The common bean (Phaseolus vulgaris L.) is the most important food legume crop worldwide. Bean production is often affected by biotic and abiotic factors (Guerrero-González et al., 2011) by microorganisms, humidity, temperature… that are detected as signals for the activation of plant response mechanisms. This crop is affected by a wide diversity of fungal pathogens (Sclerotinia spp., Fusarium spp., Phytium spp., Botrytis spp.,...) among them Rhizoctonia solani JG Kühn [Teleomorph: Thanatephorus cucumeris (AB Frank) Donk] has a remarkable importance as responsible of important economic losses in this crop (Valenciano et al., 2006). R. solani is a necrotrophic pathogen responsible for the root and hypocotyl diseases. Plant infection occurs through wounds or by the direct action of the fungal mycelium, which tears the cuticle and penetrates the epidermis (Guerrero-González et al., 2011).

As a strategy to control bean infectious diseases, mainly those caused by fungi, the use of biocontrol agents (BCA) can reduce the negative effects of plant pathogens and they also can promote positive responses in the plant (Shoresh et al., 2010). The genera Trichoderma, Gliocladium, Rhizobium, Pseudomonas, are beneficial organisms that have shown good efficiency as BCAs against pathogenic microorganisms. Trichoderma (Teleomorph: Hypocrea) is a fungal genus that is found in the soil, and it is a secondary fast growing opportunistic invasive. In addition, Trichoderma biocontrol strains are able to induce the expression of genes involved in defense response and also to promote plant growth, root development, and nutrient uptake (Hermosa et al., 2012).

The relationships established between plant and micro-organisms are very diverse. When a plant is exposed to a pathogenic microorganism, the production of molecules associated to salicylic acid is increased, being this a systemic acquired resistance (SAR) response. The response of plants against non-pathogenic microorganisms is different, resulting in activation of signaling cascades that are dependent on jasmonic acid and ethylene, such as hydroperoxide lyase, peroxidase, and phenylalanine ammonia lyase, all of which belong to an induced systemic resistance (ISR) response (Druzhinina et al., 2011). Other responses result in a rapid cell death in infected tissues, then plants activate the hypersensitive response that involves the accumulation of salicylic acid, reactive oxygen species and an increased the influx of Ca²⁺ (Guerrero-González et al., 2011).

In the tripartite interaction of bean plants with the pathogen R. solani and a biocontrol Trichoderma species, several changes are produced in the plant, such as the increase in phenolic acid and lignin, accumulation of phytoalexins (Guerrero-González et al., 2011), and down- or up-regulation of defense-related genes expression (Mayo et al., 2015). Different categories of defense-related genes whose expression is modulated by biotic stresses have been described in bean plant interacting with pathogen and non-pathogenic microorganisms (Mayo et al., 2015).

Our hypothesis is that the combination of real time PCR with “in silico” analysis is a valid strategy to identify bean defense-related genes regulated by BCAs and/or plant pathogens. The aim is develop a systematic strategy to detect bean defense-related genes regulated by the presence of Trichoderma velutinum and/or R. solani. Finally, the procedure has been validated by the analysis of expression of the selected genes in the presence or absence of these two fungi.

Plants have developed some defensive strategies to perceive pathogen attack and to translate this perception into an appropriate adaptive response. During attack, plants are able to enhance their resistance (induced, acquired, hypersensitive) (Lodha and Basak, 2012). Contact with pathogenic and non-pathogenic microorganisms triggers two mechanisms: (i) SAR that is usually triggered by local infections, it provides long-term systemic resistance to pathogen attack and requires the involvement of the signal molecule salicylic acid (Durrant and Dong, 2004), and (ii) ISR that is known to result from colonization of roots by certain non-pathogenic microorganisms and is dependent on components of the jasmonic acid and ethylene signaling pathways (Shoresh et al., 2010). Then, the combination of both types of induced resistance response can protect the plant against pathogens and can even result in additive level of induced protection against pathogens through both the jasmonic acid/ethylene and salicylic acid pathways (Verhagen et al., 2006).

In the present work we developed a strategy to select genes involved in bean defense response, which would belong to those pathways, but also genes that can contribute to plant defense by other mechanisms. In this sense several previous works have described genes involved in bean defense response (Guerrero-González et al., 2011; Mayo et al., 2015). However, in the present work, by a systematic approach, 48 genes were initially considered, and 14 finally selected, which match with the criteria set up in this work: (i) they showed significant homology with previously described plant defense genes, and (ii) were expressed in bean leaves of plants treated with Trichoderma and/or infected with R. solani.

The expression of P. vulgaris defense-related genes was analyzed in leaves, although the interaction with Trichoderma and/or R. solani is initially produced at the root level, to determine if the signals generated in roots as result of this interaction are able to systematically stimulate the bean defense along long distance from roots to the leaves. The isolate T. velutinum T028 was selected following a similar strategy to that previously described (Mayo et al., 2015), and based on its positive effect on bean growth. In this work, to select a Trichoderma isolate, the results of the in vitro membrane assays and direct confrontation assays against R. solani were analyzed. Isolate Trichoderma T019 was then selected, showing a percentage of inhibition higher than 40% in the membrane assays, and/or 20% in the direct confrontation assays. This isolate also showed the best positive effects on plant phenotype among all the analyzed isolates.

WRKY transcription factors have been involved in the regulation of plant defense gene expression (Rushton and Somssich, 1998; Singh et al., 2002). Thus, WRKY33 has a role in biotic stress defense, where it regulates the balance between necrotrophic and biotrophic pathogen responses (Lippok et al., 2007; Pandey and Somssich, 2009; Birkenbihl et al., 2012). Previous studies have pointed out the involvement of Arabidopsis WRKY transcription factors in regulating the expression of PR genes by direct binding (Chen et al., 2002; Kim et al., 2006). A rapid pathogen-induced WKRY33 expression did not require salicylic acid signaling but a downregulation of this gene involved a direct activation of jasmonic acid (Bakshi and Oelmüller, 2014). In the present case, when bean plants were in contact T. velutinum T028 without pathogen, the WRKY33 gene expression was significantly up-regulated while the PR genes expression (PR2, PR3 and PR4) was significantly down-regulated compared to expression levels in plants without Trichoderma treatment. In the present work, when R. solani was added to the substrate, expression of WRKY33 was significantly down-regulated in plants with Trichoderma inoculation, while PR2, PR3 and PR4 were down-regulated. In the study by Mayo et al. (2015), the expression of PR1, PR2, PR3, and PR4 was down-regulated when beans were inoculated with R. solani.

WRKY33 is also involved in the regulation of the expression of genes modulated by components of the ethylene signaling pathway. In this work, expression of the ERF1 and ERF5 reached similar significant values either with or without Trichoderma and or R. solani in the substrate. This result contrasts with previous reports showing that ERF5 was up-regulated and WRKY33 was down-regulated in Arabidopsis infected with Alternaria brassicicola (Son et al., 2012). WRKY33 would act as a represor of ERF1 and ERF5 expression. Thus, when the expression of WRKY33 is increased, expression of ERF1 and ERF5 is down-regulated.

CH5b encodes an endochitinase precursor and it is also related with the ethylene signaling pathway. In previous works, it has been shown that, when this gene was over-expressed the R. solani symptoms were reduced in crops like Nicotiana tabacum and Brassica napus (Broglie et al., 1991). However, in this study, when bean plants were in contact with R. solani, the expression of this gene was down-regulated but not significantly, while treatment of these infected plants with T. velutinum resulted in its significant up-regulation. These results are in agreement with previous data, showing that the pathogen represses its expression, and the presence of Trichoderma induced it (Mayo et al., 2015).

PAL plays an important role in plant defense; it is involved in the biosynthesis of salicylic acid, which is related to plant systemic resistance (Nugroho et al., 2002; Chaman et al., 2003). PAL gene expression is also regulated in response to pathogen infection. In this work, the presence of T. velutinum and R. solani in the soil resulted in a significant down-regulation of this gene compared with control plants.

Osmotins have plant protective effects against pathogen infection (Narasimhan et al., 2009). In this study, when T. velutinum or R. solani were present in the soil, the expression of OSM34 was not significantly up-regulated respect to control plants, but when both fungi were in the soil at the same time, OSM34 was slightly but significantly down-regulated.

The CNGC genes can be related to early plant defense responses due to changes in ion flux, including H⁺ and Ca²⁺ influx and K⁺ and Cl^- efflux (Atkinson et al., 1996). The up-regulation of CNGC2 can confirm the importance of ion channels for the plant resistance response (Borges et al., 2012). In this work, this gene was up-regulated when R. solani was present in the soil not significant. Conversely, CNGC2 was down-regulated in plants treated with T. velutinum. Then, the pathogen would induce an activation of hypersensitive defense mechanisms.

Hydroperoxide lyase (HPL) is involved in the production of antimicrobial and defense signaling oxylipins (Noordermeer et al., 2001; Huang et al., 2010). In this study, the presence of T. velutinum and R. solani, resulted in a down-regulation of this gene expression respect to control plants. In previous works, when tomato plants were in contact with Botrytis cinerea, HPL expression increased 24 h after gray mold infection, but after that time the expression of this gene decreased gradually (Wan et al., 2013). In the present case, after 45 days in contact with the fungus T. velutinum and/or R. solani, its expression was down-regulated, indicating that the plant identifies Trichoderma and Rhizoctonia as two invader organisms, and some of the mechanisms activated against the presence of both are similar, independently of the final response specifically activated in the plant by each one.

GSTa (2,4-D inducible glutathione S-transferase) expression also responds to pathogen attack (Mauch and Dudler, 1993) and can be induced by molecules such as salicylic acid, methyl jasmonate, abscisic acid and H₂O₂ (Dixon et al., 2002; Moons, 2005). In Gossypium arboretum, GST provides resistance to fungal pathogens and oxidative stress (Barthelson et al., 2010). GST expression was up-regulated during fungal infection in barley, Arabidopsis, and cotton (Dowd et al., 2004; Durrant and Dong, 2004; Lu et al., 2005). However, in banana GST was down-regulated following Fusarium oxysporum f specialis (f. sp.) cubense infection (Wang et al., 2013), which is in agreement with the present case, where the expression of GSTa was down-regulated when T. velutinum and/or R. solani were present in the soil.

hGS encodes a homoglutathione synthetase that is involved in response to oxidative stress. There is not much information about the behavior of this gene in the plant. In the present study, when bean plants were in contact with T. velutinum and/or R. solani, expression of this gene was significantly up-regulated compared to control plants. In other studies, treatment of Medicago truncatula plants with compounds that release nitric oxide, a key signaling molecule in plants, induced expression of GST but not hGS in roots (Innocenti et al., 2007). Similarly, common bean plants treated with H₂O₂ showed up-regulation of hGS in nodules, whereas treatments with cadmium, sodium chloride, or jasmonic acid had no effect (Loscos et al., 2008).

From 48 genes initially analyzed, 14 bean genes were selected in the present work 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 showed significant homology with described plant defense genes and they are expressed in bean leaves of plants treated with T. velutinum and/or infected with R. solani. The proposed strategy will be very useful in studies about the interaction of bean with pathogens and biocontrol fungi.

PC and SG conceived the research. SM, OG-L, and AR-G designed the research. SM, OG-L, and AR-G conducted the experiments. SM, PC, and SG analyzed data. SM, PC, SG, EC, and FS interpreted the data. SM, PC, SG, EC, and FS wrote the manuscript. All authors were agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors critically revised the manuscript. All authors approved the final version to be published.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer EVG declared a past co-authorship with one of the authors SG to the handling Editor, who ensured that the process met the standards of a fair and objective review.