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, Apple fruit, Stab inoculation, Pathogenicity assay
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
Equipment
Software
Procedure
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
Recipes
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
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