Trichoderma Counteracts the Challenge of Phytophthora nicotianae Infections on Tomato by Modulating Plant Defense Mechanisms and the Expression of Crinkler, Necrosis-Inducing Phytophthora Protein 1, and Cellulose-Binding Elicitor Lectin Pathogenic Effectors

Abstract

Decoding the mechanisms of plant defense against plant pathogens in a scenario where antagonistic activity and the plant growth-promoting effects of useful organisms intervene simultaneously is a new frontier of plant pathology. Here, we demonstrated that (i) two selected strains of Trichoderma asperellum and Trichoderma atroviride promoted tomato (Solanum lycopersicum) growth and reduced the severity of disease caused by the oomycete Phytophthora nicotianae and (ii) the genetic patterns of the components of the experimental model system tomato-Trichoderma spp.-P. nicotianae were differentially expressed. The beneficial effects in both the promotion of the growth of host plant and the biological control of the pathogen by two selected strains of different Trichoderma species were tested both in planta and in vitro. In both respects, T. atroviride demonstrated to be more effective than T. asperellum. Additionally, the simultaneous transcriptional reprogramming of several plant defense-related genes, pathogen effectors, and mycoparasitism-related genes in tomato, P. nicotianae, and Trichoderma spp., respectively, was evaluated during the three-component interaction. Results support the hypothesis that Trichoderma spp. elicit the expression of plant defense-related genes. As expected, a mycoparasitism-related gene was significantly up-regulated in Trichoderma-colonizing tomato plants infected by P. nicotianae. Finally, a marked up-regulation of the genes encoding two necrosis-inducing effectors was observed in P. nicotianae infecting tomato plants colonized by Trichoderma. In conclusion, this study is a contribution toward understanding the genetic pathways related with the ability of Trichoderma spp. to counteract the challenge of P. nicotianae infections on tomato. Additionally, the experiments revealed the beneficial effects in the tomato growth promotion of a new T. atroviride strain and its good antagonistic effectiveness in the biological control of root and crown rot incited by P. nicotianae, confirming that Trichoderma spp. can be a powerful tool in integrated pest management strategies of Phytophthora diseases of horticultural crops.

Keywords: Trichoderma asperellum; Trichoderma atroviride; antagonism; biological control; crown rot; gene expression; root rot.

FIGURE 1

Proposed model for the three-way system plant-pathogen-antagonist showing how Trichoderma species can modulate the molecular signaling in the challenge between the oomycete pathogen Phytophthora nicotianae and the host plant tomato. The colonization of the tomato rhizosphere by Trichoderma spp. triggers both growth promotional effects and plant defense mechanisms by the elicitation of salicylic acid (SA)-, ethylene (ET)-, and jasmonic acid (JA)-dependent processes. At the same time, the P. nicotianae-parasitic infection process is mediated by the secretion of pathogenic effectors (including Crinkler–CRN–proteins, the necrosis-inducing Phytophthora protein 1–NPP1, and cellulose-binding elicitor lectin–CBEL–glycoproteins) which act to suppress the ET- and JA-plant response and whose synthesis is modified by the antagonistic interaction with Trichoderma spp.

FIGURE 2

Effects of Trichoderma asperellum IMI393899 and Trichoderma atroviride TS treatments on the growth of Solanum lycopersicum cv. Cuor di bue seedlings. Weekly stem growth rate. Open and gray circles represent outliers and mean value data, respectively. Values sharing same letters are not statistically different according to Tukey’s honestly significant difference (HSD) test (P ≤ 0.05).

FIGURE 3

Effects of Trichoderma asperellum IMI393899 and Trichoderma atroviride TS treatments on the growth of Solanum lycopersicum cv. Cuor di bue seedlings. (A) Stem length and (B) fresh root weight at the end of the test. Open and gray circles represent outliers and mean value data, respectively. Values sharing same letters are not statistically different according to Tukey’s honestly significant difference (HSD) test (P ≤ 0.05).

FIGURE 4

Effects of Trichoderma asperellum IMI393899 and Trichoderma atroviride TS treatments on the growth of Solanum lycopersicum cv. Cuor di bue seedlings. Seedling total length at the end of the test. Open and gray circles represent outliers and mean value data, respectively. Values sharing same letters are not statistically different according to Tukey’s honestly significant difference (HSD) test (P ≤ 0.05).

FIGURE 5

In planta antagonistic effectiveness of Trichoderma test strains against Phytophthora nicotianae. Wilting severity of 4-month-old Solanum lycopersicum cv. Cuor di bue developed from the following: (i) untreated tomato plants transplanted in a non-infested potting mixture (NI-PM); (ii) untreated tomato plants inoculated with infested potting mixture (I-PM); (iii) tomato plants treated with Trichoderma asperellum strain IMI393899 and transplanted in NI-PM; (iv) tomato plants treated with T. asperellum strain IMI393899 and transplanted in I-PM; (v) tomato plants treated with Trichoderma atroviride strain TS and transplanted in NI-PM; and (vi) tomato plants treated with T. atroviride strain TS and transplanted in I-PM. Open and gray circles represent outliers and mean value data, respectively. Values sharing same letters are not statistically different according to Tukey’s honestly significant difference (HSD) test (P ≤ 0.05).

FIGURE 6

In planta antagonistic effectiveness of Trichoderma test strains against Phytophthora nicotianae. (A) Fresh root weight and (B) length of 4-month-old Solanum lycopersicum cv. Cuor di bue developed from the following: (i) untreated tomato plants transplanted in a non-infested potting mixture (NI-PM); (ii) untreated tomato plants inoculated with infested potting mixture (I-PM); (iii) tomato plants treated with Trichoderma asperellum strain IMI393899 and transplanted in NI-PM; (iv) tomato plants treated with T. asperellum strain IMI393899 and transplanted in I-PM; (v) tomato plants treated with Trichoderma atroviride strain TS and transplanted in NI-PM; (vi) tomato plants treated with T. atroviride strain TS and transplanted in I-PM. Open and gray circles represent outliers and mean value data, respectively. Values sharing same letters are not statistically different according to Tukey’s honestly significant difference (HSD) test (P ≤ 0.05).

FIGURE 7

Differences in the expression levels of PR1b, TomLoxC, SlyDF2, PR-P2, and TomLoxA-encoding genes (GenBank accession numbers: Y08804.1, U37839.1, NM_001346524.1, X58548.1 and U09026.1, respectively) from 7-day-old Trichoderma-treated or non-treated Solanum lycopersicum cv. Cuor di bue seedlings inoculated or non-inoculated with Phytophthora nicotianae. Columns with asterisks are statistically different according to Dunnett’s test (^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001), compared to their calibrator.

FIGURE 8

Differences in the expression levels of PpCRN4, PpCBEL4, PpNPP1.1, PpNPP1.3, and PpNPP1.4-encoding genes (GenBank accession numbers: ETM55095.1, ETM43740.1, ETM52620.1, ETM39327.1 and ETM36738.1, respectively) in Phytophthora nicotianae isolate Ph_nic from 7-day-old Trichoderma-treated or non-treated Solanum lycopersicum cv. Cuor di bue P. nicotianae-inoculated seedlings. Columns with asterisks are statistically different according to Dunnett’s test (^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001), compared to their calibrator.

FIGURE 9

Differences in the expression levels of CHI18-5 (GenBank accession number: XM_014088210) and chi42 (GenBank accession number: HM191684.1)-encoding genes, respectively, in Trichoderma atroviride strain TS (on the left) and Trichoderma asperellum strain IMI393899 (on the right) from 7-day-old Phytophthora nicotianae-inoculated or non-inoculated Solanum lycopersicum cv. Cuor di bue Trichoderma-treated seedlings. Columns with asterisks are statistically different according to Dunnett’s test (^∗P < 0.05), compared to their calibrator.