Development of a qPCR Strategy to Select Bean Genes Involved in Plant Defense Response and Regulated by the Trichoderma velutinum - Rhizoctonia solani Interaction

Abstract

Bean production is affected by a wide diversity of fungal pathogens, among them Rhizoctonia solani is one of the most important. A strategy to control bean infectious diseases, mainly those caused by fungi, is based on the use of biocontrol agents (BCAs) that can reduce the negative effects of plant pathogens and also can promote positive responses in the plant. Trichoderma is a fungal genus that is able to induce the expression of genes involved in plant defense response and also to promote plant growth, root development and nutrient uptake. In this article, a strategy that combines in silico analysis and real time PCR to detect additional bean defense-related genes, regulated by the presence of Trichoderma velutinum and/or R. solani has been applied. Based in this strategy, from the 48 bean genes initially analyzed, 14 were selected, and only WRKY33, CH5b and hGS showed an up-regulatory response in the presence of T. velutinum. The other genes were or not affected (OSM34) or down-regulated by the presence of this fungus. R. solani infection resulted in a down-regulation of most of the genes analyzed, except PR1, OSM34 and CNGC2 that were not affected, and the presence of both, T. velutinum and R. solani, up-regulates hGS and down-regulates all the other genes analyzed, except CH5b which was not significantly affected. As conclusion, the strategy described in the present work has been shown to be effective to detect genes involved in plant defense, which respond to the presence of a BCA or to a pathogen and also to the presence of both. The selected genes show significant homology with previously described plant defense genes and they are expressed in bean leaves of plants treated with T. velutinum and/or infected with R. solani.

Keywords: Phaseolus vulgaris; biocontrol agent; biotic stress; defense genes; hypersensitive response; induced systemic resistance; systemic acquired resistance.

FIGURE 1

Bean seeds of “Canela” variety of the Protected Geographical Indication “Alubia La Bañeza – León” (Spain).

FIGURE 2

Schematic representation of the work flow set up in the present work to select bean genes involved in plant defense.

FIGURE 3

Evaluation of the diameter of the hypocotyl (above left), length of root system (above right) dry weight of the aerial part (below left) and root system (below right) of bean plants grown during 45 days after sowing. [Trichoderma velutinum T028 without pathogen (CT028), T. velutinum T028 with Rhizoctonia solani (RT028), R. solani control (RC) and control without fungus (CC)]. Differences statistically significant respect to control plants (p < 0.05) are indicated with different letters.

FIGURE 4

Analysis of relative expression levels of the bean defense genes selected in the present work in bean plants infected with R. solani versus their levels of expression in control plants. The data were analyzed by the 2^-ΔΔCt method. The differences statistically significant respect to control plants (p < 0.05) are indicated with an asterisk.

FIGURE 5

Analysis of relative expression levels of the bean defense genes selected in the present work in bean plants treated with T. velutinum versus their levels of expression in control plants. The data were analyzed as indicated in the legend to the Figure 4.

FIGURE 6

Analysis of relative expression levels of the bean defense genes selected in the present work in bean plants infected with R. solani and treated with T. velutinum versus their levels of expression in control plants. The data were analyzed as indicated in the legend to the Figure 4.