Abstract
Grapevine (Vitis vinifera L.) is susceptible to an array of diseases among them the grey mold caused by the necrotrophic fungus Botrytis cinerea that decreases grape productivity and quality. To ensure a satisfactory yield and harvest quality numerous chemical fungicides are required, but they have serious drawbacks. One alternative is the use of beneficial bacteria to improve plant health. Pseudomonas fluorescens has been shown to trigger a plant-mediated resistance response in aboveground plant tissues against fungal, oomycete, bacterial, and viral pathogens. Triggered plant resistance exploits mechanisms of the plant immune system through a priming state that provides plants with enhanced capacity for rapid and strong activation of defense responses after pathogen infection, resulting in a lower fitness-cost. The primed responses by beneficial bacteria include induced expression of defense-related genes, cell wall reinforcement, and the production of secondary metabolites after pathogen infection. In this protocol, we describe the experimental design to evaluate the priming state of grapevine plants by the beneficial bacterium Pseudomonas fluorescens PTA-CT2 and their resistance level to Botrytis cinerea according to Verhagen et al. (2011) and Gruau et al. (2015).
Keywords: Biocontrol, Pseudomonas, Grapevine, Priming, Botrytis
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Constitutive Actin gene of B. cinerea in leaves of intact plants was analyzed by quantitative real-time RT-PCR using specific primers (see Grau et al., 2015). The reference genes EF1α (BQ799343, GenBank) and 60RSP (XM_002270599, GenBank) were used as internal controls and leaves of infected plantlets at zero time correspond to the reference sample (1x expression level). The results presented are means from three independent experiments. Necrosis surface provoked by B. cinerea on detached leaves was acquired with the APS Assess 2.0 software. About 30 leaves were used for each condition in one experiment and the results are means of six independent experiments. ANOVA test was performed using Duncan’s multiple range test; P < 0.05.
Recipes
Acknowledgments
This work was partially supported by the Vineal 2 program, and grants to C. Gruau and B. Verhagen from the Champagne-Ardenne Region and the City of Reims. This protocol was adapted from Gruau et al. (2015) and Verhagen et al. (2011).
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.