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Pathogenicity Assay of Penicillium expansum on Apple Fruits
扩展青霉对苹果果实的致病性测定

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Abstract

Penicillium expansum, a widespread filamentous fungus, is a major causative agent of fruit decay and leads to huge economic losses during postharvest storage and shipping. Furthermore, it produces mycotoxin on the infected fruits that may cause harmful effects to human health. This pathogenicity assay involves a stab inoculation procedure of P. expansum on apple fruit, an important experimental technique to study fungal pathogenesis. This assay can be applied to analyze the virulence of postharvest pathogen on other fruits such as orange, pear and kiwifruit.

Keywords: Penicillium expansum(扩展青霉), Apple fruit(苹果果实), Stab inoculation(穿刺接种), Pathogenicity assay(致病性测定)

Background

Penicillium expansum is a destructive postharvest pathogen that causes decay in many popular fruits, such as apple and pear, during postharvest handling and storage. It causes significant socioeconomic impacts and has implications for international trade. The pathogen can also lead to serious health problems in human since it produces toxic secondary metabolites, including patulin, citrinin, and chaetoglobosins (Andersen et al., 2004). Control of decay caused by P. expansum has become important for ensuring the quality and safety of various fruits.
   Conidia of P. expansum typically enter through wounds, which is necessary to provide sites for initiation of the pathogen development (Spotts et al., 1998). Pathogenicity of P. expansum on fruits is usually tested by needle-stab inoculation, which is also used for pathogenicity assays of Botrytis cinerea vs. tomato, Monilinia fructicola vs. peach, Colletotrichum gloeosporioides vs. mango, etc. (Liu et al., 2012; Shi et al., 2012; Zhang et al., 2014). Here, we described a protocol to assess pathogenicity of P. expansum on apple fruits based on stab inoculation method.

Materials and Reagents

  1. 90 x 15 mm Petri dish (any brand will suffice)
  2. Plastic film (polyurethane material, any department store)
  3. 1,000 µl pipette tips (Corning, Axygen®, catalog number: TF-1000-R-S )
  4. 200 µl pipette tips (Corning, Axygen®, catalog number: TF-200-R-S )
  5. 10 µl pipette tips (Corning, Axygen®, catalog number: TF-300-R-S )
  6. Polyester filter cloth cut into 8 x 8 cm squares (any fabric store)
  7. Penicillium expansum T01: was isolated by our laboratory and whole-genome sequenced (Li et al., 2015)
  8. Freshly harvested red Fuji apples
  9. Glycerol (AMRESCO, catalog number: M152 )
  10. Tween 20 (Sigma-Aldrich, catalog number: T2700 )
  11. Sodium hypochlorite (Sigma-Aldrich, catalog number: 239305 )
  12. Sterile distilled water
  13. Potato
  14. Dextrose (Macklin, catalog number: D823520 )
  15. Agar (HUAAOBIO, catalog number: HA0552 )
  16. 2% sodium hypochlorite solution (see Recipes)
  17. PDA medium (see Recipes)

Equipment

  1. Clean bench (Beijing Donglian Har Instrument Manufacture, model: SCB-1520 )
  2. Sterile nail (approximately 3 mm in diameter, manual polishing)
  3. Constant temperature incubator (TAICANG, model: THZ-C )
  4. Hemacytometer (QIUJING, catalog number: XB-K-25 )
  5. Vortexer (Select BioProducts, model: SBS100-2 )
  6. 100 µl-1,000 µl pipette (Eppendorf, catalog number: 3120000267 )
  7. 10 µl-1,00 µl pipette (Eppendorf, catalog number: 3120000240 )
  8. 0.5µl-1,0 µl pipette (Eppendorf, catalog number: 3120000224 )
  9. Optical microscope (CHONGQING OPTEC Instrument, model: B203LED )
  10. 40 x 30 x 10 cm plastic basket (any brand will suffice)
  11. Hand held sprayer (any brand will suffice)
  12. 40 x 40 x 30 cm containers (any brand will suffice)
  13. Hygrothermograph (Fisher Scientific, catalog number: 11-661-20 )

Software

  1. SPSS version 13.0

Procedure

  1. Fruit disinfection
    1. The seasonal apple fruits with uniform size and colour and without physical injuries are used as experimental materials.
    2. The fruits are surface disinfected for 2 min in a container with 2% sodium hypochlorite solution, rinsed three times with deionized water, and air dried in a clean bench.
    3. Two wounds (3 x 3 mm) are made face to face with a sterile nail on the equator of each apple prior to inoculation with pathogen (Figure 1A).


      Figure 1. Inoculation of apple fruits with P. expansum. A. Wounding of fruit with sterile nail; B. The storage condition of fruits after inoculation.

  2. Pathogen inoculum preparation
    1. Five-microliter spore suspension (5 x 106/ml in 16% glycerol, stored at -80 °C) of P. expansum is inoculated on PDA plate and cultured for 2 weeks at 25 °C in the dark.
    2. Conidia are harvested with 0.05% Tween 20 and filtered through four layers of sterile polyester filter cloth. Conidia are counted with a hemocytometer using an optical microscope and diluted to a concentration of 1 x 105 conidia/ml with 0.05% Tween 20.

  3. Inoculation
    1. Each wound site of the apple fruits is inoculated with 5 μl spore suspension (the suspension is mixed by vortexer before inoculation) using a pipette. Sterile distilled water with 0.05% Tween 20 is used as the control.
    2. Six inoculated fruits are put into plastic basket and sealed with plastic film, about 5 ml sterile water is sprayed on the inside of the plastic film with a hand held sprayer to maintain a high relative humidity (about 95%), and stored at 25 °C in the dark (Figure 1B).

  4. Disease scoring
    The lesion diameters are measured on a daily basis after three days post-inoculation. Two diameter values of each lesion in two mutually perpendicular directions are recorded. The average of the two values is defined as the diameter of the lesion (Figure 2).


    Figure 2. Pathogenicity analysis of infected apple fruits. A. Symptoms of infected apple fruits after inoculation for 4 days at 25 °C; B. Disease scoring of the decayed apple fruits.

Data analysis

Data from three independent experiments, each with 24 fruits (48 wounds), are then analyzed with a statistic software SPSS version 13.0. ANOVA test is performed using Duncan’s multiple range test; P < 0.05.

Representative data

Figure 3 shows representative data for pathogenicity assay of ∆GeneA and ∆GeneB, two gene knock-out mutants of P. expansum, on apple fruits. To compare differences in the virulence of the mutants and WT, the different strains were inoculated into wounds on apple fruits. No significant difference in lesion diameter between ∆GeneA and WT was detected at 4, 5, and 6 d after inoculation (Figures 3A and 3B). Deletion of GeneB significantly reduced the virulence of P. expansum on the fruits. Obvious lesions could be observed 5 days after inoculation, where the lesion diameter of the ∆GeneB was significantly smaller than the lesion size in the wild type (Figuers 3C and 3D). These results indicate that GeneB has a significant impact on virulence of P. expansum.


Figure 3. Pathogenicity assay of ∆GeneA and ∆GeneB of P. expansum on apple fruits. A and C. Symptoms of infected apple fruits at 5 d after inoculation; B and D. Statistical analysis of lesion diameters at 4, 5, and 6 d after inoculation at 25 °C.

Notes

  1. Use 1-2 week old culture to ensure full pathogenicity of P. expansum spores.
  2. The apple fruits must be fresh and without mechanical wounds.
  3. The cultivation temperature of infected apple fruits must be controlled at 25 °C or slightly under 25 °C.

Recipes

  1. 2% sodium hypochlorite solution (20 L)
    Add 400 ml of sodium hypochlorite into a 40 x 40 x 30 cm container and then bring volume up to 20 L with distilled water
  2. PDA medium (1 L)
    200 g potato
    20 g dextrose
    15 g agar
    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 at 121 °C for 20 min

Acknowledgments

This protocol was adapted from Li et al. (2015) and Zhang et al. (2014). The work was supported by Chinese Ministry of Science and Technology (grant number 2016YFD0400902).

References

  1. Andersen, B., Smedsgaard, J. and Frisvad, J. C. (2004). Penicillium expansum: consistent production of patulin, chaetoglobosins, and other secondary metabolites in culture and their natural occurrence in fruit products. J Agric Food Chem 52(8): 2421-2428.
  2. Li, B., Zong, Y., Du, Z., Chen, Y., Zhang, Z., Qin, G., Zhao, W. and Tian, S. (2015). Genomic characterization reveals insights into patulin biosynthesis and pathogenicity in Penicillium species. Mol Plant Microbe Interact 28(6): 635-647.
  3. Liu, J., Sui, Y., Wisniewski, M., Droby, S., Tian, S., Norelli, J. and Hershkovitz, V. (2012). Effect of heat treatment on inhibition of Monilinia fructicola and induction of disease resistance in peach fruit. Postharvest Biol Tec 65:61-68.
  4. Shi, X., Li, B., Qin, G. and Tian, S. (2012). Mechanism of antifungal action of borate against Colletotrichum gloeosporioides related to mitochondrial degradation in spores. Postharvest Biol Tec. 67:138-143.
  5. Spotts, R., Sanderson, P. G., Lennox, C. L., Sugar, D. and Cervantes, L. A. (1998). Wounding, wound healing and staining of mature pear fruit. Postharvest Biol Tec 13(1): 27-36.
  6. 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.

简介

广泛的青霉菌是广泛的丝状真菌,是果实腐烂的主要致病因素,并在收获后储存和运输中导致巨大的经济损失。此外,它对感染的水果产生霉菌毒素,可能对人体健康造成有害影响。这种致病性测定涉及P的刺刺接种程序。膨化在苹果果实上,是一种重要的实验技术,用于研究真菌发病机制。该测定可用于分析其他水果如橙子,梨和猕猴桃的收获后病原体的毒力。

背景 青霉菌是一种破坏性的采后病原体,在采后处理和储存过程中,许多流行的水果如苹果和梨都会腐烂。造成重大的社会经济影响,对国际贸易产生影响。病原体也可能导致严重的人体健康问题,因为它会产生有毒的次级代谢物,包括patulin,citrinin和球形青霉素(Andersen等,2004)。控制由P引起的腐蚀。膨胀对于确保各种水果的质量和安全性至关重要。
&NBSP; &nbspConidia的 P。膨胀通常通过创伤进入,这是为提供病原体开发起点所必需的(Spotts等人,1998)。 P的致病性通常通过针刺接种来测试果实上的膨胀,其也用于灰葡萄孢与番茄相比,桃花生的致病性测定, 与芒果,相关的小菜蛾(Colletotrichum gloeosporioides) (Liu等人,2012; Shi等人,2012; Zhang等人,2014)。在这里,我们描述了一种评估P的致病性的方案。基于刺刺接种方法的苹果果实膨胀。

关键字:扩展青霉, 苹果果实, 穿刺接种, 致病性测定

材料和试剂

  1. 90 x 15毫米培养皿(任何品牌都可以满足)
  2. 塑料薄膜(聚氨酯材料,任何百货公司)
  3. 1,000μl移液器吸头(Corning,Axygen ®,目录号:TF-1000-R-S)
  4. 200μl移液器吸头(Corning,Axygen ®,目录号:TF-200-R-S)
  5. 10μl移液器吸头(Corning,Axygen ®,目录号:TF-300-R-S)
  6. 聚酯滤布切成8×8厘米的方形(任何织物店)
  7. 通过我们的实验室和全基因组测序分离出青霉菌T01 :(Li等人,2015)
  8. 新鲜收获的红富士苹果
  9. 甘油(AMRESCO,目录号:M152)
  10. 吐温20(Sigma-Aldrich,目录号:T2700)
  11. 次氯酸钠(Sigma-Aldrich,目录号:239305)
  12. 无菌蒸馏水
  13. 土豆
  14. 葡萄糖(Macklin,目录号:D823520)
  15. 琼脂(HUAAOBIO,目录号:HA0552)
  16. 2%次氯酸钠溶液(见配方)
  17. PDA媒体(见食谱)

设备

  1. 洁净台(北京东联哈尔仪器制造,型号:SCB-1520)
  2. 无菌指甲(直径约3毫米,手动抛光)
  3. 恒温培养箱(TAICANG,型号:THZ-C)
  4. 血细胞计数器(QIUJING,目录号:XB-K-25)
  5. Vortexer(选择BioProducts,型号:SBS100-2)
  6. 100μl-1,000μl移液器(Eppendorf,目录号:3120000267)
  7. 10μl-1,100μl移液器(Eppendorf,目录号:3120000240)
  8. 0.5μl-1,0μl移液管(Eppendorf,目录号:3120000224)
  9. 光学显微镜(重庆OPTEC仪器,型号:B203LED)
  10. 40 x 30 x 10厘米塑料篮(任何品牌都可以满足)
  11. 手持喷雾器(任何品牌都可以满足)
  12. 40 x 40 x 30厘米集装箱(任何品牌都可以满足)
  13. Hygrothermograph(Fisher Scientific,目录号:11-661-20)

软件

  1. SPSS版本13.0

程序

  1. 水果消毒
    1. 作为实验材料,使用具有均匀尺寸和颜色,没有身体伤害的季节性苹果果实
    2. 将水果在2%次氯酸钠溶液的容器中进行表面消毒2分钟,用去离子水冲洗3次,并在干净的工作台中空气干燥。
    3. 在接种病原体之前,每个苹果的赤道上用无菌指甲面对面的两个伤口(3×3mm)(图1A)。


      图1.苹果果实的接种。膨胀。 A.无菌指甲水果的伤口B.接种后果实的贮藏条件
  2. 病原菌接种物准备
    1. 将5微升孢子悬浮液(16×甘油中的5×10 6/ml,储存于-80℃)。将膨胀接种在PDA板上,并在25℃下在黑暗中培养2周。
    2. 用0.05%吐温20收获分生孢子,并通过四层无菌聚酯滤布过滤。使用光学显微镜用血细胞计数器计数分生孢子,并用0.05%吐温20稀释至浓度为1×10 5 /分钟/分钟。
  3. 接种
    1. 苹果果实的每个伤口部位用5μl孢子悬浮液接种(悬浮液在接种之前通过涡旋混合)用移液管接种。用0.05%吐温20的无菌蒸馏水作为对照
    2. 将六个接种的水果放入塑料篮中并用塑料膜密封,用手持式喷雾器将约5ml无菌水喷在塑料膜的内部,以保持高的相对湿度(约95%),并储存在25° C在黑暗中(图1B)
  4. 疾病评分
    病变直径在接种3天后每天进行测量。记录两个相互垂直方向上的每个病变的两个直径值。两个值的平均值定义为病变的直径(图2)

    图2.感染的苹果果实的致病性分析。 A.感染的苹果果实在25℃下接种4天后的症状; B.腐烂苹果果实的疾病评分。

数据分析

然后用统计软件SPSS 13.0版分析来自三个独立实验的数据,每个实验共24个果实(48个伤口)。使用Duncan的多范围测试进行ANOVA测试; 0.05。

代表数据

图3显示了ΔGeneA和ΔGeneB两种基因敲除突变体的致病性测定的代表性数据。膨胀,在苹果果实上。为了比较突变体和WT的毒力差异,将不同的菌株接种到苹果果实的伤口中。在接种后4,5和6天检测到ΔGeneA和WT之间的病变直径没有显着差异(图3A和3B)。删除 GeneB 显着降低了P的毒力。水果的膨胀。在接种后5天可以观察到明显的损伤,其中ΔGeneB的损伤直径明显小于野生型的损伤尺寸(Figuers 3C和3D)。这些结果表明,GeneB 对P的毒力有显着的影响。 expansum 。


图3.PG的ΔGeneA和ΔGeneB的致病性测定。苹果果实上的膨胀 .A和C.感染苹果果实在接种5 d后的症状。 B和D.在25℃下接种后4,5和6天的病变直径的统计学分析。

笔记

  1. 使用1-2周龄的培养物确保P的完全致病性。膨胀孢子。
  2. 苹果水果必须是新鲜的,没有机械伤口。
  3. 感染苹果果实的培养温度必须控制在25°C或稍低于25°C。

食谱

  1. 2%次氯酸钠溶液(20升)
    将400毫升次氯酸钠加入到40 x 40 x 30厘米的容器中,然后用蒸馏水将体积提高到20升。
  2. PDA媒体(1升)
    200克土豆
    20克右旋糖
    15克琼脂
    将200克切片的土豆在1升蒸馏水中煮沸30分钟,然后通过干酪布滗析肉汤,并在肉汤中加入20克葡萄糖和15克琼脂粉末。加入蒸馏水组成1升,培养基通过121℃高压灭菌20分钟灭菌

致谢

该协议由Li等人改编。 (2015)和张等人。 (2014)。这项工作得到了中国科技部的支持(授权号码2016YFD0400902)。

参考

  1. Andersen,B.,Smedsgaard,J. and Frisvad,JC(2004)。  Penicillium expansum :培养中的patulin,chaetoglobosins和其他次生代谢产物的一致生产及其在水果产品中的天然存在。农业食品化学> 52(8):2421-2428。
  2. Li,B.,Zong,Y.,Du,Z.,Chen,Y.,Zhang,Z.,Qin,G.,Zhao,W。和Tian,S。(2015)。基因组表征揭示了青霉属物种中patulin生物合成和致病性的见解。Mol Plant Microbe Interact 28(6):635-647。
  3. Liu,J.,Sui,Y.,Wisniewski,M.,Droby,S.,Tian,S.,Norelli,J.and Hershkovitz,V。(2012)。< a class ="ke-insertfile"href ="http://www.sciencedirect.com/science/article/pii/S0925521411002535"target ="_ blank">热处理对桃果实抑制的影响和诱导桃果实的抗病性 Postharvest Biol Tec 65:61-68。
  4. Shi,X.,Li,B.,Qin,G.and Tian,S。(2012)。硼酸盐对孢子中线粒体降解有关的球菌抗真菌作用的机理。 Postharvest Biol Tec 。 67:138-143。
  5. Spotts,R.,Sanderson,PG,Lennox,CL,Sugar,D。和Cervantes,LA(1998)。成熟梨果的伤口,伤口愈合和染色。 Postharvest Biol Tec 13(1):27-36。 >
  6. Zhang,Z.,Qin,G.,Li,B.and Tian,S。(2014)。在Botrytis cinerea 中敲除 bcsas1 影响细胞外蛋白质的增长,发育和分泌,从而降低毒力。 Mol Plant Microbe Interact 27(6):590-600。
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引用:Chen, Y., Li, B., Zhang, Z. and Tian, S. (2017). Pathogenicity Assay of Penicillium expansum on Apple Fruits. Bio-protocol 7(9): e2264. DOI: 10.21769/BioProtoc.2264.
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