Trichoderma Applications on Strawberry Plants Modulate the Physiological Processes Positively Affecting Fruit Production and Quality

Many Trichoderma spp. are successful plant beneficial microbial inoculants due to their ability to act as biocontrol agents with direct antagonistic activities to phytopathogens, and as biostimulants capable of promoting plant growth. This work investigated the effects of treatments with three selected Trichoderma strains (T22, TH1, and GV41) to strawberry plants on the productivity, metabolites and proteome of the formed fruits. Trichoderma applications stimulated plant growth, increased strawberry fruit yield, and favored selective accumulation of anthocyanins and other antioxidants in red ripened fruits. Proteomic analysis of fruits harvested from the plants previously treated with Trichoderma demonstrated that the microbial inoculants highly affected the representation of proteins associated with responses to stress/external stimuli, nutrient uptake, protein metabolism, carbon/energy metabolism and secondary metabolism, also providing a possible explanation to the presence of specific metabolites in fruits. Bioinformatic analysis of these differential proteins revealed a central network of interacting molecular species, providing a rationale to the concomitant modulation of different plant physiological processes following the microbial inoculation. These findings indicated that the application of Trichoderma-based products exerts a positive impact on strawberry, integrating well with previous observations on the molecular mechanisms activated in roots and leaves of other tested plant species, demonstrating that the efficacy of using a biological approach with beneficial microbes on the maturing plant is also able to transfer advantages to the developing fruits.


Figure legends
Supplementary Figure S1. HPLC-DAD chromatogram of an exemplificative strawberry dried extract recorded at 520 nm. Putative compound identification was performed according to the scientific literature (Holzwarth 2012;Carbone et al., 2009). (1) Table S3). Data are reported as log 2 transformed abundance ratio values.
Supplementary Figure S3. Heat-map representation and hierarchical clustering analysis of proteins involved in carbon and energy metabolism that were differentially represented in strawberry fruits produced by plants subjected to the treatments with Trichoderma strains (T22, TH1 and GV41), as compared to control (Ctr). Shown are proteins presenting abundance fold changes ≥1.50 or ≤0.66 with respect to control (P≤ 0.05) (Supplementary Table S3). Data are reported as log 2 transformed abundance ratio values.
Supplementary Figure S4. Heat-map representation and hierarchical clustering analysis of proteins involved in stress response that were differentially represented in strawberry fruits produced by plants subjected to the treatments with Trichoderma strains (T22, TH1 and GV41), as compared to control (Ctr). Shown are proteins presenting abundance fold changes ≥1.50 or ≤0.66 with respect to control (P≤ 0.05) (Supplementary Table S3). Data are reported as log 2 transformed abundance ratio values.
Supplementary Figure S5. Heat-map representation and hierarchical clustering analysis of proteins involved in amino acid metabolism (upper panel), coenzyme metabolism (middle panel), nucleotide metabolism (middle panel) or lipid metabolism (lower panel), which were differentially represented in strawberry fruits produced by plants subjected to the treatments with Trichoderma strains (T22, TH1 and GV41), as compared to control (Ctr). Shown are proteins presenting abundance fold changes ≥1.50 or ≤0.66 with respect to control (P≤ 0.05) (Supplementary Table  S3). Data are reported as log 2 transformed abundance ratio values.
Supplementary Figure S6. Heat-map representation and hierarchical clustering analysis of proteins involved in RNA biosynthesis (upper panel), RNA processing (middle panel) and protein biosynthesis (lower panel), which were differentially represented in strawberry fruits produced by plants subjected to the treatments with Trichoderma strains (T22, TH1 and GV41), as compared to control (Ctr). Shown are proteins presenting abundance fold changes ≥1.50 or ≤0.66 with respect to control (P≤ 0.05) (Supplementary Table S3). Data are reported as log 2 transformed abundance ratio values.
Supplementary Figure S7. Heat-map representation and hierarchical clustering analysis of proteins involved in protein modification (upper panel), protein translocation (middle panel) and protein degradation (lower panel), which were differentially represented in strawberry fruits produced by plants subjected to the treatments with Trichoderma strains (T22, TH1 and GV41), as compared to control (Ctr). Shown are proteins presenting abundance fold changes ≥1.50 or ≤0.66 with respect to control (P≤ 0.05) (Supplementary Table S3). Data are reported as log 2 transformed abundance ratio values.  Table S3). Data are reported as log 2 transformed abundance ratio values.

Supplementary
Supplementary Figure S9. Heat-map representation of hierarchical clustering analysis of proteins with unknown function that were differentially represented in strawberry fruits produced by plants subjected to the treatments with Trichoderma strains (T22, TH1 and GV41), as compared to control (Ctr). Shown are proteins presenting abundance fold changes ≥1.50 or ≤0.66 with respect to control (P≤ 0.05) (Supplementary Table S3). Data are reported as log 2 transformed abundance ratio values. Results related to proteins with unknown function are shown.