Trichoderma virens is the most effective antagonist for Colletotrichum species in the dual-culture assay
In the dual-culture assay (Fig. 1), there was a significant interaction of the antagonism of the Trichoderma species on Colletotrichum. The inhibitory effect was variable and determined by the intrinsic characteristics of each interaction, varying from 7.5–52.3% (Table 2). All Trichoderma species significantly inhibited the mycelial growth of Colletotrichum in relation to the control, evidencing its efficacy in the in vitro control of Colletotrichum species isolated from pecan. The antagonism of Trichoderma virens stood out, inhibiting the mycelial growth of the largest number of isolated Colletotrichum species (Table 2).
Table 2
Percentage of inhibition of Colletotrichum in paired-culture test with Trichoderma after seven days of culture
Pathogen | Control | T. harzianum Ecotric® | T. koningiopsis IIOT1 | T. asperellum QualityWG® | T. tomentosum T2s | T. virens TF1 | Mean |
Paired-culture test |
C. karstii - FAR2 | 0.0 aC | 7.5 cB | 21.5 bA | 15.8 cA | 20.7 cA | 14.3 cA | 15.9 |
C. nymphaeae - SAJ2 | 0.0 aD | 20.0 bB | 11.8 bC | 27.8 bA | 29.8 cA | 35.8 bA | 25.0 |
C. kahawae - PIB1 | 0.0 aC | 14.7 bB | 17.8 bB | 33.6 bA | 27.7 cA | 33.5 bA | 25.4 |
C. gloeosporioides - CZA | 0.0 aC | 27.9 aB | 20.4 bB | 30.1 bB | 35.4 bA | 40.8 bA | 30.9 |
C. fioriniae - CDL | 0.0 aD | 32.6 aB | 24.9 bC | 36.3 bB | 40.0 bB | 52.3 aA | 37.2 |
C. siamense - ADM3 | 0.0 aC | 34.3 aB | 43.9 aA | 46.3 aA | 48.0 aA | 47.2 aA | 43.9 |
Mean | 0.0 | 22.8 | 23.4 | 31.7 | 33.6 | 37.3 | |
Inhibitory effect (%) of volatile metabolites |
C. karstii - FAR2 | 0.0 aC | 26.7 aB | 34.3 aB | 26.8 aB | 19.9 aB | 41.0 aA | 29.7 |
C. nymphaeae - SAJ2 | 0.0 aC | 15.4 bA | 9.3 bB | 21.7 aA | 22.6 aA | 29.4 bA | 19.7 |
C. kahawae - PIB1 | 0.0 aB | 20.3 bA | 21.7 bA | 30.4 aA | 21.1 aA | 18.5 bA | 22.4 |
C. gloeosporioides - CZA | 0.0 aB | 26.4 aA | 35.7 aA | 25.4 aA | 36.0 aA | 27.6 bA | 30.2 |
C. fioriniae - CDL | 0.0 aC | 14.8 bB | 19.1 bB | 23.3 aB | 28.7 aA | 31.9 bA | 23.6 |
C. siamense - ADM3 | 0.0 aB | 27.1 aA | 26.0 aA | 21.0 aA | 24.6 aA | 29.7 bA | 25.7 |
Mean | 0.0 | 21.8 | 24.4 | 24.7 | 25.5 | 29.7 | |
* Means followed by the same lowercase letter do not differ in the column and means followed by the same uppercase letter do not differ in the row by the Scott-Knott test at 5% probability of error. |
Trichoderma koningiopsis, T. asperellum, T. tomentosum, and T. virens promoted from 14.3–48.0% of inhibition for C. karstii and C. siamense. For C. nymphaeae and C. kahawae, the highest control was in the presence of T. asperellum, T. tomentosum, and T. virens (27.7–35.8%). For C. gloeosporioides the highest inhibition occurred with T. tomentosum and T. virens (35.4% and 40.8%, respectively), while the highest percentage of inhibition was observed for C. fioriniae (52.3%) by the antagonist T. virens (Table 2).
Inhibitory effects ranging from 9.5–61.2% were demonstrated by Trichoderma species (T. harzianum, T. virens, T. harzianum, T. asperellum, and T. koningiopsis) in C. nymphaeae, C. gloeosporioides, and C. siamense, the causal agents of anthracnose in strawberries, pepper, and mountain guava, respectively (Karimi et al. 2017; De La Cruz-Quiroz et al. 2018; Fantinel et al. 2018).
As an effect of the antagonism, there was a tendency towards a decrease in the speed of mycelial growth of Colletotrichum from the third day of evaluation (Fig. 2). In the control group, such a decrease was not observed, and constant growth was maintained until the seventh day of evaluation. Such growth interruption on the third day of direct confrontation with Trichoderma was also observed for Fusarium sambucinum, Rhizopus stolonifera, and C. lindemutianum (Maciel et al. 2014; Bomfim et al. 2010; Christmann et al. 2019).
Based on the scores of the antagonism scale employed, the best results were found for T. virens, which presented mean scores lower than 2.0 in all confrontations (Table 3), demonstrating a growth throughout the Petri dish, exerting strong antagonism against the pathogen. Trichoderma tomentosum also presented mean scores below 2.0, except for the confrontation with C. fioriniae. Trichoderma asperellum presented intermediate and variable scores depending on the interaction (1.6 to 2.8), while T. harzianum and T. konigiopsis, had the lowest overall performance, with scores ranging from 2.0 to 4.0 (Table 3).
Table 3
Classification of Trichoderma species regarding the antagonism to Colletotrichum. The given score is related to the growth of pathogens and antagonists in the Petry dish. Antagonist grows throughout the Petri dish and onto the pathogen disc = 1; Antagonist grows throughout the Petri dish not overlapping the pathogen disc = 2; Antagonist grows over 3/4 of the plate = 3; Antagonist grows over 2/3 of the plate = 4; Antagonist and pathogen grow to the half of the plate = 5; Pathogen grows over 2/3 of the plate = 6; and Pathogen grows throughout the Petri dish = 7.
Pathogen | Control | T. harzianum | T. koningiopsis | T. asperellum | T. tomentosum | T. virens | Mean |
C. karstii | 6.0 aD | 3.6 bC | 2.0 aA | 2.8 bB | 1.6 aA | 1.4 aA | 2.3 |
C. nymphaeae | 6.0 aD | 3.0 aB | 3.8 bC | 1.6 aA | 1.6 aA | 1.6 aA | 2.3 |
C. kahawae | 6.0 aC | 2.8 aB | 2.8 aB | 2.2 bA | 1.8 aA | 1.8 aA | 2.3 |
C. gloeosporioides | 6.0 aD | 3.0 aB | 4.0 bC | 1.4 aA | 1.6 aA | 2.0 aA | 2.4 |
C. fioriniae | 6.0 aC | 2.8 aB | 2.4 aB | 2.6 bB | 2.6 bB | 1.4 aA | 2.4 |
C. siamense | 6.0 aD | 3.8 bC | 2.8 aB | 2.0 aA | 2.0 aA | 2.0 aA | 2.5 |
Mean | 6.0 | 3.2 | 3.0 | 2.1 | 1.9 | 1.7 | |
Means followed by the same lowercase letter do not differ in the column and means followed by the same uppercase letter do not differ in the row by the Scott-Knott test at 5% probability of error. CV: 8.7% |
The inhibitory capacity of many Trichoderma species on fungal pathogens in in vitro dual-culture assays is promoted by different mechanisms that act individually or together, such as competition for space and nutrients, parasitism, and antibiosis by the production of volatile and non-volatile secondary metabolites. The competition mechanism is dependent on the rate of growth of the biocontrol agent in relation to the pathogen, when both are associated in a site, so that, when there is rapid colonization of the site by the biocontrol agent, it prevents the establishment and development of pathogenic agents by unavailability of free area and nutrients (Medeiros et al. 2018). As for the mechanism of parasitism, the biocontrol agent parasitizes the structures of the pathogen by penetrating and colonizing the hyphae, interfering with its development, while antibiosis consists of the production of secondary metabolites by the biocontroller with direct action in reducing the growth of the pathogen (Medeiros et al. 2018).
Trichoderma species can produce more than 40 different secondary metabolites that can contribute to their antagonistic action, including gliotoxin, viridine, harzianic acid, trichodermine, suzucacillin, alamethicin, dermadine, pyrones, daucanas, siderophores, among others (Dennis and Webster 1971; Ribas et al. 2014; Reino et al. 2008). The mycoparasitism of Trichoderma is also accompanied by the production of chitinases, cellulases, and proteases, lytic enzymes that degrade the host cell wall (Markovich and Kononova 2003).
The reduction or blockage of growth and sporulation, reduction in spore germination, hyphal distortions, and endolysis are effects caused by secondary metabolites of the antagonist on phytopathogens (Bomfim et al. 2010). In the present study, some of these effects were observed during the confrontation process, such as the formation of an inhibition halo between antagonists and pathogens (indication of antibiosis, suppression of mycelial growth of the pathogen by non-volatile metabolites; Fig. 3A), degradation of the pathogen's hyphae (Fig. 3B), and coiling of the pathogen's hyphae by the antagonist (mycoparasitism, which works when the organisms are in contact; Fig. 3C).
Volatile metabolites from Trichoderma inhibit the mycelial growth of Colletotrichum
In the antibiosis test by volatile metabolites, all tested Trichoderma species provided a significant inhibitory effect against the different Colletotrichum species (Table 2 and Fig. 4).
The antagonist T. tomentosum provided an inhibition index of up to 41.0% when confronted with C. karstii and, for the other pathogens, the percentage of inhibition varied from 18.5–31.9%. Trichoderma virens showed slightly lower inhibition rates, ranging from 19.9–36.6%. The antagonists T. koningiopsis, T. harzianum, and T. asperellum also inhibited the mycelial growth of the pathogens, in smaller percentages (Table 2 and Fig. 4).
Analogously to this study, several other tests have observed the antifungal effect of volatile metabolites from Trichoderma. An in vitro mycelial growth inhibition of C. nymphaeae of up to 6.25% was observed using T. asperellum as an antagonist agent (Karimi et al. 2017). Joshi and Misra (2013) observed an inhibition rate of up to 14.7% for C. falcatum using different species of Trichoderma, while Guimarães et al. (2016) observed a 50% reduction in the mycelial growth of Cladosporium herbarum when confronted with T. harzianum.
All strains representing the species of Trichoderma evaluated in this study showed potential for the in vitro control of Colletotrichum. The tests aimed to select isolates of Trichoderma capable of exerting an antagonistic action against the etiological agents of anthracnose in pecan. However, tests in a greenhouse and in the field have also to be performed to confirm the antagonism since some conditions of temperature and humidity can interfere with the development of both the biocontrol agent and the pathogen (Bell et al. 1982).
The available literature emphasizes that fungi of the genus Trichoderma have wide possibilities for application in agriculture, such as the biocontrol of pathogens in seeds (Junges et al. 2016), the control of post-harvest diseases in fruits (Moreira et al. 2002), the biocontrol of root phytopathogens in soil (Louzada et al. 2009), the biocontrol of pathogens from the aerial part of plants (Martins et al. 2007), for biomolecular production (Gómez-García et al. 2019) as well as for promoting growth and inducing resistance in plants (Silva 2012). Here we show the antagonistic potential of five different Trichoderma species against the etiological agents of anthracnose in pecan trees. Trichoderma virens and T. tomentosum were the species with the greatest in vitro antagonistic effect against Colletotrichum, but in vivo tests are recommended to confirm the effectiveness of the biocontrol of the anthracnose in pecan. It is worth noting that biocontrol tends to be more efficient when inserted within the integrated management of diseases, where the various techniques converge to promote plant health and suppress pathogens.