fruits were put into plastic basket (40 cm x 30 cm x 10 cm) and sealed with plastic film, about 5mL sterile water was sprayed into the plastic film to maintain a high relative humidity (about 95%), and stored at 25 °C.

  4. Disease scoring
    Two days after inoculation, the lesion diameters were measured daily. Two diameter values of each lesion in two mutually perpendicular directions were recorded. The average of the two values was defined as the diameter of the lesion (Figure 2).


    Figure 2. Lesions caused by B. cinerea and buffer. A. lesions on apple fruits (four days after inoculation). B. lesions on tomato fruits (3 days after inoculation). Buffer, KH2PO4-Glucose solution. (Source: Zhang et al., 2014)

Recipes

  1. PDA medium (1 L)
    Potato 200 g
    Dextrose 20 g
    Agar 15 g
    Boiling 200 g of sliced potatoes in 1 L distilled water for 30 min, then decanting the broth through cheesecloth and adding 20 g dextrose and 15 g agar powder in the broth. Add distilled water to make up 1 L, and the medium is sterilized by autoclaving for 20 min.
  2. 2% sodium hypochlorite (5 L)
    100 ml sodium hypochlorite
    4.9 L dH2O
  3. KH2PO4-glucose solution (100 ml)
    KH2PO4 0.136 g (10 mM)
    Glucose 0.198 g (10 mM)
    Tween-80 50 μl (0.05%, v/v)

Acknowledgments

This protocol was adapted from Zhu et al. (2010) and Chan et al. (2007). The work was supported by the 973 program (grant number 2013CB127103), by National Natural Science Foundation of China (grant number 31030051) and by the Chinese Academy of Sciences (grant number KSCX2-EW-G-6).

References

  1. Chan, Z., Qin, G., Xu, X., Li, B. and Tian, S. (2007). Proteome approach to characterize proteins induced by antagonist yeast and salicylic acid in peach fruit. J Proteome Res 6(5): 1677-1688.
  2. Zhang, Z., Qin, G., Li, B. and Tian, S. (2014). Knocking out Bcsas1 in Botrytis cinerea impacts growth, development, and secretion of extracellular proteins, which decreases virulence. Mol Plant Microbe Interact 27(6): 590-600.
  3. Zhu, Z., Zhang, Z., Qin, G. and Tian, S. (2010). Effects of brassinosteroids on postharvest disease and senescence of jujube fruit in storage. Postharvest Biol Technol 56(1): 50-55.

简介

(灰葡萄孢菌(Botrytis cinerea))是侵袭性的真菌病原体,其感染超过200种植物物种。 此外,病原体可攻击一些植物的果实,例如番茄和苹果。 B。 灰霉病已经成为分子植物病理学的模型系统之一,因为其经济重要性和复杂的遗传操作方法。 毒力测定在真菌发病机理的研究中非常重要。 该协议详述了B的人工接种程序。 西红柿和苹果果实上的灰霉菌。 它还可用于分析收获后真菌病原体对其他果实如梨,桃,枣等的毒力。

材料和试剂

  1. 新鲜收获的番茄和苹果(完全成熟)
  2. <灰色葡萄孢(Botrytis cinerea)(B05.10):由Paul Tudzynski Westfaelische Wilhelms-Universitaet Muenster教授提供,德国

  3. (北京化工公司,目录号:HG/T 1274-1993)</sub>
  4. 葡萄糖(北京化工厂,目录号:HG/T 3475-1999)
  5. 2%次氯酸钠(西隆化学,目录号:7681-52-9)
  6. 土豆
  7. 葡萄糖
  8. PDA介质(参见配方)
  9. 2%次氯酸钠(见配方)
  10. KH sub 2 PO 4葡萄糖溶液(参见配方)

设备

  1. 玻璃棒
  2. 血球计(QIUJING,型号:XB-K-25)
  3. 奶酪
  4. Vortexer(Select BioProducts,型号:SBS100-2)
  5. 光学显微镜(重庆Optec仪器,型号:B203LED)
  6. 洁净台(东联电子科技开发,型号:SCB-1520)
  7. 移液器
  8. 塑料篮
  9. 无菌指甲(直径约3 mm)

程序

  1. 水果消毒
    1. 将具有均匀性和无物理伤害的果实用作实验材料
    2. 将这些果实浸入2%次氯酸钠溶液中2 min,用无菌自来水冲洗,并在干净的工作台中风干 (约2小时)。
    3. 空气干燥后,果实 用无菌指甲在赤道处伤口(深度约4mm) (用酒精灯灭菌),然后接种病原体 (图1)。


      图1.苹果果实A和番茄果实B用无菌指甲伤口

  2. 病原体接种物准备
    1. 将5微升孢子悬浮液(5×10 6个/ml,在15%甘油中, -80℃)。 灰葡萄孢接种在PDA平板上并培养2天 周在22℃
    2. 通过淹没收获真菌孢子 表面的培养物与含有0.05%KH 2 PO 4 - 葡萄糖溶液的KH 2 PO 4 - (v/v)Tween-80,然后通过四层无菌过滤 干酪布。 通过测定孢子悬浮液的浓度 使用血球计在光学显微镜下观察。 然后, 将孢子悬浮液的浓度调节至5×10 4 /毫升   通过KH 2 2 PO 4 - 葡萄糖溶液。

  3. 联系
    使用移液管,在每个受伤部位分别用10μl孢子悬浮液(接种前通过Vortexer混合悬浮液)接种番茄和苹果果实,用KH sub 2 PO 4 - 葡萄糖溶液用作对照。将10个接种的果实放入塑料篮(40cm×30cm×10cm)中并用塑料膜密封,将约5mL无菌水喷射到塑料膜中以保持高的相对湿度(约95%),并在25℃ ℃。

  4. 疾病评分
    接种后两天,每天测量损伤直径。记录两个相互垂直方向上的每个损伤的两个直径值。两个值的平均值定义为损伤的直径(图2)。


    图2.由 B 和缓冲区造成的损伤。 A.苹果果实上的病变(接种后四天)。 B.番茄果实上的损伤(接种后3天)。缓冲液,KH 2 PO 4 - 葡萄糖溶液。 (资料来源:Zhang et al。,2014)

食谱

  1. PDA介质(1 L)
    土豆200克
    葡萄糖20克
    琼脂15克
    将200g切片马铃薯在1L蒸馏水中煮沸30分钟,然后通过干酪包布滗析肉汤,并在肉汤中加入20g葡萄糖和15g琼脂粉末。 加入蒸馏水补足1 L,培养基通过高压灭菌20分钟灭菌。
  2. 2%次氯酸钠(5L) 100 ml次氯酸钠
    4.9 L dH 2 O
  3. KH 2 PO 4 - 葡萄糖溶液(100ml)
    KH 2 PO 4 4 0.136g(10mM) 葡萄糖0.198g(10mM)
    Tween-8050μl(0.05%,v/v)

致谢

该协议改编自Zhu等人(2010)和Chan等人(2007)。 这项工作得到中国国家自然科学基金(拨款号31030051)和中国科学院(拨款号KSCX2-EW-G-6)的973项目(拨款号2013CB127103)的支持。

参考文献

  1. Chan,Z.,Qin,G.,Xu,X.,Li,B.and Tian,S。(2007)。 蛋白质组学方法,用于表征桃蚜中由拮抗剂酵母和水杨酸诱导的蛋白质。 em Proteins Res 6(5):1677-1688。
  2. Zhang,Z.,Qin,G.,Li,B.and Tian,S.(2014)。 敲除Botrytis cinerea中的Bcsas1会影响细胞外蛋白质的生长,发育和分泌,从而降低毒力。 Mol Plant Microbe Interact 27(6):590-600。
  3. Zhu,Z.,Zhang,Z.,Qin,G。和Tian,S。(2010)。 油菜素类固醇对收获后疾病和枣树果实的衰老的影响。 Postharvest Biol Technol 56(1):50-55
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zhang, Z., Qin, G., Li, B. and Tian, S. (2014). Infection Assays of Tomato and Apple Fruit by the Fungal Pathogen Botrytis cinerea. Bio-protocol 4(23): e1311. DOI: 10.21769/BioProtoc.1311.
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