The capacity as a biological control agent is due to multiple mechanisms, such as competition for nutrients, for space and myco-parasitism. While the fungus of the Trichoderma genus has several advantages as a Biological Control Agent; it takes nutrients from the fungi that it degrades and from the organic matter helping its decomposition. Therefore, the objective of this work was to determine through microcultures the mycoparasitic capacity of native strains of Trichoderma spp. against Verticillum dahliae, etiological agent of olive verticillium wilt. The action and type of parasitism of three native strains of Trichoderma against the phytopathogen V. dahliae was determined using the microculture technique. A trial with a completely randomized design with a 3x2x2 factorial arrangement (3 antagonists x 2 pH levels x 2 lighting levels) was performed. The capacity of each native strain of Trichoderma to exert different types of parasitism under the established conditions was determined. This being considered of great interest since the biocontrol of the phytopathogen is evidenced through physical contact with the native strain of Trichoderma. Therefore, it is of great importance to continue the antagonism studies of different strains of Trichoderma to determine the efficiency of the control of V. dahliae.

Keywords: microcultures, olive Verticillium wilt, pH, photoperiod

The determination of Biological Control Agents requires carrying out specific antagonism studies on the various phytopathogens.¹

Trichoderma is the genus of a free-living, facultative aerobic, plant symbiont, avirulent fungus that belongs to the Hypocraceae family and has more than 30 species, all with beneficial effects for agriculture² and is characterized by not present a certain sexual state.^3,4 Trichoderma reproduces asexually with mycelium and an abundant amount of green conidia (or spores), formed from the naked cells of its fruiting body, it is fast growing and has extensive enzyme production.⁵ The conidia are ovoid in shape, formed from highly branched and septate conidiophores. It also has resistance structures called chlamydospores formed on the mycelium with a thick and rough cell wall. The species most used for Biological Control are T. atroviride; T. harmatum; T. asperellum and T. harzianum.⁶

This genus can act as a biocontrol agent in an effective way, being low-cost in production, does not cause environmental damage and does not harm useful organisms that contribute to the balance of the environment.^7,8 According to studies carried out, Trichoderma was a fungus with efficiency to reduce phytopathogens that cause infections, such as those that cause vascular diseases or foliar diseases in plants.^5,9 These characteristics mean that the correct choice of a promising strain of Trichoderma in accordance with the desirable qualities that it presents, generates benefits in the modes of action and ecological aptitudes, these being specific to the species or strain to develop its full potential in a successful commercial product.^3,5 Its capacity as a biocontroller is due to the multiple mechanisms it possesses it generates competition for nutrients, space and myco-parasitism, ^3,8,10-13 among others. In turn, Trichoderma has several advantages as a Biological Control Agent; it takes nutrients from fungi (which it degrades) and from organic matter helping its decomposition, for which the incorporation of organic matter and composting favor it.²

The antagonistic action mechanism of Trichoderma was described in the 1970s by Weindling,¹⁴ currently indirect and direct actions are known, the latter regulate the development of phytopathogenic fungi^15-17 such as: mycoparasitism, antibiosis, competition (nutrient and space), being for Guedez et al.¹⁸ mycoparasitism the main mechanism of action of these fungi and the best known.¹⁰ The antagonists can present more than one way of acting; this is of great interest at the time of choosing the Biological Control Agent,^16,19 in this way the resistance of the strains.²⁰ So, the mechanisms depend on each strain of Trichoderma and the environmental conditions. Among them is Mycoparasitism, which is the direct impact of Trichoderma on another species of fungus. There are currently 75 species of Hypocrea/Trichoderma known to have this ability. The processes described below occur sequentially and continuously:^8,15,21,22

Chemotrophic growth: It is the direct growth towards a chemical stimulus. In the host localization stage, Trichoderma can detect the phytopathogen from a distance and its hyphae grow towards it.

Recognition: Recognition is carried out through lectin-carbohydrate interactions, finding carbohydrates present in the cell wall of Trichoderma, while lectins are present in phytopathogens. Then the process continues with the development of hyphae and appressoria. According to research, these are effective only against specific phytopathogens, so molecular recognition between Trichoderma and the host (phytopathogen) is essential for antagonism.

Adhesion and coiling: After recognition, the Trichoderma hyphae adhere to those of the host through hyphae and appressoria that coil around the host, the adherence of the Trichoderma hyphae occurs through the carbohydrate-lectin association.

Lytic activity: In this stage there is production of extracellular lytic enzymes (chitinase, cellulase, glucanase and proteases), which degrade the host cell wall and resistance structures that allow the penetration of the antagonist's hyphae, as well as facilitate the insertion of specialized structures (hyphae, haustoria) for the absorption of nutrients from the interior of the phytopathogen. Finally, mycoparasitism ends with the loss of the cytoplasmic content of the host cell. The remaining cytoplasm is found surrounding the invading hyphae, with symptoms of disintegration, retraction of the plasma membrane and disorganization of the cytoplasm.^12,15,16,18

Some of the investigations show the action of Trichoderma against Rhizoctonia solani, Alternaria alternata; Sclerotinia sclerotiorum, Fusarium spp., Botrytis cinerea, Pythium spp. and Ustilago maydis where the deterioration of phytopathogens is confirmed.⁸ In studies through microscopic observations, they highlight that it is not always feasible to visualize these interactions, since it depends on the Trichoderma isolate and the phytopathogenic agent in question.¹⁶ Therefore, the objective of this work was to determine through microcultures the mycoparasitic capacity of native strains of Trichoderma spp. against V. dahliae, etiological agent of olive verticillium wilt.

The action and type of parasitism of three native strains of Trichoderma against the phytopathogen V. dahliae was determined using the microculture technique. A trial with a completely randomized design was carried out with a 3x2x2 factorial arrangement, the first factor "antagonists" with three strains of Trichoderma, a second factor "pH" with two pH levels (6.5 and 4.5) and the third “photoperiod” factor with two lighting levels (8 and 16 h of light).

The microcultures were carried out following the technique of Martins Corders and de Melo.²³ Petri dishes of 15 cm in diameter were used as a humid chamber, in which sterile slides were conditioned and on them a drop of sterile ADP culture medium with the established pH was placed, on which the phytopathogen contained in a 5 mm disc was sown. of Ø of ADP in active growth, a coverslip was placed and after 48 h of incubation, the strains with antagonistic potential were planted in front: Trichoderma asperellum (VL1 and PaM3) and T. hamatum (M5A), contained in a 5 ADP disc. mm of Ø in active growth. Subsequently, it was incubated under controlled conditions with a T° of 25 ± 1°C and under the determined lighting conditions. Daily evaluations were carried out to determine the type of parasitism and the mycoparasitic characteristics they presented, for which cotton blue was added, and it was observed with an optical microscope with a 40x objective.

The results obtained are presented in Table 1. In the treatment with T. hamatum (Vert + M5A) it was observed that in a photoperiod of 8 h light with pH 6.5 both strains presented fruiting (Figure 1), while with the same pH and at 16 h light in the combined treatment with T. asperellum (Vert + VL1) only fruiting was observed in the phytopathogen. Meanwhile, with a photoperiod of 8 h light and pH of 4.5, the treatment with the phytopathogen and the strain with T. asperellum, (Vert + PaM3), also presented fruiting. The action exerted by the M5A strain on V. dahliae is observed in Figure 2.

Figure 1 Whorls and conidia of V. dahliae under conditions of 8 h light and pH 6.5.

Figure 2 Strain M5A: A, coiling; B, mycoparasitism, emission of haustoria.

The similar results obtained by Alonso Bahena²⁴ with V. dahliae were observed in the two strains of T. asperellum (VL1 and PaM3) under the evaluated conditions, where he also observed recognition, adhesion, parasitic symbiosis, and penetration with haustoria. Also, Rajani et al.²⁵ found results like those of this study. This coincides with what was stated by García Velasco et al.,²⁶ and according to what was stated by Zin, and Badaluddin,²⁷ the antagonist recognizes the phytopathogen, binds to the hyphae by appressoria and subsequently degrades the cell wall by secreting different enzymes. While other authors^26,28,29 in studies carried out only found rolling. This could be due to the lack of specificity of the Trichoderma strains evaluated against the phytopathogen in question, that it is not in the optimal incubation conditions to express its antagonistic potential, or another possibility that it presents a good antagonistic behavior through other properties. not expressed through these results as the metabolites, both volatile and diffusible.

The ability of each native strain of Trichoderma under study to exert different types of parasitism under the established conditions was verified. This being considered of great interest since the biocontrol of the phytopathogen is evidenced through physical contact with the native strain of Trichoderma. Therefore, it is of great importance to continue the antagonism studies of different strains of Trichoderma to determine the efficiency of the control of V. dahliae.

None.

Authors declare there are no conflicts of interest.

1. González Basso V, Di Barbaro G, Felicetti J, et al. Biological control, an important tool for sustainable agricultura. J Appl Biotechnol Bioeng. 2022;9(5):176‒180.
2. Valdes Rios EL. Main characters, advantages and agricultural benefits provided by the use of Trichoderma as biological control. Agroecosystems Magazine. 2014;2(1):254–264.
3. Moreno Velandia CA, Cotes AM, Beltrán Acosta C, et al. Biological control of soil phytopathogens. In: Santos Díaz AM et al. Ed. Biological control of phytopathogens, insects and mites. Vol. I: Biological Control Agents. Editor: Alba Marina Cotes. 2018;144–221.
4. Juan C Sanchez–Hernandez J. del Pino N, Capowiez Y, et al. Soil enzyme dynamics in chlorpyrifos–treated soils under the influence of earthworms. Sci Total Environ. 2018;612:1407–1416.
5. Martínez B, Infante D, Reyes Y. 2013a. Trichoderma spp. and its role in pest control in crops. Veg Protection. 28(1):1–11.
6. López Mondejar R. Detection and quantification of Trichoderma harzianum and detection of its biocontrol activity against fusarium vascular blight of melon through the application of molecular tools. PhD thesis. Alicante. University of Alicante. 2011.
7. Michel Aceves AC, Otero Sánchez MA, Martínez Rojero RD, et al. Mass production of Trichoderma harzianum Rifai in different organic substrates. Chapingo Magazine Horticulture Series. 2008;14(2):185–191.
8. Sood M, Kapoor D, Kumar V, et al. Trichoderma: The “secrets” of a multitalented biocontrol agent. Plants(Basel). 2020;9(6):762.
9. Guerrero R. Selection and effectiveness of the use of isolates of Trichoderma spp. for the control of Bacterial Canker (Clavibacter michiganensis subsp. michiganensis) of tomato (Lycopersicum esculentum Mill.) Master's thesis. The Silver. National University of La Plata. 2016.
10. Howell CR. Mechanisms Employed by Trichoderma Species in the Biological Control of Plant Diseases: The History and Evolution of Current Concepts. Plant Dis. 2003;87(1):4–10.
11. Benítez T, Rincón AM, Limón MC, et al. Biocontrol mechanisms of Trichoderma strains. Int Microbiol. 2004;7:249–260.
12. Harman GE, Howell CR, Viterbo A, et al. Trichoderma species opportunistic, avirulent plant symbionts. Nat Rev Microbiol. 2004;2(1):43–56.  
13. López Escudero FJ, Mercado Blanco J. Verticillium wilt of olive: a case study to implement an integrated strategy to control a soil borne pathogen. Plant Soil. 2011;344:1–50.
14. Weindling R. Trichoderma lignorum as a parasite of other soil fungi. Phytopath. 1932;22(10):837–845.
15. Infante D, Martínez B, González N, et al. Mechanisms of action of Trichoderma against phytopathogenic fungi. Rev Protection Veg. 2009;24(1):14–21.
16. Infante Martínez D. Morpho–Physiological Pathogenic, Biochemical and Molecular Characterization of Trichoderma sp. Doctoral Thesis. National Center for Agricultural Health Havana, Cuba. 2014.
17. Morán Díez ME. Isolation, Characterization and Analysis of the Thpg1 Gene from Trichoderma harzianum, PhD thesis. Hispanic–Portuguese Center for Agricultural Research Dept. of Microbiology and Genetics – University of Salamanca. 2008.
18. Guédez C, Cañizález L, Castillo C, et al. Antagonistic effect of Trichoderma harzianum on some post–harvest pathogenic fungi of strawberries (Fragaria spp.). Rev Soc Ven Microbiol. 2009;29(1):34–38.
19. Martínez B, Pérez J, Infante D, et al. Antagonism of isolates of Trichoderma spp. against Didymella bryoniae (Fuckel) Rehm. Rev Protection Veg. 2013;28(3):192–198.
20. Vero S. Biocontrol mechanisms. In: Mondino P, Vero S. Biological Control of pathogens in plants. Editor: Uriarte G. 2006;49–74.
21. Hoyos Caravajal L, Chaparro P, Abramsky M, et al. Evaluation of isolates of Trichoderma spp. against Rhizoctonia solani and Sclerotium rolfti under in vitro and greenhouse conditions. Colombian Agronomy. 2008;26(3):451–458.
22. Erazo JG, Palacios SA, Pastor N, et al. Biocontrol mechanisms of Trichoderma harzianum ITEM 3636 against peanut brown root rot caused by Fusarium solani RC 386. Biological Control. 2021;104774.
23. Martins Corder MP, de Melo I S. In vitro antagonism of Trichoderma spp. to Verticillium dahliae Kleb. Scientia Agricola. 1998;55(1).
24. Alonso Bahena A. 2020. In vitro antagonistic activity of native strains of Trichoderma spp. against Rosellinia necatrix, Verticillium dahliae and Botrytis cinerea, pathogens of the rose crop. Thesis. Tenancingo, State of Mexico University Center UAEM Tenancingo. Autonomous Mexico State University.
25. Rajani P, Rajasekaran C, Vasanthakumari MM, et al. Inhibition of plant pathogenic fungi by endophytic Trichoderma spp. through mycoparasitism and volatile organic compounds. Microbiol Res. 2021;242:126595.
26. García Velasco R, Alonso Bahena A, Domínguez Arizmendi G, et al. Antagonistic effect of native strains of Trichoderma spp. against the phytopathogenic fungus Rosellinia necatrix in Mexico. Tropical Agronomy. 2021;71:e4605221.
27. Zin NA, Badaluddin NA. Biological functions of Trichoderma spp. for agriculture applications. Annals of Agricultural Sciences. 2020;65(2):168–178.
28. Guédez C, Cañizalez L, Castillo C, et al. In vitro evaluation of Trichoderma harzianum isolates for the control of Rhizoctonia solani, Sclerotium rolfsii and Fusarium oxysporum in tomato plants. Journal of the Venezuelan Society of Microbiology. 2012;32:44–49.
29. Maza M, Allori Stazzonelli E, Yasem de Romero MG. In vitro evaluation of native Trichoderma isolates as biocontrol agents and early growth promotion in soybean. Revta Agron No Argent. 2012;32(1–2):55–62.