elocation-id: e3987
Diseases represent one of the primary causes of loss in agricultural production. Although chemicals are a common alternative for controlling them, they generate adverse effects on human health and the environment. Therefore, sustainable options are required, such as the use of soil microorganisms with biocontrol activity, such as Trichoderma, a fungus that has multiple mechanisms of action against phytopathogens, stimulates the soil microbiota, improves nutrient absorption, and activates plant defense mechanisms. This research aimed to evaluate the antagonism of eleven native Trichoderma species against Botrytis cinerea, Fusarium oxysporum, Rhizoctonia solani and Sclerotinia sclerotiorum using the percentage of radial growth inhibition, the percentage of growth area, the degree of antagonism, and the presence of mycoparasitism. Dual confrontation was performed under in vitro conditions in the phytopathology laboratory of the Faculty of Agricultural Sciences of the Autonomous University of the State of Mexico. The isolates Trichoderma atroviride TH3 and T. asperellum Th11 stood out for their inhibition capacity, reduction of pathogenic growth, high degree of antagonism, and mycoparasitism. The degree of antagonism was assessed with coverage of up to two-thirds of the growth area over the pathogen. Rhizoctonia solani showed the lowest percentage of growth area and was the main target of mycoparasitism. The results suggest that Trichoderma species have potential for use in sustainable agricultural practices.
antagonism, growth area, inhibition, mycoparasitism, pathogens, Trichoderma.
There is a serious risk of economic losses in agricultural production, as well as a major challenge to food security, due to the significant impact of pests, which cause global losses of between 17.2% and 21.5% in crops such as wheat, rice, corn, potatoes, and soybeans (Savary et al., 2019; Skendžić et al., 2021). The massive use of phytosanitary products in pest control has generated negative consequences, such as biodiversity loss, the development of resistance in pest organisms, and damage to human health (Vinchira-Villarraga and Moreno-Sarmiento, 2019).
In response, more sustainable alternatives have been sought, including the use of antagonistic microorganisms, which are considered a viable option to ensure the production of healthy food (Gutiérrez-Ramírez et al., 2013; Companioni et al., 2019; Vinchira-Villarraga and Moreno-Sarmiento, 2019). Microorganisms with biocontrol activity are characterized by rapid growth, high reproductive capacity, competitive efficiency, and environmental adaptation (Viera-Arroyo et al., 2020).
Currently, there is a large number of microorganisms with profitable applications in agriculture as control agents, with Trichoderma spp. standing out, a cosmopolitan fungus highly competitive for space and nutrients and that produces bioactive metabolites (Amerio et al., 2020; Andrade-Hoyos et al., 2023; Cortés-Hernández et al., 2023). More than 200 species of the genus Trichoderma have been described as biological control agents (Garrido et al., 2019; Sood et al., 2020; Allende-Molar et al., 2022). In Mexico, 42 species have been reported, with T. asperellum and T. viride being the most common (Allende-Molar et al., 2022).
Species such as T. asperellum, T. atroviride, T. hamatum, T. harzianum, T. longibrachiatum, T. koningii, and T. viride, among others, have shown antagonistic and mycoparasitic effects against soil phytopathogens such as Phytophthora capsici, Fusarium oxysporum, Macrophomina phaseolina, Rhizoctonia solani, Sclerotium rolfsii, and Phymatotrichopsis omnivora (Andrade-Hoyos et al., 2019; Camacho-Luna et al., 2021; Allende-Molar et al., 2022). Other species act on pathogens due to the presence of various secondary metabolites (Montes-Vergara et al., 2022).
In the biotechnological area, Trichoderma species are of interest for the production of enzymes, generously used in the food industry (Allende-Molar et al., 2022); in addition, other species are reported as bioinducers of plant growth (Hernández-Melchor et al., 2019; Matas-Baca et al., 2023) and as responsible for activating the systemic response in plants through the expression of genes such as Epl1 and Sm1 (Martínez-Canto et al., 2021).
Although there are numerous studies on the biological control of pathogens by Trichoderma spp., it is necessary to implement studies on autochthonous strains, adapted to local environmental conditions (Amerio et al., 2020). This research aimed to evaluate the antagonistic activity of eleven native Trichoderma species against the phytopathogens Botrytis cinerea, Fusarium oxysporum, Rhizoctonia solani and Sclerotinia sclerotiorum under in vitro conditions.
The fungal strains used in this study belong to the collection of the Phytopathology Laboratory of the Faculty of Agricultural Sciences of the Autonomous University of the State of Mexico. The pathogens used were: Botrytis cinerea (strawberry), Fusarium oxysporum (avocado), Rhizoctonia solani (potato), and Sclerotinia sclerotiorum (sunflower). The antagonist strains of Trichoderma spp. were collected in different regions of the State of Mexico in 2023, from rhizosphere samples of various agricultural crops, and molecularly identified in 2024 (in the process of publication).
The pathogen and antagonist strains were activated in PDA BD Difco™ medium (potato dextrose agar) for seven days at 25 °C, using the same medium for dual seeding. The confrontation assays consisted of antagonist-pathogen dual seeding, following the methodology of Dennis and Webster (1971). To do this, a 7 mm mycelial disc of the pathogen was placed on the inner margin of the Petri dish containing the culture medium, and the antagonist was seeded at the opposite end.
The control consisted of seeding the pathogen alone at the margin of the Petri dish. Petri dishes were incubated at 25 °C in darkness for 10 days, until the mycelia made contact and the control fully colonized the Petri dish. The mean values of the percentage of radial growth inhibition (PRGI) of the mycelium were calculated according to the formula: PRGI= [(R1 - R2) / R1] × 100 (Ezziyyani et al., 2004). Where: PRGI= percentage of radial growth inhibition of mycelium (mm); R1= mycelial growth of the pathogen (control); R2= mycelial growth of the pathogen in confrontation with the antagonist.
The experiment was conducted under a completely randomized design with three replications per pathogen in dual seeding with eleven Trichoderma species.
The percentage of the pathogen’s growth area due to the antagonist’s effect in dual seeding was estimated using the AutoCAD® technological application, for use in engineering and architecture (Khoroshko, 2020). This software allows areas to be estimated, using dynamic commands, which is helpful with circular or irregular shapes, as in the case of fungal growth.
The degree of Trichoderma antagonism was determined using the scale by Bell et al. (1982), which describes the antagonist’s colonization capacity on the surface of the culture medium in dual seeding with the pathogen, with five levels ranging from the antagonist’s supremacy to its absence.
Semipermanent preparations stained with lactophenol blue (Corrales et al., 2020) were used from dual cultures of 10 days of age. These samples were observed under the optical microscope at 40X and 100X magnifications. Ten fields from each sample were examined to determine the presence of mycoparasitism, following the methodology described by Andrade-Hoyos et al. (2019), with modifications. Mycoparasitism was considered when coiling of the antagonist’s hyphae and deformation or fragmentation of the pathogen’s mycelium were observed.
The results of PRGI and GAP were transformed using the arcsine function, followed by an analysis of variance (Anova) and a multiple comparison of means with Tukey’s test (p ≤ 0.05), under a completely randomized design with three replications. The statistical analysis was performed using InfoStat version 2020.
The percentage of radial growth inhibition (PRGI) evaluated in dual seeding with the eleven native species of Trichoderma and the phytopathogens Botrytis cinerea, Fusarium oxysporum, Rhizoctonia solani, and Sclerotinia sclerotiorum showed inhibition of mycelial growth with significant differences (p ≤ 0.05) relative to the control (Table 1).
The isolates that showed the highest inhibitory activity against pathogens were: T. atroviride Th3 (57.46%), T. scalesiae Th9 (57.31%), and T. atroviride Th1 (57.22%). As for the pathogens most affected in their mycelial development by the effect of the antagonist, they were B. cinerea (59.32%), F. oxysporum (55.88%), and R. solani (52.67%); in contrast, the lowest level of control was observed in S. sclerotiorum (47.41%).
According to Matas-Baca et al. (2023), the use of native species with natural antagonistic activity is highly efficient in controlling various phytopathogens, and their use is recognized in sustainable agriculture plans (Companioni et al., 2019; Cortés-Hernández et al., 2023). The Trichoderma species considered in this study showed biocontrol activity against soil phytopathogens, as indicated by Yao et al. (2023), with T. viride and T. harzianum limiting the growth of 29 species of phytopathogenic fungi, among which Botrytis cinerea, Fusarium oxysporum, Rhizoctonia solani, and Sclerotinia sclerotiorum stand out.
The inhibition of pathogens by Trichoderma isolates varies with the native strain and the phytopathogen species used. In this study, the response of the PRGI of T. atroviride Th3 on the pathogen Rhizoctonia solani was 57.51%, exceeding that reported by Pérez et al. (2020) for this same species. Similarly, Geng et al. (2022) report a significant inhibition of 68% and a rapid development of T. harzianum on B. cinerea in dual culture in vitro, a percentage comparable to those of the autochthonous isolates T. scalesiae Th9 (65.02%) and T. atroviride Th2 (64.98%) (Figure 1).
A) B. cinerea in confrontation with T. scalesiae; B) F. oxysporum in confrontation with T. atroviride; C) R. solani in confrontation with T. asperellum; D) S. sclerotiorum in confrontation with T. scalesiae. E) Trichoderma atroviride coiling around Rhizoctonia solani; F) Trichoderma atroviride invades the mycelium of Rhizoctonia solani.
On the other hand, Silva et al. (2022) report a 12.2% inhibition of S. sclerotiorum growth when using T. lentiforme; in contrast, T. hamatum Th6 and T. atroviride Th10 quadrupled this percentage, reaching PRGI values of 51.45% and 51.19%, respectively. As for F. oxysporum, Sallam et al. (2019) report high PRGI using T. atroviride as an antagonist, a result that agrees with the findings of this study. The analysis of variance indicated a significant effect of treatments with different native Trichoderma species on the PRGI of the different pathogens, with a coefficient of determination R2= 0.98. Likewise, Tukey’s multiple comparisons test (p ≤ 0.05) identified significant differences among the isolates, grouping them into different levels of antifungal effectiveness.
Using AutoCAD® software for the percentage of growth area (GAP) of phytopathogenic fungi and their respective antagonists allowed us to measure colony area (cm2) more efficiently than with manual methods (Khoroshko, 2020). The pathogen with the lowest GAP was Rhizoctonia solani (9.54%), and the highest was Fusarium oxysporum (16.95%), showing significant differences between isolates (Tukey, p ≤ 0.05), as described in (Table 2).
The need to estimate the percentage of fungal growth area in inhibition studies is confirmed by Camacho-Luna et al. (2021), who evaluated the action of Trichoderma sp. on the percentage of growth area of Fusarium oxysporum and F. proliferatum, reaching values of 20.4% and 33.3%, respectively. In contrast, the results obtained in this study showed GAPs ranging from 10.9% to 12.9%. These results highlight the use of computational tools to estimate areas in biological phenomena, allowing precise and quantifiable data to be obtained.
The Trichoderma attributes recognized in antagonist success include its ability to compete for space and nutrients, rapid development, and mycoparasite ability (Martínez-Martínez, 2020). In general, the results showed that the native species of Trichoderma reached degree 2 on the pathogens evaluated, which represents the colonization of two-thirds of the culture medium, thereby limiting the growth of phytopathogens. This behavior was particularly evident against Botrytis cinerea and Fusarium oxysporum.
In contrast, the strains Trichoderma viridarium TH4 and Trichoderma viride TH5 showed low antagonistic activity against Rhizoctonia solani, presenting a degree 3, which indicated competition between both organisms; on the other hand, for Sclerotinia sclerotiorum, degrees 3 and 4 were observed, respectively, reflecting a limited effectiveness of the antagonist. However, Rodríguez and Flores (2018) reported values of 1 to 3 using T. harzianum against Rhizoctonia solani.
The genus Trichoderma has been recognized with mycoparasitism activity, defined as the ability to act on the host cell wall (Matas-Baca et al., 2023). In this study, it was possible to document the type of mycoparasitism carried out by autochthonous strains of Trichoderma on the pathogen Rhizoctonia solani, because it presents a thick mycelium and dark coloration that contrasted with the antagonist. The Trichoderma species that showed mycelium coiled around the pathogen were Trichoderma atroviride Th2 and Trichoderma viride Th5; those with mycelium invading the pathogen internally were T. atroviride Th3, Trichoderma scalesiae Th9, and Trichoderma asperellum Th11; and the strains that fragmented the mycelium of the pathogen were T. atroviride Th7 and T. atroviride Th10 (Figure 1).
It has been documented that, after successful penetration, Trichoderma is able to degrade the cell wall of its host by producing enzymes on different pathogen species; it can even be located in the lumen of the fungus once it has penetrated its interior (Sood et al., 2020). Tyskiewicz et al. (2022) recognize the action of Trichoderma spp. by degrading the cell wall of fungi. In this sense, Fernández (2022) reports parasitic initiation with the activation of recognition genes, followed by coiling and, finally, the disintegration of hyphae.
The potential of native species of Trichoderma spp. as biocontrol agents against the phytopathogens Botrytis cinerea, Fusarium oxysporum, Rhizoctonia solani, and Sclerotinia sclerotiorum is confirmed, given their PRGI, GAP, DA, and mycoparasitism characteristics, with the isolates Trichoderma atroviride Th3 and Trichoderma asperellum Th11 standing out. The mycoparasitic activity of Trichoderma was outstanding with Rhizoctonia solani, including mycelium coiling, penetration into the pathogen, and degradation of its cell wall.
Finally, this study contributed to the knowledge about the use of native Trichoderma spp. in the control of pathogens in in vitro cultures, providing valuable information for their future applications in disease management programs.
The research received support from the Comecyt Fund, Edomex-Ficdtem-2022-01: funding for research by women scientists. Project: FICDTEM-2023-115.
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