Abstract
Botrytis cinerea (B. cinerea) attacks many crops of economic importance, represents one of the most extensively studied necrotrophic pathogens. Inoculation of B. cinerea and phenotypic analysis of plant resistance are key procedures to investigate the mechanism of plant immunity. Here we describe a protocol for B. cinerea inoculation on medium and planta based on our study using the tomato-B. cinerea system.
Keywords: Botrytis cinerea, Tomato, Inoculation
Background
B. cinerea causes serious loss in more than 200 crops worldwide, including many important vegetables and small fruit crops. The broad-spectrum pathogen can infect plant stem, leaf, flower and fruit to produce spores (Dean et al., 2012; van Kan et al., 2017), which prefer to occur under high humidity (Elad et al., 2007). The produced spores pose long lasting threat to diverse hosts (Elad et al., 2007). Based on its scientific and economic importance, B. cinerea was ranked as the second most important plant-pathogenic fungus (Dean et al., 2012). Among B. cinerea host plants, tomato (Solanum lycopersicum), an economically valuable species, also serves as a classic model to study plant immunity (Ryan, 2000; Sun et al., 2011; Rosli and Martin, 2015). To investigate the molecular basis of plant immunity to B. cinerea, we employ a routine procedure to produce B. cinerea spores on artificial media. In addition, we provide detailed methods to infect tomato plants or detached leaves with a controlled strength using the collected spores and quantify disease development. This protocol has been successfully used to reveal the transcriptional regulation of master regulator MYC2 in Jasmonate-mediated plant immunity (Du et al., 2017).
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Images of the diseased leaflets were imported to software ImageJ. “Polygon selections” was used to select diseased area on the image. Then chose “measure” function from “Analyze” menu in software imageJ to calculate the lesion area. Data analysis was conducted in software Microsoft Excel. Three independent experiments were conducted, each experiment included at least six replicates. All data from independent experiments were analyzed. The statistical test included average, STDEV and Student’s t-test calculation in excel. The t-test value of 0.05 is considered as significant difference and 0.01 for very significant difference. In the experiment, a droplet of spore suspension on leaf surface should develop to a diseased lesion with round shape. In some cases, the droplet spreads along with leaf vein and developed to an irregular shaped lesion, which should be avoided for analysis. Otherwise, all samples were collected for data analysis.
Notes
Recipes
Acknowledgments
This work was supported by the National Key Research and Development Program of China (2016YFD0100603-10), by Tai-Shan Scholar Program from the Shandong Province. This protocol was adapted appropriately from previous work (Du et al., 2017). The authors declare that they have no competing interests.
References
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