Biological Control of Fusarium oxysporum in Tomato Seedling Production with Mexican Strains of Trichoderma Biological Control of Fusarium oxysporum in Tomato Seedling Production with Mexican Strains of Trichoderma

The problems and limitations of the control of diseases caused by phytopathogens through the use of fungicides, make the biological control present as an alternative method in the production of tomato plants in greenhouse, which is limited by the incidence of Fusarium oxysporum Schlechtend.:Fr., being the most worldwide destructive disease. The objective of the present investigation was to evaluate the effect of three Mexican strains of the genus Trichoderma against F. oxysporum on the production of tomato seedlings under greenhouse conditions, as well as to determine the antagonistic effect of the strains used. The Trichoderma harzianum strain had the highest antagonistic activity (81.50%) and the highest growth rate (1.25 cm/day), proving to be the most aggressive strain to control F. oxysporum . in addition the results of the interaction of the dual cultures paired, presented a visible overgrowth zone with hyphae of Trichoderma spp. Seeds inoculated with T. harzianum showed a survival of 84% and a mortality of 16%, lower than the control group, which present a mortality of 58%; however, the treatment inoculated with F. oxysporum had the highest incidence of “disease” with 83%, the lowest survival (17%) and a decrease of the green biomass with respect to the control.


Introduction
The need to reduce the use of fungicides in phytosanitary control makes it necessary to develop technologies that allow easy, economical and effective ways to obtain products from endogenous microorganisms with sufficient quality and quantity to their application in the crops areas [1]. In the soil there are microorganisms with antagonistic capacity, the most studied in the world is Trichoderma spp [2]; due to its ubiquity, its ability to isolate and present rapid growth on a large number of substrates [3,4].
In the last 10 years, research work has been carried out, and native species of Trichoderma spp. have been isolated, selected and evaluated, with the potential to establish a biological control against different diseases, which have proposed several mechanisms of innovation for the implementation of this fungus with satisfactory results [5][6][7]. These mechanisms of action may act synergistically on various phytopathogens such as Septoria triticii in wheat, Sclerotinia sclerotiorum in soybean and lettuce, Rhizoctonia solani in soybean, Sclerotium rolfsii in cucumber cohombro, Fusarium oxysporum in tomato and Pythium splendens in beans [8,9].
The genus Trichoderma presents several mechanisms by which they easily move to the phytopathogen, but the most important is based on three types: (a) Direct competition for space or nutrients [10][11][12][13], (b) Production of antibiotic metabolites, whether of a volatile or nonvolatile nature [14,15] and (c) Direct parasitism of some species of Trichoderma spp [16,17].
The fungus F. oxysporum Schlechtend.: Fr. cause root and neck rot in tomato plants (Lycopersicon esculentum M.), causing severe losses that affect the quality and quantity of the production [18]. The most noticeable symptoms produced by using F. oxysporum occur in the transplantation of seedlings and at the beginning of flowering [18]. If a transverse section of the stem is made, it is possible to observe a vascular necrosis of brown color, particularly on the smaller lateral roots; which accelerates foliage wilting; after the plant dies and the fungus fructifies on the surface of the stem, under conditions of a humid environment [19]. The vascular wilt of the tomato by F. oxysporum was first described by Masse in 1885, on the Isle of Wight and Guernsey, located in the English Channel. In the year 1899, the disease was already in the United States of America, causing severe losses in the areas dedicated to growing tomato in the north of the state of Florida. In 1940 they reported that the disease was disseminated throughout the world and F. oxysporum was given greater importance [20].
The tomato (L. esculentum M.), is grown in all types of soils for family and commercial use [21]; for the 2016 year, occupied the first place with a total area planted of 4734 million hectares with a production of 163 million tons [22]. To date China is the first producer with 50 million tons, followed by India with 18 million tons, the United States with 12 million tons and Mexico is in the tenth position with 3282 million tons [23]. In Mexico the statistics of the Sistema de Información Agropecuaria [24], reported that in the 2016 year, 52,374 thousand hectares of tomato were planted with a production of 2875.164 tons, with a value of 15,735 million of pesos. While data from the Sistema Producto, they indicated that exports amounted to 20 billion pesos, with the United States and Canada being the main buyers; where the main producers were Sinaloa with 867,832.04 tons, San Luis Potosí with 196,011.25 tons and Michoacán with 169,768.98 tons [25]. Tomato production under greenhouse conditions during 2016 represented 26.2% of the national production, with average yields of 171.82 tons/ ha, where Puebla ranked 14th with 75,219.09 tons of tomato [24].
In Mexico, few investigations have been carried out for the biological management of phytopathogens with soil origin through the use of native strains of Trichoderma spp [26]. Biocontrol of phytopathogenic fungi and biofertilization using the genus Trichoderma is a method used in various crops in different parts of the world; however, the use of commercial strains presents difficulties with their persistence in the soil, due to factors such as the genetics of the isolates, the environmental conditions and others characteristic of phytopathogenic species [27]. For the aftermentioned, the objective of this research was to characterize three native Trichoderma strains from the municipality of Tetela de Ocampo, Puebla-Mexico and to evaluate its antagonistic effect on the incidence of root and neck rot in tomatoes caused by F. oxysporum in the production of tomato seedlings in greenhouse.

Strains
Strains native from the state of Puebla-Mexico were used, Th-T4 (3) from Trichoderma harzianum, Tav-T7 (2) from Trichoderma atroviridae, Tv-T3 (1) from Trichoderma viridae and the strain (Fo-A) from Fusarium oxysporum, which belong to the Centro de Recursos Genéticos del Centro de Agroecología del Instituto de Ciencias-BUAP and are in culture medium PDA (Potato Dextrose Agar).

Rate of development and speed of growth
The rate of development and rate growth of Th-T4 (3), Tav-T7 (2), TV-T3 (1) and (Fo-A) strains, was determined in Petri dishes (4.5 cm in diameter) in culture medium (PDA), incubated at room temperature for 7 days; the growth rate was measured every 24 h until the culmination of the total colonization of the strains, the macroscopic morphological characteristics of the colonies were recorded in texture, density, aerial mycelium and color. The rate of development and growth rate were determined using the following formula [28]:

Antagonistic activity of the strains of Trichoderma spp. on F. oxysporum in vitro
To evaluate the antagonistic activity of Trichoderma spp., Cherif and Benhamou technique was used [29]. For each of the treatments that were performed in Petri dishes with PDA (Agar, Dextrose and Potato) culture medium, was place at one end of the Petri dish a 5 mm diameter agar disc with mycelium of the pathogenic fungus, in this case F. oxysporum, due to its slow growth was allowed to develop for 2 days and then another 5 mm disc with mycelium of Trichoderma spp. was inoculated at the opposite end, (natives) at a distance of approximately 5 cm between them [30,31]. The controls consisted of mycelium of the pathogens and the antagonist, separately cultured in Petri dishes with PDA medium described above. Petri dishes were incubated at a temperature of 26°C. To measure the inhibitory effect of antagonistic fungus to the pathogen, measures colony diameter was recorded every 24 h; until standoff and formation of an inhibition zone between the colonies was formed. Ten repetitions were considered for each comparison, in this case will be three strains of Trichoderma spp., a single strain of F. oxysporum, evaluating a total of 30 experimental units. The percentage growth inhibition are calculated using the formula given by [32]: where: PICR: Percent inhibition of radial growth.
Additionally, the strain was compared respect to the antagonistic capacity according to the scale proposed by Bell [33] in 1982 ( Table 1).

Greenhouse antagonism tests
Seeds of tomato (L. esculentum Mill) var. Ramses were used, these were sterilized in 20% (v/v) sodium hypochlorite solution for 20 min, followed by three 5-minute washes in sterile distilled water. Subsequently the sowing was carried out in disinfected trays of 27 × 17 × 4 cm, containing 60 g of sterilized vermiculite in an autoclave at 121°C for 1 h. In each tray 100 tomato seeds were sown. Three strains of Trichoderma spp. were selected in the laboratory for their antagonistic response [Th-T4 (3), Tav-T7 (2), Tv-T3 (1)] plus two commercial strains (Perkins-C21 and Tricovel-25) and a control inoculated with F. oxysporum without Trichoderma spp. plus an absolute control; for a total of seven treatments, where each tray with 100 tomato seeds represented an experimental unit. The inoculations were carried out at the time of planting the seeds in the trays with 1 mL of a suspension of F. oxysporum at a concentration of 5 × 10 6 conidia mL −1 per well. The trays were installed in a culture chamber at 25 ± 2°C, were irrigated with 80 mL of distilled water per tray every 2 days. After 15 days, 1 mL of the five strains of Trichoderma spp. was applied at a total concentration of 83 × 10 4 conidia with a viability percentage of 96%.

1
Overgrowth of Trichoderma that colonized the entire medium surface and reduced colony pathogen.

2
Overgrowth Trichoderma colonized at least 2/3 of the medium surface. 3 Trichoderma and pathogen colonized medium to medium (more than 1/3 and less than 2/3).

4
Pathogenic fungus colonized at least 2/3 of the medium surface and resist invasion by Trichoderma.

5
Overgrowth of the pathogenic fungus that colonized the entire surface of the medium. The variables evaluated were incidence and severity of disease at 30 days after sowing, both for the radical part and for the aerial part ( Table 2) using the scale proposed by Amaro-Leal [34]. The percentage of mortality and survival of tomato seedlings at 45 days, as well as height, stem thickness and total dry biomass were evaluated.
The results obtained were subjected to an analysis of variance (ANOVA) and test of separation of means by Tukey (p < 0.05), sing the SPSS version 17 (Statistical Package for Social Sciences) to determine differences between treatments.

Growth rate and rate of development
The Tv-T3 (1) and Th-T4 (3) strains of Trichoderma spp. presented a cottony texture with abundant density and abundant mycelium, a dark green and white coloration, whereas the Tav-T7 (2) strain presented a regular density, regular mycelium and a green/yellow coloration. For the F. oxysporum strain in this case (Fo-A) presented a velvety texture with a regular density and mycelium of pink white color, as described by Guzman [35], in addition its mycelium is formed by septate hyphae and the conidiophores present clusters of macroconidia where chlamydospores are observed.
T. harzianum presented the highest growth rate with a mean of 1.25 cm/day, followed by T. viridae with 0.75 cm/day, with the T. atroviridae strain being the lowest growth rate with 0.64 cm/day. For F. oxysporum the growth was 0.83 mm/day, results similar to those found by Amaro-Leal [36], with a speed between 70 and 73 mm/day in PDA, results similar to those of the present investigation.

Confrontation of Trichoderma spp. on F. oxysporum in vitro
The results of the percentage of inhibition of Trichoderma spp. strains on F. oxysporum by the dual culture method are shown in Figure 1, the Mexican strains of Trichoderma spp. inhibited the growth of the pathogenic fungus, where they presented a percentage of inhibition of radial significant growth (PIRG) [p = 0.056] at the Fo-A strain of F. oxysporum, with T. harzianum which showed higher antagonistic activity, with an average value of 81.50% (PIRG), followed   [37]. The values obtained from inhibition are higher than those obtained by Michel [38], who at evaluating the antagonistic effect of native Trichoderma spp., on mycelial growth and reproductive potential of F. oxysporum and Fusarium subglutinans, presented inhibition of 47.6 and 73.0%, respectively. Snyder and Hansen [39], reported a percentage inhibition of 77.8% for F. oxysporum, when compared with Trichoderma viridae isolates, results lower than those reported in the present investigation.
The results of the interaction of the most representative paired cultures are presented in Figure 2. The F. oxysporum Fo-A strain was given 2 days advantage because of its slow growth compared to Trichoderma; the days after the first contact between hyphae, the behavior was determined, which was very heterogeneous and highly significant (P = 0.0001). Most of the Trichoderma isolates showed a visible overgrowth zone with the hyphae of F. oxysporum; the greater the area of overgrowth, the greater the aggressiveness of the antagonistic fungus [29].
In this sense, Michel [36], reported antagonism 1, 2 and 3 of F. subglutinans and F. oxysporum, similar results in the present investigation.

Greenhouse antagonism tests
The treatment inoculated with F. oxysporum showed symptoms of the disease in the root and aerial part ( Table 4), presenting the highest values in incidence and severity, this in comparison with the other treatments evaluated in this study. These results coincide with that observed by Kim [40], who point out the damage caused by Phytophthora sp., at the root and crown of the stem of chile plants under greenhouse conditions, similar results in this research.
In the present investigation, the lowest incidence and severity was obtained in the treatment based on T. harzianum with 6%, presenting slight dry circular lesions in the root and without symptomatology in the aerial part. T. harzianum has the ability to produce enzymes such as cellulases, β-1,3-glucanase and chitinases, which degrade the cell wall of phytopathogens [41].
Treatments based on T. harzianum, T. atroviridae and T. viridae. used in this research work, present antagonistic efficacy against F. oxysporum with a survival ranging from 62.7 to 76.4% in comparison to the control treatment (Figure 3), which had a survival rate of 46%; while For the variables height, stem thickness and dry biomass of each treatment, showed significant differences (p = 0.043) among the strains of Trichoderma spp.; being the treatments based on Mexican strains Trichoderma spp. those that presented better results in the evaluated    Table 5). Romo [42], mention that Trichoderma spp. has antifungal properties, thanks to the production of substances such as: trichodermine, dermadina, sequisterpeno, suzukacillina, alamethicina, trichotoxina, acetaldehyde, as well as extracellular enzymes such as β-1,3 glucanase, chitinase and cellulase that degrade The host cell walls and allow the penetration of the antagonist's hyphae, reducing its propagation in the root.  Harman [43], argues that T. harzianum stimulates the growth of plants by producing metabolites that promote developmental processes, which allow greater root development and absorbent hairs, which favors the mobilization of nutrients in the soil, thus improving nutrition and water absorption; also accelerates the decomposition of organic matter and minerals [44].
The native strains of Trichoderma spp. presented higher biomass (green), emphasizing the treatment based on T. harzianum, which showed a root height of 10.58 cm and a green biomass of 0.97 g; denoting a significant increase in comparison with the control " Figure 4", which showed an average root height of 4.53 cm, and a green biomass of 0.19 g, while the treatment inoculated with the F. oxysporum strain Fo-A, presented the lowest root height averages, with 3.70 cm and 0.18 g in green biomass.

Conclusions
Plants affected by F. oxysporum reduce their growth due to the pathogen's ability to colonize roots, which prevents proper nutrition of the seedling and leads to death, causing losses in the producer in the first stage of tomato crop.
The evaluation of Mexican strains of Trichoderma spp. and its antagonistic effect on F. oxysporum on tomato seedlings (L. esculentum Mill) was determinant to verify their potential for biological control in a crop of great importance in the economy and the country's food.
The T. harzianum strain presented the highest growth rate with a mean of 1.25 cm/day, proving to be the most aggressive strain to control F. oxysporum with a development rate of 3.80 mm/day.
The three native strains of Trichoderma spp. present inhibition of radial growth on the Fo-A (F. oxysporum) strain, with T. harzianum being the most antagonistic (81.50%), in addition, the results of the interaction of the paired dual cultures to F. oxysporum, was very heterogeneous and highly significant statistic differences, where the Trichoderma isolates showed a zone of visible overgrowth with the F. oxysporum hyphae, which shows more aggressiveness on the part of the antagonistic fungus.
The efficacy shown by the native strains of Trichoderma spp. evaluated in this study against F. oxysporum, applied to tomato seedlings (Lycopersicon esculentum Mill), showed that T. harzianum obtained higher height, greater stem thickness and greater production of dry biomass, likewise, the treatment inoculated with F. oxysporum obtained the highest incidence (83%) and the lowest survival (17%) of germination in greenhouse conditions.