Trichoderma harzianum favours the access of arbuscular mycorrhizal fungi to non-host Brassicaceae roots and increases plant productivity

Abstract

The family Brassicaceae includes plants that are non-host for arbuscular mycorrhizal fungi (AMF) such as the model plant Arabidopsis thaliana (arabidopsis) and the economically important crop plant Brassica napus (rapeseed). It is well known that Trichoderma species have the ability to colonize the rhizosphere of Brassicaceae plants, promoting growth and development as well as stimulating systemic defenses. The aim of the present work is to ascertain that Brassicaceae plants increase productivity when AMF and Trichoderma are combinedly applied, and how such an effect can be ruled. This simultaneous application of a Trichoderma harzianum biocontrol strain and an AMF formulation produces a significant increase in the colonization by Trichoderma and the presence of AMF in arabidopsis and rapeseed roots, such colonization accompanied by improved productivity in both Brassicaceae species. Expression profiling of defense-related marker genes suggests that the phytohormone salicylic acid plays a key role in the modulation of the root colonization process when both fungi are jointly applied.

Figure 1

Number of siliques produced by arabidopsis (a) and rapeseed (b) plants. Arabidopsis (ARA) and rapeseed (BN) inoculated with T. harzianum T34 (-T34) and AMF (-AMF). Each value represents the average of 45 plants from 3 independent experiments (15 plants each), with their standard deviation. Two-way analysis of variance (ANOVA) was performed, followed by Sidak’s multiple comparison test, indicating significant differences as follows: *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2

Real time reverse transcription polymerase chain reaction (qRT-PCR) analysis of some defense genes in roots of 3 week-old A. thaliana plants inoculated with T. harzianum T34 and/or AMF. Genes of the isochorismate synthase 1 (ICS1), pathogenesis-related protein 1 (PR-1), callose synthase 5 (CALS5), lipoxygenase 1 (LOX1), vegetative storage protein (VSP2) and plant defensin 1.5 (PDF1.5). Values correspond to relative measurements against arabidopsis grown in the absence of the fungus (2^−ΔΔCt = 1). The arabidopsis actin gene was used as an internal reference. Bars represent standard deviations of the means of three root pools of five plants each one collected from three independent experiments. Two-way analysis of variance (ANOVA) was performed, followed by Sidak’s multiple comparison test, indicating significant differences as follows: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 3

Real time reverse transcription polymerase chain reaction (qRT-PCR) analysis of some defense genes in roots of 5 week-old A. thaliana plants inoculated with T. harzianum T34 and/or AMF. Genes of the isochorismate synthase 1 (ICS1), pathogenesis-related protein 1 (PR-1), callose synthase 5 (CALS5), lipoxygenase 1 (LOX1), vegetative storage protein (VSP2) and plant defensin 1.5 (PDF1.5). Values correspond to relative measurements against arabidopsis grown in the absence of the fungus (2^−ΔΔCt = 1). The arabidopsis actin gene was used as an internal reference. Bars represent standard deviations of the means of three root pools of five plants each one collected from three independent experiments. Two-way analysis of variance (ANOVA) was performed, followed by Sidak’s multiple comparison test, indicating significant differences as follows: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 4

Real time reverse transcription polymerase chain reaction (qRT-PCR) analysis of expression of some defense genes in roots of 4 weeks-old tomato plants inoculated with T. harzianum T34 and/or AMF. Genes of the isochorismate synthase 1 (ICS1), pathogenesis-related protein 1 (PR-1), lipoxygenase 1 (LOX1) and ethylene signaling protein (EIN2). Values correspond to relative measurements against tomato grown in the absence of the fungus (2^−ΔΔCt = 1). The tomato actin gene was used as endogenous reference control. Bars represent standard deviations of the means of three root pools of five plants each one collected from three independent experiments. Two-way analysis of variance (ANOVA) was performed, followed by Sidak’s multiple comparison test, indicating significant differences as follows: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 5

Real time reverse transcription polymerase chain reaction (qRT-PCR) analysis of expression of some defense genes in roots of 7 weeks-old tomato plants inoculated with T. harzianum T34 and/or AMF. Genes of the isochorismate synthase 1 (ICS1), pathogenesis-related protein 1 (PR-1), lipoxygenase 1 (LOX1) and ethylene signaling protein (EIN2). Values correspond to relative measurements against tomato grown in the absence of the fungus (2^−ΔΔCt = 1). The tomato actin gene was used as endogenous reference control. Bars represent standard deviations of the means of three root pools of five plants each one collected from three independent experiments. Two-way analysis of variance (ANOVA) was performed, followed by Sidak’s multiple comparison test, indicating significant differences as follows: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.