Study location

This work was carried out in the Biology Laboratory of the Intercultural University of the State of Puebla, Huehuetla, Puebla, Mexico. Its geographical coordinates are the parallels 20º 01’ 48” and 20º 09’ 12” of north latitude and the meridians 97º 35’ 00” and 97º 40’ 24” of west longitude.

Fungal material

Three species of Trichoderma were isolated from soil in potato dextrose-agar medium (PDA). 1 g of wet soil was dispersed in boxes with PDA medium and incubated at 25 ±2 °C in the dark for two days, then removed from the incubator and kept under a white light lamp for two days to induce sporderma sporulation sp., from the development of Trichoderma, a portion was taken to transfer to PDA culture medium and incubate at 25 ±2 °C, this was done until obtaining axenic and pure growths, free of other fungi or bacteria. After the last sporulation in this medium, they were preserved in sterile water and mineral oil.

The pathogens Rhizoctonia sp., Fusarium sp. and Phytophthora capsici were isolated from chili roots, the roots were washed with running water; subsequently, the tissue portion was deflated by immersion for 1 min in 1.5% solution of sodium hypochlorite and rinsed with sterile distilled water, the roots were drained on sterile sanitas, finally 1 cm fragments were seeded in PDA medium for isolation and purification of Rhizoctonia sp. and Fusarium sp.

In the case of the Phytophthora capsici oomycete, 1.5 cm long chili roots were seeded in agar-agar^® medium with V8^® juice (8 vegetable juice) and antibiotics were added (Pimaricin 10 µg L^-1, Ampicillin 292 µg L^-l, Rifampicin 10 µg L^-1, Pentachloronitrobenzene 0.1 g L^-1 and Himexazol 0.25 µg L^-1), subsequently incubated at 28 ±2°C for three days (^{Andrade et al., 2012}), previously purified pathogens from the chili root was kept under conservation in sterile water for the purpose of preservation (^{Molina-Gayosso et al., 2016}). Prior to dual confrontation, microorganisms were activated in PDA culture medium plus lactic acid (Figure 1).

Figure 1 Growth in PDA culture medium of antagonists and pathogens of the wilting of chili. A= T. viride; B= T. harzianum; C= T. asperellum (C2); D= Rhizoctonia sp.; E= P. capcisi; and F= Fusarium sp. 

Morphological characterization

To study the morphological characteristics, both pathogen and Trichoderma isolates were grown in PDA medium and incubated at 24 ±2 °C for 12 h under white light (^{Rivera-Jiménez et al., 2018}). In the identification of the genus Fusarium sp. the description of ^{Burgess et al. (1994)} and the identification of the species was made using the keys of ^{Seifert (1996)}. Rhizoctonia species was identified based on ^{Sneh et al. (1991)} and that of the oomycete Phytophthora sp. It was characterized by the keys of ^{Erwin and Ribeiro (1996)}; ^{Gallegly and Hong (2008)}.

Finally, in the morphological characterization of the isolates of Trichoderma spp. the taxonomic keys of ^{Barnett and Hunter (1972)} were used. For the Trichoderma isolates, monoconidial cultures were used in combination with the wet chamber technique (^{Harris, 1986}). A 5 mm diameter PDA disc was inoculated with the Trichoderma spore and placed between a slide and coverslip and this preparation on a sterile rod triangle inside a petri dish and incubated at 25 ±2 °C for 24 h.

The macro and microscopic characteristics to be considered were colony texture, presence of mycelium, concentric rings, colony staining on the reverse, shape and size of conidia and phyloid (^{Chaverri et al., 2015}; ^{Jang et al., 2018}; ^{Nawaz et al., 2018}; ^{Du Plessis et al., 2018}), the structures of the Trichoderma spp. at 40X in an optical microscope (Zeiss Axiosiop plus).

Growth rate

In determining the growth rate of mycelium in antagonists and pathogens, grown in PDA, measurements were made every eight hours.

Trichoderma antagonistic activity on pathogens

The dual culture technique was used to determine the antagonistic activity of the different Trichoderma isolates on Rhizoctonia sp., Fusarium sp. and P. capsici (^{Sonnenbichler et al., 1983}). A completely randomized design was used (with two factors: Trichoderma and pathogens), with eight repetitions.

The tests were carried out evaluating dual cultures of Trichoderma spp., Contrasted with three phytopathogens, three isolates of Trichoderma against Rhizoctonia sp., Fusarium sp. and P. capsici. In the test of dual cultures, petri dishes with antibiotic-free PDA were used, a 5 mm diameter disc with active mycelium of the phytopathogen (previously grown in PDA for five days) was placed on one end and an equidistant end was placed Mycelium disc (previously grown in PDA for five days) of 5 mm in diameter with Trichoderma spp., the boxes were incubated at 28 ± 2 °C and observed every 8 h to record the number of days at the first contact between the antagonist and the phytopathogen, the growth of both colonies (cm) was measured and the percentage of inhibition of radial mycelial growth was evaluated, based on the formula proposed by ^{Ezziyyani et al. (2007)}, calculating as follows:

Where: PICR= percentage of radial growth inhibition; R1= radial growth (mm) of the pathogen without Trichoderma spp., R2= radial growth (mm) of the pathogen with Trichoderma spp.

Trichoderma mycoparasitism on pathogens

To know the presence of parasitism of the Trichoderma strains towards Rizoctonia sp., Fusarium sp. and P. capsici, the Riddel microculture technique described by ^{Paul (1999)} was used. This technique consists of placing a V-shaped glass rod inside a petri dish and on top of it, a sterile slide, on which a 10 mm diameter PDA disc was placed. The arrangement of each fungus was made in two cardinal points, after placing the mycelium of both fungi on the PDA, with a difference of 24 h, the pathogens were first placed with the help of a sterile needle, which have a slow growth compared with Trichoderma and a sterile coverslip was placed, it was incubated at 25 ±2 °C.

When the agar disk was covered with the growth of both fungi, the slide was removed from the petri dish to prepare the smears. The coverslip was placed on another clean slide and provided with a drop of dye known as cotton blue. On the other hand, the agar disc was removed from the original slide, a drop of the dye and a coverslip were placed. Once the excess dye was removed, the preparations were sealed and evaluated microscopically, that is, 10 samples were observed to determine whether or not there was parasitism by the Trichoderma sp. strains, 40X was observed in an optical microscope (Zeiss Axiosiop plus).

Statistic analysis

The PICR data, expressed as a percentage, were transformed with the angular arcsine √x+1 (^{Steel et al., 1986}) and in the mycelial growth in the biocontrol test, a completely randomized design was used (three strains of Trichoderma sp and three phytopathogens) with eight repetitions. The data obtained for each of the trials were subjected to an analysis of variance and a comparison test of Tukey means (p≤ 0.05). These data were analyzed with the statistical package SAS version 9.0 for Windows (^{SAS, 2002}).

Morphological characterization

The pathogens causing the wilting of chili corresponded with what was reported by ^{Rivera-Jimenez et al. (2018)}. Similarity was found with F. oxysporum, the growing colonies in PDA had abundant aerial mycelium and pale pink to deep purple to magenta with a cottony texture. The macro-conidia presented one to five septa, the conidia measured 23-54 × 3-4.5 μm, respectively. In the identification of Rhizoctonia solani, it corresponded with that reported by ^{Sneh et al. (1991)} and ^{Lozano et al. (2015)}, R. solani was characterized by forming aerial mycelium, brown, in PDA culture medium formed mycelium hyaline.

The hyphae generally showed a right angle and after this a septum formed, the diameters of the hyphae presented 4.1 to 8.9 μm, which corresponds to ^{Barnett and Hunter (1998)}. The isolation of Phytophthora capsici was characterized by forming ovoid, elongated, ellipsoid sporangia with one or two papillae, the size of the sporangia was 35.8 to 64.7 x 19.6 to 25.2 μm., finally the apical thickness was 1.85 to 4.3 μm (^{Erwin and Ribeiro, 1996}; ^{Gallegly and Hong, 2008}).

According to the taxonomic identification criteria of ^{Barnett and Hunter (1972)}, Trichoderma species were compared with taxonomic keys and identified by their morphological characteristics. The species of T. harzianum presented light-to-dark green colonies with a dusty-cottony texture, with mycelium, conidophore and green-looking conidia (Figure 2, Table 1) show various branches in pyramidal form, in some cases the formation of two to three lateral branches and presents aerial mycelium. In T. asperellum and T. viride, an absent coloration can be seen on the back of the petri dish.

Figure 2 Growth rate, A) T. harzianum, T. viride and T. asperellum; and B) P. capsici, R. solani and F. oxysporum. 

The spores and phyloids, characteristic structures correspond to Trichoderma species, presented forms and sizes typical of the species of T. harzianum, T. asperellum and T. viride corresponding to what was reported by different authors (^{Barnett et al., 1972}; ^{de Hoog, 2000}; ^{Romero-Arenas et al., 2009}; ^{Chaverri et al., 2015}; ^{Jaklitsch et al., 2015}).

Growth rate

The evaluation of the initial growth rate of the antagonists and pathogens turned out to be completely related at the beginning of mycelial growth at 8 h; however, the three species of Trichoderma have the ability to antagonize the completion of the filling of the petri dish in 80 h. These results indicate the importance of knowing the growth rate of pathogens, as well as the antagonists of T. harzianum, T. viride and T. asperellum.

The isolates of Trichoderma spp. showed a faster growth than the pathogens, this behavior is promising in the control of pathogens of the root, also allowed this trial allowed to consider it for the PICR tests and give Fusarium oxysporum, Rhizoctonia solani and Phytophthora capsici two days advantage.

Antagonistic activity of Trichoderma on pathogens

The antagonistic activity of T. viride against P. capsici showed a significant difference (p≤ 0.05), because T. viride presented 0.516 mm in comparison to P. capsici, which obtained 0.241 mm of growth in a petri dish with culture medium, is say T. viride is suitable for use as a biological control agent, as it is effective against P. capsici, since hyperparasitism was also observed by the pathogen (Figure 3). The foregoing is consistent with that reported by ^{Bouziane et al. (2016)}, when evaluating T. viride against P. infestans in in vitro tests, they found that the beneficial fungus showed inhibitory capacity between 58 and 68%, similar results were presented when evaluating in plants.

Figure 3 Dual growth, in vitro confrontations with Trichoderma against pathogens, A) Trichoderma viride vs. P. capsici; B) Trichoderma asperellum vs Fusarium oxysporum; and C) Trichoderma harzianum vs Rhizoctonia solani. 

Similarly, ^{Zegeye et al. (2011)}, found that T. viride showed a complete inhibition in the radial growth of P. infestans in in vitro tests. In addition, they mentioned that the foliar application of T. viride has a good potential to control P. infestans in greenhouse conditions. On the other hand, the evaluation of dual antagonism of T. viride against F. oxysporum showed growth of 0.656 mm of the antagonist and 0.221 mm by the pathogen, this evidences how T. viride is suitable for use in the control of F. oxysporum.

In the dual confrontation T. viride against R. solani obtained a growth of the 0.746 mm antagonist and the 0.112 mm pathogen, the results of growth by the antagonist were higher compared to the pathogen (Figure 3). In both cases similar results have been found where the two fungi are mycoparasited or destroyed by T. viride (Figure 3) since it has an anti-fungal effect with this pathogen (^{Jhon et al., 2010}; ^{Perveen, 2012}; ^{Sánchez-García et al., 2017}).

It is important to mention that in the first 8 h the growth of the antagonists showed an accelerated growth that facilitates the encounter with the pathogen in less time, indicative that the antagonist grew or covered more surface in culture in vitro.

Similar results mentioned several authors where they demonstrated that Trichoderma spp. and T. viride are used for the biological control of R. solani (^{Sánchez-García et al., 2017}). An antifungal effect was observed, in which it inhibits growth and causes lysis of this pathogen. In the confrontations of T. viride against the causative agents of the wilting of chili. The evaluation of the antagonism of T. harzianum against P. capsici, F. oxysporum and R. solani (Figure 4) showed greater growth by the antagonist compared to the pathogen, denoting hyperparasitism by completely invading the pathogen, a similar result was obtained by ^{Osorio-Hernández et al. (2011)} in which he observed that Tricoderma spp., is an effective antagonist for the control of P. capsici, inhibiting this pathogen up to 48%.

Figure 4 Mycelial growth (mm) in the biocontrol test of A) T. viride vs P. capsici, F. oxysporum and R. solani; B) T. asperellum. vs P. capsici, F. oxysporum and R. solani; and C) T. harzianum vs P. capsici, F. oxysporum and R. solani. 

Also, in the confrontation of T. viride against F. oxysporum and R. solani, the same tendency was found. A significant value (p≤ 0.05) was found in the susceptibility assessment based on the PICR of the three pathogens responsible for the wilting of chili; however, the highest susceptibility was presented by Fusarium oxysporum with 92.68%, followed by Rizoctonia solani and P. capsici (Table 2), results that prove the effectiveness of the antagonists (^{Osorio et al., 2016}) against pathogens that affect the root of the chilli from seedlings and plants in production (^{Romero et al., 2017}).

In the evaluation of the percentage of radial growth inhibition (PICR) of the pathogens causing the wilting of the chili, there were no significant differences between the antagonists T. asperellum, T. viride and T. harzianum; therefore, it is suggested to use any of the three Trichoderma as a biological control agent; that is, they are effective in inhibiting more than 85% of the pathogens causing the wilting of chili (Table 2), the response of inhibition of the growth of phytopathogens is due to the synthesis of secondary metabolites and to the different mechanisms of action of mycoparasitism (^{Harman, 2006}; ^{Infante, 2009}).

The similar inhibition behavior of the three treatments are favorable results found by those who report that above 80% inhibition is acceptable in the biocontrol of pathogens that are transmitted by the soil and by the efficacy of fighting root diseases (^{Hermosa et al., 2012}; ^{Sabbagh et al., 2017}).

The results of mycoparasitism of the antagonists frequently had a more favorable growth, it should be mentioned that the first 8 h were significant (p≤ 0.05) showing that the antagonist had an accelerated growth that favors that the encounter can be had in a short time. Likewise, it was observed that not only did it present the inhibition of the growth of these pathogens, but it also presented a total invasion (hyperparasitism) of the antagonist towards the pathogen even sporulating on the pathogen. In a study with selections of 25 Trichoderma strains including T. harzianum, of which they presented inhibition in F. oxysporum on mycelium growth in 33 and 35%.

Similarly, ^{Sinuco et al. (2017)} indicate that volatile organic compounds of T. viride affected the growth halos of Fusarium spp. The growth of T. asperellum showed significant differences (p≤ 0.05) when confronted with the different pathogens causing the wilting of chili caused by the pathogens of P. capsici, F. oxysporum and R. solani (p≤ 0.001) since at to analyze the data, it was observed that the growth rate of the pathogen was reduced by almost half when confronted with T. asperellum.

In addition, this antagonist overgrown up to 100% on the pathogens, covering the box completely and thus causing hyperparasitism, thus showing a very efficient antagonistic ability to each of the pathogens.

These results coincide with other authors who mentioned that Trichoderma ssp. overgrowth Phytophthora causing hyperparasitism (^{Bae et al., 2016}). Similarly, Trichoderma spp. it develops against R. solani, causing the inhibition of in vitro growth of R. solani from 58 to 86%. Trichoderma spp., has the ability to induce the expression of genes related to the defense of plants and produce a higher level of ergosterol, indicating its ability to grow at a higher rate in the soil, this explains its positive effects on the growth and defense of the plant in the presence of the pathogen (^{Mayo et al., 2015}).

Trichoderma isolates confronted with P. capsici exerted a highly significant antagonistic effect (p≤ 0.001), causing inhibition in oomycete growth under dual conditions. T. harzianum, T. viride and T. asperellum, also had a similar behavior against F. Oxysporum and R. solani (Figure 4). The antagonists continued to grow up to six days later, showing hyperparasitism by invading or growing completely on the pathogen. In this regard, ^{Segarra et al. (2013)}, mentioned that T. asperellum strain T34, is able to reduce P. capsici up to 71%, when applied to pepper plants at different stages of growth.

Mycoparasitism

In vitro tests the inhibition of the three species of Trichoderma was evaluated, the results showed the efficacy in competition for space and nutrients, in addition to appreciating the parasitic ability against Rhizoctonia solani, Fusarium oxysporum and P. capsici. In addition, was observed by light microscopy and corroborate in microcultures and double-confrontation cultures at the crossing point between the native isolates of Trichoderma spp. and pathogens causing the wilting of chili the different types of mycoparasitism (Figure 5).

Figure 5 Mycoparasitism of Trichoderma sp. A) T. harzianum surrounding the mycelium of Rhizoctonia solani; B) T. viride and T. asperellum surrounding the mycelium of P. capsici; and C) massive winding of T. asperellum covering Fusarium oxysporum. 

In some early investigations, myco-parasitism, antibiosis has been observed, in addition to Trichoderma having a growth behavior parallel to the pathogen until it is wound avoiding the different survival mechanisms of the pathogen (^{Chet et al., 1981}; ^{Harman, 2006}; ^{Infante, 2009}).