扩展青霉对苹果果实的致病性测定

Yong  Chen; Boqiang Li; Zhanquan Zhang; Shiping Tian

doi:10.21769/BioProtoc.2264

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Peer-reviewed

Pathogenicity Assay of Penicillium expansum on Apple Fruits

扩展青霉对苹果果实的致病性测定

YC Yong Chen

BL Boqiang Li

ZZ Zhanquan Zhang

Shiping Tian email

发布: 2017年05月05日第7卷第9期 DOI: 10.21769/BioProtoc.2264 浏览次数: 10137

评审: Zhaohui LiuTohir BozorovAnonymous reviewer(s)

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Cover of Molecular Plant Microbe Interactions, featuring study using the protocol.

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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

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

Equipment

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

Software

SPSS version 13.0

Procedure

English

中文翻译

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如何引用

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.