In vitro Antagonism of strains of Trichoderma spp., on Pathogenic Fungi of Nopal Vegetable

In vitro antagonistic effect of different strains of 4 Trichoderma sp. namely T. harzianum, T. viride and T. atroviride and T. hamatum were evaluated against pathogenic fungi isolated from Nopal vegetable crops in San Sebastian Villanueva region Puebla, Mexico. The pathogenic fungi were Pythium sp., Fusarium sp. and Colletotrichum sp. Present investigation revealed T. harzianum inhibited maximum 73.7 and 68% radial growth of Fusarium sp. and on Colletotrichum sp. Trichoderma hamatum inhibited maximum 71.6 and 73.8% radial growth of Colletotrichum sp. and Pythium sp., respectively.

its use are due to mechanisms of action, such as competition, mycoparasitism, antibiosis and the production of volatile compounds that reduce the infection of causal agents in plants 11 . Trichoderma species have a high reproductive capacity and ecological plasticity coupled with the presence of microbial inhibitors, which give it great antagonistic activity 12 . The Nopal is a vegetable that is mainly consumed fresh and it is necessary to obtain healthy and innocuous products, which is why an etiological determination is essential for good crop management. Consequently, the objective of this work was to determine the antagonistic capacity and the percentage of inhibition of the radial growth in vitro of 4 strains of Trichoderma spp., on fungal isolates of the Nopal vegetable crop in San Sebastian Villanueva, Acatzingo, Puebla-Mexico.

Isolation zone
The samples were collected in the months of May-June 2019, from commercial crops of prickly pear cactus on an area of 1,000 m 2 with 5-year-old plants belonging to the community of San Sebastian Villanueva, municipality of Acatzingo Puebla-Mexico, located between the parallels 19°07'94' 'north latitude and 97°70'93'' west longitude at a height of 2,400 masl. 16 transects of 10 x 6 m were made on cladodes presenting symptoms of fungal diseases; of which 0.5 cm 2 tissue were cut and placed in humid chambers then transfer to the laboratory. The samples with symptoms of fungal attack (phytopathogens) were disinfected with 1.5% sodium hypochlorite. These samples were washed three times with sterile distilled water, dried with sterile paper, 100 µL of dilution 10 6 were plated with antibiotic PDA agar (gentamicin 2 mL/Lt), pH 5.5 and incubated at 26 ± 2°C for 7 days. Once the fungal colonies had grown, they were purified by tip hyphae 13 .

Morphological characterization
The generic identification of purified fungi from tissue was carried out by comparing the morphological characteristics of the colony and the type of structures with taxonomic identification keys 14 , in a microculture system using an optical microscope (Carl Zeiss, Jena, Germany) at 1,000x magnifications. The colonies of the different fungi characterized in PDA plates (Agar-Papa-Dextrose, Bioxon ™) were used to evaluate growth and development rate using the formula TD = VCF-VCI / Number of days; measuring the diameter in mm of the colonies every 24 hours for 10 days 9 . Evaluation of the antagonism of 4 strains of Trichoderma spp., against phytopathogenic fungi The evaluation of the antagonism of 4 different Trichoderma strains with 3 repeats each, of which the T. hamatum strain SAH3, was used as a control and the variables to measure where the development rate and the antagonistic capacity of the treatments on the isolated phytopathogenic fungi. The dual culture technique 8 was used, whereby, for each treatment, a 5 mm diameter disk with active mycelium of 8-day old fungal colonies of selected pathogenic fungus were deposited in one end of Petri dishes with PDA and allowed to develop for 3 days, for its slow growth. Subsequently, at the other end of the box, were deposited disks of 5 mm discs of the involved strains of Trichoderma spp., incubating at 25°C with a photoperiod of 12 h and 40% relative humidity. Mushroom growth readings were taken every 24 h with a Vernier; to determine the number of days at the first contact between the hyphae of the two fungi, the intersection zone after planting Trichoderma spp., with these data the "Percentage of inhibition of radial growth" was determined, applying the formula (PICR). To complement the evidence of antagonism, each trial was compared and classified in one of the 5 levels of the Bell scale 15 (Table 1).
Where: R1: diameter of the control (average of the radial growth of the triplicates of each strain). R2: diameter of the tested organism.

Biological material
The evaluation of the antagonism was made with the TH-T4 strain of Trichoderma harzianum, TH-T3 Trichoderma viride, SAH3 Trichoderma hamatum, and TH-T1 of Trichoderma atroviride belonging to the Genetic Resources Center of the Agroecology Center of the Benemerita Universidad Autonoma de Puebla (BUAP).

Statistical analysis
Data were analyzed with ANOVA (twoway) in the IBM SPSS Statistics version 25 statistical package. Growth rate, development rate, and PICR were the response variables with three replications. The experiment was repeated twice for validation. A comparison of means was made by the Tukey-Kramer method with a probability level of p≤0.05.

Characterization of phytopathogens from the Nopal vegetable crop
The pathogenic fungi were from infected plant tissues of the Nopal vegetable crop, from the municipality of Acatzingo Puebla-Mexico. The pathogenic fungi were Colletotrichum sp. (M7), Fusarium sp. (M4) and Pythium sp. (M6).
Among the main diseases caused by phytopathogenic fungi in the cultivation of prickly pear vegetables in the community of San Sebastian Villanueva, black spot is of greater importance, due to its high incidence during the production cycle as well as being favored by the presence of moisture and damage mechanical, as reported by Mendez-Gallegos 16 , who mentioned that the presence of C. gloesporoides is associated with black spot in an advanced stage of the disease. The isolation of the phytopathogen was made in cladodes that had sunken black lesions, in a kind of concentric rings that developed in a limited area, these symptoms are currently described as anthracnose 17 . The M7 strain presented radial colonies of gray color with a cottony appearance, septate mycelium, conidiomata, conidiophores and setae formed directly on hyphae. Conidia hyaline, smooth-walled, aseptate, the apex rounded, the base rounded to truncate, their size varies between 16-18 µ long and 3-4 µ wide 18 (Fig. 1A). The morphological characteristics corresponding to Colletotrichum sp., Found in this work, are similar to those reported by Montero 17 . De la Torre 23 quotes Colletotrichum sp. as a cause of anthracnose in the genus Opuntia. Quezada 4 report as a saprophyte, taking advantage of the conditions set by Pseudocercospora sp. to develop in diseased tissue; However, in the isolates carried out in this work, this fungus was not found, being C.
gloesporoides the one with the highest incidence in the cladode isolates that presented the black spot. Montero 17 , have reported that Fusarium sp. Along with the aforesaid fungi, it acts as a causal agent of this disease, causing stains and rots in the cladode tissue, contrasting with what was found in this research. Likewise, Villa 19 reported Fusarium oxysporum causing root rot in most agricultural crops, initially penetrating the root asymptomatically and later colonizing vascular tissue and triggering massive wilt, necrosis and chlorosis of the aerial parts.
The M4 strain was characterized as a species belonging to the genus Fusarium. Fungal colonies developed abundant aerial mycelium, with a cottony texture and a white-pink color, and  stained the agar with shades between purple and violet, aerial and abundant, fusiform macronidia with transverse concepts and ellipsoidal and piriform microconidia 20 (Fig. 1B), can form masses (simulate heads) but never chains. In addition to macroconidia with the typical sickle or moon shape and have from one to five septa 21 .
The species of the genus Pythium are particularly destructive to the root system of some agricultural crops 18 , they produce a disease known as drowning and wilting (Damping off) in a large number of seedlings, and it also causes rotting of fruits and stems 22 , in addition, it is associated with fungi of the genus Phytophthora and Fusarium 18 . Pythium sp., it was identified in this work, which is reported by De la Torre 23 , as a cause of rots in Opuntia sp. According to its macroscopic and microscopic characteristics, two different species were found of this pathogen, and it is convenient to carry out a molecular identification.
The M6 strain was characterized as a species belonging to the Pythium genus. It presented thin whitish hyphae with a tree-like upward growth, white-black mycelium, aerial and abundant. Oogonia equinulate and reticulatedwalled (Fig. 1C); asexual reproduction structures that are represented by zoospores that produce intercalary zoosporangia.

Phytopathogen development rate
According to the analysis of variance, a significant difference is found between the growth rate of the different fungi, for which one isolate represented a higher development rate compared to the rest (Fig. 2). Of the isolated phytopathogenic fungi, Colletotrichum sp., was the one that presented a higher rate of development with an average of 51 mm/day in PDA culture medium, a value that is similar to that quoted by Rojas 24 in his research, where C. gloesporoides under a range of 25-30°C it grew between 44 and 53 mm/day. This result showed to be high compared to that reported by Morales 25 , where C. gloesporoides under conditions similar to those of this experiment had a maximum growth of 24 mm/day, similar to that reported by Montero 17 , who mentions a growth rate between 24 and 31 mm/day. However, Akhter 26 reports a radial growth of 63 mm/day, being greater than what was found in this investigation.
For Fusarium sp. the growth was 48 mm/day, which differs from that reported by other authors, where Fusarium sp. it presents between 70 and 73 mm/day in PDA 27 , unlike what was mentioned by Neagu and Borda 28 , where in barley and rice media it grows up to 98 and 99 mm/day. For sample M6 (Pythium sp) 40 mm/ day, representing the lowest growth values of the total isolated fungi. Colletotrichum sp., grows faster compared to the other isolated fungi, which shows its greater aggressiveness and this can also explain the higher frequency of isolated colonies as well as the highest incidence in the field.

Antagonistic capacity of strains of Trichoderma spp. on nopal vegetable culture phytopathogens
In the present investigation, the Trichoderma species used showed various degrees of inhibition on the isolated fungi. The criterion for selecting the best strains was considering the highest percentage of inhibition, which varied from 8 to 73.8% (Fig. 3), so that the strains corresponding to T. harzianum and SAH3 (T. hamatum) which served as control, are those that showed the best results according to the Tukey test (p <0.05). Inhibition of radial growth in dual culture is attributed to substances released by one or both organisms or by competition 29 .  The parasitic action exerted by one fungus on another is called hyperparasitism, this is because some fungi produce cellulases and antibiotics and can metabolize chitin; Tapwal 29 report than T. harzianum, T. viride and T. malignorum, as biological control agents on phytopathogenic fungi such as R. solani, Pythium sp., Fusarium spp., Alternaria spp., C. gloesporoides, Rhizopus arrhizus, Rhizovtonia endophytica and other pathogenic fungi that reduce agricultural production.
The SAH3 strain had the highest inhibition values with 73.8 and 71.6% over the Pythium sp. T. harzianum stopped the growth of Fusarium sp., by 73.7% and Colletotrichum sp., by 68.9%. The strain with the lowest inhibition value was T. atroviride on Colletotrichum sp., with 8%.
Some authors cite T. harzianum and T. viride with potential for the control of fungi of the genera Pythium and Fusarium in various agricultural crops 30,31 , in the case of the present work, the Trichoderma strains exerted a percentage of inhibition greater than 50% for Pythium and Fusarium, except for T. hamatum, whose value was lower.
The values of T. harzianum, T. viride and T. atroviride with the exception of T. hamatum had values above 50% on Fusarium sp., this is similar to that reported by Sundaramoorthy and Balabaskar 32 , who mention T. harzianum with potential for the biocontrol of F. oxysporum in tomato, by inhibiting it's in vitro growth by 53%. The results of this investigation are high compared to that reported by Sierra 33 for F. subglutinans, where the highest percentages are in correspondence with T. atroviride with 48%.
According to the classification of Bell 15 antagonisms at 7 days of observation, the isolates were located between classes I, II and III. Most of them remained under scale I were Trichoderma inhibited the total growth of the phytopathogens, such as T. harzianum case. The strain of T. atroviride had the lowest percentage of inhibition on Colletotrichum sp., Remaining on scale III, where both strains were developed in 50% of the culture media (Fig. 4).
Rodriguez and Veneros 34 pointed out that T. harzianum showed class 2 antagonistic capacity according to the Bell scale on isolated fungi of C. gloesporoides, Stemphylium lycopersici, F. oxysporum, A. alternata, indicating that the antagonist invaded ½ of the surface of the colony of the pathogenic fungus, similar results in the present investigation. Reyes 35 reported that one of the significant characteristics of the genus Trichoderma is its high growth rate, which gives it the possibility of being a good competitor for space and nutrients, fundamentally, in vitro conditions against pathogens that cause plant diseases.

CONClUsiON
Three pathogens were identified in the nopal vegetable crop, being Colletotrichum sp. the most representative in San Sebastian Villanueva, Puebla, followed to a lesser extent by Fusarium sp. and Pythium sp.
The strains of the species T. harzianum and T. hamatum showed the highest percentage of inhibition (70%) which represents a potential in biological control of the phytopathogens found in the community of San Sebastian Villanueva, municipality of Acatzingo Puebla-Mexico.
The analyzes of antagonism tests through in vitro bioassays, constitute an important parameter to know the competitive capacity of the species in relation to the phytopathogenic fungi and to establish a selection strategy for their control.