Tomato is one of the most consumed vegetables worldwide. Corynespora cassiicola is a pathogen that attacks tomato plants after the seedling stage, causing lesions on stems, flowers, and fruits. It is distributed in tropical areas, where it causes significant production losses. It is controlled mainly through the use of synthetic fungicides that require several applications during the crop cycle, causing environmental contamination and leading to the emergence of resistant strains (^{Rodríguez and Sandoval, 1998}; ^{Junxiang et al., 2019}).

Trichoderma spp. is a saprophytic fungus common in ecosystems and highly interactive in root, soil and leaf environments. It is known as a plant growth promoter and as a biological control agent against phytopathogens, an alternative to the use of synthetic fungicides. Its success is due to its mechanisms of action, which include antibiosis, emission of volatile antifungal compounds, production of defense enzymes, systemic resistance, and mycoparasitism through cell wall perforation and nutrient absorption (^{Bhat, 2017}; ^{Imran et al., 2020}; ^{Wonglom et al., 2020}; ^{Zin and Badaluddin, 2020}). Its endophytic capacity allows it to establish symbiotic relationships and induces the production of bioactive metabolites that successfully regulate root architecture and improve nutrient absorption and plant growth (^{Ying-Tzu et al., 2017}; ^{Segaran and Sathiavelu, 2019}).

The present work aimed to evaluate the growth promotion induced by the inoculation of different concentrations of T. asperellum Ta13-17 and its effectiveness in the biocontrol of C. cassiicola, which causes leaf spot in S. lycopersicum plants.

The study was conducted at the National Technological Institute of Mexico, Campus Conkal. The crop was established in a tunnel-type greenhouse between October 2019 and February 2020 with an average minimum temperature of 19 °C and an average maximum temperature of 30 °C. Laboratory evaluations were carried out in the Phytopathology laboratory of the same institute. The native strain T. asperellum Ta13-17, which belongs to the strain collection of the Phytopathology laboratory, was used as biostimulant for plant growth and biocontrol, isolated in an endophytic form from the root and stem of chili pepper (Capsicum annuum) cv. Creole. The strain was molecularly identified, showing 100% homology and coverage with the reference sequences KC479809.1 and JF501661.1 from the Gene Bank of the National Center For Biotechnology Information (http://blast.ncbi.nlm.nih.gov). The phytopathogen evaluated was C. cassiicola. It was inoculated naturally and was molecularly identified in subsequent works (unpublished data) with 100% homology and access number: ON815356 (http://blast.ncbi.nlm.nih.gov). The model plant was S. lycopersicum cv. Hybrid DRD 8551 type saladette, with determined growth, vigorous, resistance to heat and tolerant to: tomato yellow leaf curl disease (Tomato yellow leaf curl disease-TYLCV), tomato mosaic virus (Tomato mosaic virus-ToMV), F. oxysporum f. sp. lycopersici race 1 and 2, Verticillium dahliae, Meloidogyne arenaria, M. incognita, and M. javanica (Seminis, 2022; https://n9.cl/brb4j).

A 5 mm disc of mycelium with seven-days growth of T. asperellum Ta13-17 was sown in four 500 mL Roux flasks with culture medium of dextrose potato broth for 21 days. The strain presented abundant mycelial growth, cottony, dark green in color, and abundant production of spores. These characteristics are associated with the species under study. After 21 days, the medium was filtered with sterile gauze. The filtrate was placed in 50 mL Falcon tubes and centrifuged three times for 20 minutes at 3,000 rpm. The spores were recovered and used as the mother solution. Dilutions of the stock solution were made until concentrations of 1x10⁵, 1x10⁶, 1x10⁷ and 1x10⁸ conidia mL^-1 were obtained. To corroborate the number of conidia per dilution, 10 µL of each solution were taken and counted in a Neubauer chamber, applying the formula NE =(X/0.1) (1,000) (9), where NE: No. of spores, X: average of quadrants recorded in the Neubauer camera (^{Gómez-Ramírez et al., 2013}).

To evaluate the promotion of plant growth, tomato seeds were placed in flasks with 30 mL of the corresponding spore solution plus 1 mL of Tween 20, then shaken for 10 min. The seeds were then drained and sown in polystyrene trays with 75 cavities. Two reinforcements of the treatments were applied 15 and 45 days after germination. During the latter, the plants were transplanted in 4 kg plastic bags with sterile soil (120 °C for 15 min). Irrigation was applied with a nutrient solution (20N-20P-20K 1 g L^-1). The experiment was maintained under protected conditions and the treatments were applied using a completely randomized experimental design: 1x10⁰, 1x10⁵, 1x10⁶, 1x10⁷ and 1x10⁸ conidia mL^-1 of T. asperellum Ta-13-17 and Fithan® (commercial control based on T. harzianum, T. fasciculatum, and T. viride). Ten repetitions per treatment were used, with each plant taken as a repetition (^{Candelero et al., 2015}).

A SPAD 502 meter (Minolta, Tokyo, Japan) was used to measure the physiological variables 65 days after transplantation (dat) to determine the influence of the T. asperellum Ta13-17 strain on the plants. SPAD units were estimated (a quantitative evaluation of the intensity of the green of leaves) and an infrared gas analyzer (LICOR 6400XT, Nebraska, United States) was used to assess the following variables: photosynthesis, stomatal conductance (SC), intercellular carbon (IC), transpiration and water use efficiency (USA). These variables were evaluated 65 days after transplantation. Three plants were taken at random from each treatment; five readings were made per leaf and four leaves were read per plant (^{Garruña-Hernández et al., 2014}).

At the end of the experiment (130 days after sowing), a destructive sampling was carried out, taking 10 plants per treatment. Plant height (PH) and stem diameter (SD) were measured. The displacement method was used to estimate root volume (VR). The dry weight of each organ (leaves, stem and root) and the partial dry weight of plants were measured in plants dried in a convection oven for 96 hours at 60 °C. Normality tests were performed on the data, giving a p-value <0.0001. An analysis of variance was performed and a comparison of means was performed using Tukey’s method (p≤0.05).

At 80 dat, the presence of C. cassiicola was detected naturally in the culture. The biocontrol effect of T. asperellum Ta13-17 was estimated by measuring the severity of the disease using a six-class scale where: 1=1%, 2= 5%, 3=10%, 4=20% and 5=40% and 6=60% or more damage (^{Costa et al., 2015}). Three evaluations were made at 80, 90 and 100 dat. With the severity data, disease progress curves were constructed, and the intensity of the disease was estimated using epidemiological models. The area under the disease progress curve (AUDPC) was built using the trapezoidal integration method. The apparent infection rate was estimated using the inverse parameter of b (1/b) from the Weibull model. The final severity was calculated using Y_final (^{Mejía-Bautista et al., 2016}).

The analysis of variance showed significant differences between treatments for the SPAD variable. The highest average of SPAD units (40.3) was estimated in the treatment inoculated with T. asperellum Ta13-17 at a concentration of 1x10⁵ conidia mL^-1. The control treatment showed 37.8, which indicated a higher concentration of nitrogen in the leaves of these treatments (Table 1). The values of SPAD units found in the present study were lower than those reported by ^{Mendoza et al. (1998}) without the incorporation of fungal inoculants.

There was no relationship between SPAD units and photosynthesis. The treatments with 1x10⁶, 1x10⁸ and Fithan^® obtained the highest photosynthetic rates with 2.8, 2.7 and 1.7 µmol m^-2 s^-1, respectively, higher than the control. The Fithan® treatment showed the highest stomatal conductance, which was associated with its photosynthetic activity. A greater CO₂ uptake was estimated; however, there was also a greater accumulation of intercellular carbon with 323.4 µmol mol^-1, as in the control treatment without fungal inoculant. This indicated that the Fithan® treatment had low photosynthetic activity (Table 1), which means that the carbon molecules in the intercellular spaces were not efficiently assimilated and started to accumulate. The control treatment had the lowest transpiration value, which is associated with better efficiency in the use of water. This suggests a stable relationship between stomatal opening and transpiration in this treatment.

The highest transpiration values were found in treatments 1x10⁶ (13.2 mmol m^-2 s^-1) and 1x10⁸ (12.9 mmol m^-2 s^-1). This means that a greater content of water was lost for each molecule of CO₂ that was fixed for the photosynthesis process. The treatments of the plants inoculated with T. asperellum Ta 13-17 presented lower efficiency in the use of water. Studying wheat plants inoculated from the seed with Trichoderma spp., ^{Mulu et al. (2020}) reported increased photosynthesis, decreased stomatal conductance, intercellular CO₂ and transpiration, which improved water use efficiency under salt stress conditions.

There were no statistical differences between treatments with respect to plant height and root dry weight. The 1x10⁵ conidia mL^-1 treatment had the highest biomass production in leaves, as indicated by having the highest average dry weight of this organ, statistically equal to the 1x10⁶ conidia mL^-1 treatment, 45.9 and 30.1 g higher values, respectively, than the values obtained with the Fithan® treatment plants, which had the lowest dry weight of leaves. Regarding the dry weight of the stem, the treatments were statistically equal except for the 1x10⁶ conidia mL^-1 treatment, which had the lowest dry weight of the stem. The 1x10⁵ conidia mL^-1 treatment obtained the highest mean dry weight, 47.2 g higher than the Fithan® treatment. Statistically, the 1x10⁵ conidia mL^-1 treatment was equal to the 1x10⁶ and 1x10⁷ treatments, as week as to the control treatment without fungal inoculant. The highest fruit production was obtained with the 1x10⁸ conidia mL^-1 treatment with 1347.0 g per plant, 259.4 g higher than the control treatment without the presence of T. asperellum Ta13-17. This result was statistically equal to the 1x10⁷ conidia mL^-1 treatment (Table 2).

^{Cetz-Chi et al. (2018}) reported increases in the growth of tomato plants, with height gains between 4.4 and 14.2% when inoculated with the native species T. virens (Th33-59 and Th26-52) and T. simmonsi (Th33-58). The Th33-59 strain of T. virens increased root volume and root dry weight with respect to the control. ^{Ruiz-Cisneros et al. (2018)} observed increases in plant height, stem diameter, and root length in tomato plants inoculated with T. asperellum, T. harzianum, and T. longibrachiatum at concentrations of 10⁶ conidia mL^-1. ^{Marquez-Benavidez et al. (2017}) reported increases in root length in seedlings and greater production of fresh biomass of leaves and roots in the flowering stage of Phaseolus vulgaris plants inoculated with T. harzianum.

The success in promoting growth in plants by inoculating them with Trichoderma spp. is associated with the symbiotic relationship. Trichoderma takes advantage of compounds produced by plants such as carbohydrates, organic acids and vitamins, while plants use phytohormones and secondary metabolites secreted by fungi, which also facilitate the decomposition and mineralization of organic matter and improve the availability of nutrients in the soil (Ortiz -Castro et al., 2009). Other mechanisms that promote plant growth activity are the ability of Trichoderma to produce indoleacetic acid, which acts as a catalyst for primary meristematic tissues and the activation of plant plasma membrane enzymes that promote cell growth and division as well as plant growth (^{Moo-Koh et al., 2017}; ^{González-Marquetti et al., 2019}).

Some Trichoderma isolates can solubilize nutrients close to the roots, which allows these substances to be assimilated by the plant. It has been suggested that T. asperellum enhances the uptake of Fe in deficient environments. Moreover, the protein QID74 present in the cell wall modifies the root architecture, increases the total absorption surface and the translocation of nutrients in the shoots, resulting in an increase in biomass through an efficient use of N, P, K and micronutrients (^{Zhao et al., 2014}; ^{González-Marquetti et al., 2019}).

The Fithan^® treatments and the control treatment without fungal inoculant presented the highest rates of disease progress, with 0.0082 and 0.0085% per day. They also presented higher AUCPE and higher percentages of final severity. The disease decreased in plants treated with 1x10⁸ conidia mL^-1 with 0.0078 unit% per day. Statistically, the treatments inoculated with T. asperellum Ta13-17 showed an equally low accumulation of AUCPE during the evaluation time. The treatment with 1x10⁸ conidia mL^-1 presented the lowest AUCPE with 275.8 unit% day^-1, less than the control treatment without fungal inoculant. The final severity was 18.74% lower than the control (Table 3).

^{Baiyee et al. (2019}) reported a significant decrease in the severity of leaf spot disease caused by C. cassiicola and C. aeria in lettuce plants inoculated with T. asperellum T1. ^{Wonglom et al. (2020}) attributed resistance against C. cassiicola and C. aeria to volatile organic compounds emitted by T. asperellum T1.

Antagonistic microorganisms in association with plants reduce the severity of the disease by inducing responses that are triggered by the production of defense-related enzymes and enzymes that hydrolyze the cell wall (^{Baiyee et al., 2019}).