A Complex Interaction System for Understanding the Ability of Trichoderma to Trigger Defenses in Tomato Plants Challenged by Phytophthora nicotianae ^†

Abstract

In this study, the early activation of plant-defense-related genes during a three-way plant–antagonist–pathogen interaction in a tomato–Trichoderma–Phytophthora nicotianae model system was evaluated. Thirty-day-old tomato seedlings were treated at the root systems with a suspension of germinated conidia of two selected strains of T. asperellum and T. atroviride and then inoculated with zoospores of P. nicotianae. The defense mechanisms activated by tomato plants upon the simultaneous colonization of the root systems by Trichoderma spp. and P. nicotianae were evaluated 72 h post-inoculation by analyzing the transcriptomic profiles of genes involved in the pathways of salicylic acid (i.e., pathogenesis-related proteins—PR1b1 andPR-P2-encoding genes), jasmonic acid (i.e., lipoxygenases enzymes—TomLoxC- and TomLoxA-encoding genes), and the tomato plant defensin protein (i.e., SlyDF2-encoding gene). The results showed that PR1b1 was more strongly up-regulated in the three-way system including T. asperellum, while the gene PR-P2 was up-regulated in the system including T. atroviride. TomLoxA was significantly up-regulated only in the three-way system including T. asperellum, while TomLoxC was significantly up-regulated in neither of the analyzed three-way systems. Finally, the gene SlyDF2 was significantly up-regulated in tomato seedlings in both three-way systems.

1. Introduction

The European Directive 2009/128/EC, which established a framework for community action to pursue the sustainable use of pesticides, prescribes the adoption of measures aimed at reducing their use. The National Action Plans (NAPs) represent the instruments available for each member state to implement the European Directive. NAPs are directed toward setting quantitative objectives, targets, measures, timetables, and indicators to reduce the risks and impacts of pesticide use on human health and the environment and to encourage the development and introduction of integrated pest management strategies and alternative approaches or techniques devoted to reducing dependency on the use of pesticides. In recent decades, the use of biological control agents (BCAs) for plant pathogens has become an effective alternative to conventional practices based on the use of chemicals for the management of plant diseases [1,2,3].

Among BCAs, selected strains of fungi belonging to the Trichoderma genus have shown high effectiveness in the control of fungal and oomycete plant pathogens [3,4,5,6,7,8,9]. The success of Trichoderma strains such as BCAs is due to their direct antagonistic activity toward pathogens [4,5,6] and to their efficiency in the elicitation of plant defense by the triggering of mechanisms of both systemic acquired resistance (SAR) and induced systemic resistance (ISR). The activation of these mechanisms is mediated by the synthesis of salicylic acid and jasmonic acid or ethylene, respectively [2,3,10,11,12].

In recent years, the research has been focused on the investigation of the role of Trichoderma spp. in the elicitation of SAR and ISR mechanisms in experimental three-way model systems including a plant, Trichoderma, and a pathogen [13]. Together with maize, cucumber, and pepper, tomato (Solanum lycopersicum) is one of the most studied model organisms used in three-way model systems including Trichoderma spp. [2,3,14,15,16,17]. The sequencing of the tomato genome [18] together with the susceptibility of this crop to numerous diseases [19] has made this plant a suitable model for transcriptomic-based studies aimed at elucidating the mechanism by which Trichoderma spp. stimulate plant defense mechanisms to counteract infections by fungi and oomycetes. A very recent study provided preliminary information on the role of Trichoderma spp. in activating genetic pathways of plant defense mechanisms at an advanced stage of root infection in three-way systems of tomato–T. asperellum–P. nicotianae and tomato–T. atroviride–P. nicotianae (Figure 1) [3].

It should be determined whether the elicitation of tomato defenses by Trichoderma spp. is an ability of specific strains or if it is conserved in the population of a species; furthermore, the early activation of the defense response in tomato plants under the simultaneous Trichoderma spp. colonization and P. nicotianae root infection has not yet been investigated. To gain a better insight into the transcriptomic mechanisms involved in the complex three-way plant–antagonist–pathogen interaction, this study describes how two recently selected strains of T. asperellum and T. atroviride modulated salicylic acid, jasmonic acid, and antifungal defensin in the genetic pathways of tomato plants in an early stage of the infection process, using the root and crown pathogen Phytophthora nicotianae in a tomato–Trichoderma–P. nicotianae system.

2. Experiments

2.1. Selection and Culture of Test Microrganisms

T. asperellum strain T_asp_1, T. atroviride strain T_atr_6, and P. nicotianae isolate Ph_nic [3] were selected from the collection of the Molecular Plant Pathology laboratory, Di3A, University of Catania.

For the experiment, Trichoderma spp. and P. nicotianae strains were preliminarily cultured on potato dextrose agar (PDA) for 7 days at 25 °C and on V8 agar Petri dishes for one week at 28 °C in the dark, respectively.

2.2. Plant Material

Tomato test seedlings were cultured in accordance with La Spada et al. [3]. Tomato seeds (Solanum lycopersicum var. Cuor di Bue, Vilmorin Italia S.R.L.) were sterilized in 2% NaClO for 20 min, rinsed in sterile distilled water, and sown in an alveolar tray containing sterile vermiculite soaked in a nutrient solution (NS) prepared with 20-20-20 fertilizer (Asso di Fiori-Cifo, S. Giorgio di Piano, Bologna, Italy) (0.1634 g/L), MgSO₄ × 7H₂O (0.15 g/L), FeNa-EDTA (40 mg/L). Trays were kept for 3 days in the dark at 23 °C and 80% relative humidity. Then, seedlings were transferred in a walk-in growth climatic chamber (16 h of light and 8 h of dark) and kept under the same temperature conditions and relative humidity for 30 days. Here, 30 mL of NS was provided weekly to renew the contents of mineral salts; tomato plantlets were also watered twice a week. Seedlings were then transferred into plastic tubes containing 30 mL of NS.

2.3. Trichoderma Colonization and Phytophthora Nicotianae Infection Assays

Once in the plastic tubes, tomato seedlings were colonized by T. asperellum or T. atroviride test strains as well as infected by P. nicotianae isolate Ph_nic according to the method reported by La Spada et al. [3]. For each Trichoderma species, the colonization of the root system was established by treating the tomato seedlings with 300 μL of a dispersion of germinated conidia (100 conidia/mL) prepared as follows. First, 100 mL of a synthetic medium, consisting of the NS amended with 15 g/L of sucrose, was autoclaved and then inoculated with 1 mL of conidial dispersion (10⁶ conidia/mL) obtained from 7-day-old cultures grown on PDA medium. Next, flasks were shaken at 150 rpm for 24 h at 25 °C to allow spore germination.

After 48 h of incubation, each seedling colonized by T. asperellum or T. atroviride was inoculated with zoospores of P. nicotianae isolate Ph_nic (concentration: 100 zoospores/mL). P. nicotianae inoculum was prepared as follows: mycelial plugs from a 7-day-old culture of the pathogen grown on V8 agar were flooded with 20 mL of sterile distilled water and incubated at 25 °C for 48 h under constant fluorescent light. Zoospores were released in sterile distilled water by placing mycelial plugs at 6 °C for 1 h and then for another hour at 25 °C. The zoospore concentration was measured using a hemocytometer. Controls were inoculated with sterile distilled water.

2.4. Experimental Design

Overall, the scheme of the experiment consisted of the treatments reported in

Table 1. Scheme of the treatments included in the experiment.

Treatment ID	T. asperellum Strain T_asp_1	T. atroviride Strain T_atr_6	P. nicotianae Isolate Ph_nic
1 (control)	1 -	-	-
2	-	-	2 X
3	-	X	X
4	X	-	X

Note: ¹ unreceived treatment; ² received treatment.

. Each treatment included 5 biological replicates. All the seedlings from each treatment were collected 72 h post-inoculation. Stems and roots from each plant were then ground into a fine powder with liquid nitrogen. The powders were stored at −80 °C until RNA extraction. All of the microorganisms used in the test were re-isolated and then sequenced from additional seedlings from respective treatments.

2.5. RNA Isolation, cDNA Synthesis, and Quantitative Real-Time PCR (qRT-PCR)

Total RNA samples were extracted by using the RNeasy Plant Mini Kit (Qiagen) from 100 mg of powder from both stems and roots of tomato seedlings, following manufacturer instructions, which were treated with the TURBO DNA-free™ Kit. The RNA concentration was adjusted to 200 ng/μL and its quality was verified using a RNA electrophoresis gel in TAE agarose [20,21].

The cDNA synthesis was performed by using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems™) following the manufacturer’s instructions.

Each qRT-PCR process was performed in a total volume of 20 μL by mixing 10 ng of cDNA with 1 μL of 10 μM of each primer and 10 μL of PowerUp™ SYBR™ Green Master Mix (2x) (Applied Biosystems). The qRT-PCR experiments were carried out in triplicate. The thermocycling conditions were 2 min at 50 °C (UDG activation), 2 min at 95 °C (Dual-Lock™ DNA polymerase), followed by 40 cycles of two steps: 95 °C for 15 s (denaturation) and 59 or 60 °C (annealing/extension) for 1 min. The amplified target genes were PR1b1, PR-P2, TomLoxA, TomLoxC [2], and SlyDF2 [22]. The actin-7-like LOC101262163 [2] was used as housekeeping. For each gene, the primer pair sequences and annealing temperatures were in accordance with La Spada et al. [3].

2.6. Gene Expression Profile

The quantification of gene expression was carried out using the 2^−ΔΔCt method [23], where ΔΔCt = (Ct of target gene − Ct of reference gene)_sample − (Ct of target gene − Ct of reference gene)_calibrator and Ct is the threshold cycle for each transcript, defined as the point at which the amount of amplified target reaches a fixed threshold above the background fluorescence. The calibrator sample was representative of five biological replicates of untreated tomato seedling control samples (i.e., treatment 1 in Table 1).

Data were analyzed using one-way ANOVA and Dunnett’s multiple comparisons test using R software. Differences at p ≤ 0.1 were considered significant.

3. Results

A generalized up-regulation was observed for all analyzed genes in all treatments compared to the control. Both genes involved in the pathway of the salicylic acid, namely PR1b1 and PR-P2, showed significant up-regulation in all treatments (Table 1). The relative expression levels of PR1b1 ranged around 4.10 (treatment 2), 2.50 (treatment 3), and 4.70 (treatment 4) times more than those of plants from the calibrator (i.e., treatment 1, Table 1), while the levels were ca. 23.8 (treatment 2), 15.8 (treatment 3), and 14.00 (treatment 4) times greater than those of plants from the calibrator for the PR-P2-encoding gene. Similarly to previous genes, SlyDF2 was significantly up-regulated in plants from all treatments, with ca. 7.30 (treatment 2), 4.30 (treatment 3), and 5.70 (treatment 4) times greater expression than the calibrator (Figure 2).

Compared to the other genes, the up-regulation of the genes encoding for lypoxygenases, namely TomLoxA and TomLoxC, was weakly activated. TomLoxA was significantly up-regulated exclusively in treatments 2 (i.e., seedlings inoculated with P. nicotianae) and 4 (i.e., seedlings treated with T. asperellum and inoculated with P. nicotianae), while TomLoxC was significantly transcribed only in seedlings from treatment 2 (Figure 2).

4. Discussion

In the present study, we have described the response of tomato plant cv. Cuore di Bue, in terms of the activation of genetic pathways related with plant defenses, during a three-way tomato–Trichoderma spp.–Phytophthora nicotianae interaction via the application of two recently selected strains of T. asperellum (T_asp_1) and T. atroviride (T_atr_6) and the pathogen P. nicotianae isolate Ph_nic [3]. Previous studies have widely investigated the role of Trichoderma species in the elicitation of plant defense mechanisms [17,24,25,26], including three-way plant–Trichoderma spp.–pathogen systems [2,3,16]; however, there is a lack of information about the early activation of the defense responses in tomato plants under simultaneous Trichoderma colonization and P. nicotianae infection. To fill this gap in the knowledge, in this study the expression profiles of the genes involved in the pathways of salicylic (PR1b1 and PR-P2) and jasmonic acids (TomLoxA and TomLoxC) [2] and of the antifungal defensin SlyDF2 [22] were evaluated 72 h post-inoculation in T. asperellum- or T. atroviride-treated tomato seedlings inoculated with P. nicotianae and compared with seedlings that received only the pathogen P. nicotianae.

The results obtained here showed that 72 h post-treatment, a generalized trend of up-regulation was observed for all evaluated genes in tomato seedlings from both of the three-way systems. Both genes involved in the genetic pathway of salicylic acid, namely PR1b1 and PR-P2, were significantly up-regulated in seedlings from both three-way systems, with stronger up-regulation for PR-P2 compared to PR1b1. In detail, PR1b1 was more strongly up-regulated in the three-way system including T. asperellum strain T_asp_1 (treatment 4), while the PR-P2 gene was more strongly up-regulated in the system one including T. atroviride strain T_atr_6 (treatment 3). With reference to the genes involved in the pathway of jasmonic acid, namely TomLoxA and TomLoxC, these were both up-regulated on a smaller scale compared with PR1b1 and PR-P2. TomLoxA was significantly up-regulated only in the three-way system including T. asperellum strain T_asp_1 (treatment 4), while TomLoxC was not significantly up-regulated in either of the analyzed three-way systems. Finally, similarly to PR1b1 and PR-P2, the SlyDF2 gene encoding for an antifungal defensin was statistically significantly up-regulated in tomato seedlings from both the three-way systems. The transcriptional response of the defensive pathway of tomato plants in a three-way system including Trichoderma spp. and a plant pathogen was previously described under Botrytis cinerea [2] or P. nicotianae infections [3]. Tucci et al. [2] showed that at the early stages of the three-way interaction, the transcriptional profiles of genes PR1b1, PR-P2, TomLoxA, and TomLoxC can range from up- to down-regulation, depending on several aspects, including the tested tomato cultivar, the Trichoderma species selected, as well as the tested antagonistic strain. La Spada et al. [3] observed that among two different three-way systems, namely tomato–P. nicotianae–T. asperellum strain IMI393899 [27,28] and tomato–P. nicotianae–T. atroviride strain TS, 7 days post-inoculation of the pathogen the gene PR1b1 was significantly up-regulated only in the system including T. asperellum, PR-P2 was significantly up-regulated in both three-way systems, while TomLoxA- and TomLoxC-encoding genes were not up-regulated in either of the systems. In the same study, La Spada et al. [3] also observed a marked up-regulation of the SlyDF2-encoding gene in plants from both the tested three-way systems. Consistent with the above-cited studies [2,3], the results obtained here support the hypothesis that the effects due to the colonization of roots by Trichoderma spp. occur mainly during the first phases of the three-way interaction, and as reported by La Spada et al. [3], they run out after a short time.

Previous studies have supported the hypothesis that when plants are challenged with a pathogen after the establishment of interaction with Trichoderma spp., they are primed to react more strongly, increasing defense gene expression sooner and to higher levels than in untreated plants [15,24,25]; however, in the present study, the transcription of all of the evaluated genes was more strongly activated in plants that received only the inoculum rather than in Trichoderma-pre-colonized and inoculated ones. This result is in agreement with those obtained in other similar pathogen–plant systems. A decreasing trend in the expression of PR-encoding genes at 48 h post-inoculation with B. cinerea was reported from different lines of tomato plants pre-treated with T. atroviride strain P1 or T. harzianum strain T22 [2]. Additionally, the level of the CTR1-encoding gene, which is involved in the signaling pathway of jasmonic acid, was significantly higher in the roots of cucumber plants inoculated with only the Pseudomonas syringae pv. lachrymans rather than in plants inoculated and pre-colonized by T. asperellum T203 [17]. Furthermore, La Spada et al. [3] observed that 7 days post-inoculation with P. nicotianae, the expression level of the SlyDF2 gene in tomato plants pre-colonized by T. asperellum strain IMI393899 or T. atroviride strain TS was lower than in plants that received only the inoculum of the pathogen. The results obtained in this study support the hypothesis that the promotion of plant defenses by Trichoderma spp. is a complex mechanism affected by a variety of responses that could depend both on the tested plant species and inoculated pathogen [3].

5. Conclusions

In conclusion, this study indicates the genetic defense pathways in tomato related tosalicylic acid, jasmonic acid, and antifungal defensins in a three-way tomato–Trichoderma spp.–P. nicotianae interaction system are activated from the first stages of the infection process. Moreover, the results support the hypothesis that the ability to elicit a defense response in tomato plants challenged with a root pathogen, such as P. nicotianae, is a conserved feature within the same species of Trichoderma.