Application of Trichoderma asperellum in apple trees as a growth regulator and antagonist for the control of Alternaria sp.


  • Melissa MADRID-MOLINA Universidad Autónoma de Chihuahua, Facultad de Ciencias Agrotecnológicas, Campus 1. C.P. 31200. Correo postal 24, Chihuahua, Chihuahua (MX)
  • Sandra PÉREZ-ÁLVAREZ Universidad Autónoma de Chihuahua, Facultad de Ciencias Agrícolas y Forestales, Km 2.5 carretera a Rosales, Campus Delicias, C. P. 33000, Delicias, Chihuahua (MX)
  • César M. ESCOBEDO-BONILLA Instituto Politécnico Nacional-CIIDIR Unidad Sinaloa, Juan de Dios Bátiz Paredes No. 250, CP 81101 Guasave, Sinaloa (MX)
  • Crescencio URÍAS-GARCÍA Universidad Vizcaya de las Américas, Calz. Del Charro 301, Col. Centro, Campus Delicias, CD. Delicias Chihuahua, CP 33000 (MX)



Alternaria tenuissima, antagonsim, biological control, chemical control, growth regulation, Malus domestica


Chihuahua state is the main apple producer in Mexico. The present study aimed to evaluate the effectiveness of Trichoderma asperellum as a plant growth regulator and antagonist against Alternaria sp. Three treatments were used: T1 = control, T2 = 200 mL of T. asperellum per tree; T3 = 100 mL of T. asperellum per tree. Agronomic variables were evaluated including number of leaves, shoots and flowers, disease incidence, and trunk thickness. Alternaria sp. was isolated from apple leaves at the experimental site in Guerrero County, Chihuahua, Mexico, and it was grown on solid PDA medium for morphological characterization. The molecular characterization was done by PCR using primers ITS1 and ITS4 producing products of 700 bp which were sequenced, submitted to GenBank (acc. no. OQ344593) and used for further phylogenetic analysis through Bayesian inference approach. Three clades were identified and the polytome topography recovered from clade 2 indicates a high genetic similarity with A. tenuissima (100% similarity according to BLAST). The analysis of T. asperellum as growth regulator only showed significant differences in trunk thickness and displayed higher values with T3 (p≤0.05). The presence of A. tenuissima was only observed in the control, which indicated the ability of Trichoderma to control the fungus. In this study T. asperellum was not an efficient plant growth regulator, but it was a good antagonist, and hence it can be recommended to control A. tenuissima. This is the first record of A. tenuissima in apple trees in Mexico. These results indicate that T. asperellum showed no benefit as plant growth regulator when applied to apple trees of the ‘Granny Smith’ variety.


Akaike H (1973). Information theory and an extension of the maximum likelihood principle. In: Petrov BN, Csaki F (Eds). Second international symposium on information theory. Budapest, Hungary.

Andrzejak R, Janowska B, Renska B, Kosiada T (2022). Effect of Trichoderma spp. and fertilization on the flowering of Begonia × tuberhybrida Voss. ‘Picotee Sunburst’. Agronomy 11:1278.

Benavides ME, Fernandez VP, Pose G (2019). Alternaria species associated with apple and pear crops in the region of Alto Valle del Río Negro, Argentina. Cultura Científica 17:18-31.

Berlian FS, Sutarman I, Rahma IN (2021). The effect of Trichoderma and onion extract on the success of grafting in mango seedlings. Earth and Environmental Science 819: 1-7.

Cabrefiga J, Salomon MV, Vilardell P (2023). Improvement of Alternaria leaf blotch and fruit spot of apple control through the management of primary inoculum. Microorganisms 11:101.

Cai W-J, Ye T-T, Wang Q, Cai B-D, Feng Y-Q (2016). A rapid approach to investigate spatiotemporal distribution of phytohormones in rice. Plant Methods 12:47.

Camacho-Luna V, Flores-Moctezuma HE, Rodríguez-Monroy M, Montes-Belmont R, Sepúlveda-Jiménez G (2021). Inducción de la respuesta de defensa de plantas de cebolla en la interacción con Trichoderma asperellum y Alternaria porri [Induction of the defense response of onion plants in the interaction with Trichoderma asperellum and Alternaria porri]. Revista Mexicana de Ciencias Agrícolas 12(4): 685-698.

Castedo-Dorado F, Crecente-Campo F, Alvarez-Alvarez P, Barrio-Anta M (2009). Development of a stand density management diagram for radiata pine stands including assessment of stand stability. Forestry 82:1-16.

Chagas LFB, Castro HG, Colonia BSO, Carvalho-Filho MR, Miller LO, Chagas-Junior AF (2016). Efficiency of the inoculation of Trichoderma asperellum UFT-201 in cowpea production components under growth conditions in field. Revista de Ciências Agrárias, 39(3):413-421.

Chang YCh, Chang YCh, Baker R, Kleifeld O, Chet I (1986). Increased growth of plants in the presence of the biological control agent Trichoderma harzianum. Plant Disease 70:145-148.

Cooley DR, Autio WR (2011). Summer pruning of apple: impacts on disease management. Advances in Horticultural Science 25:199-204.

Darriba D, Taboada GL, Doallo R, Posada D (2012). jModelTest 2: more models, new heuristics and parallel computing. Nature Methods 9(8):772.

Díaz G, Rodríguez G, Montana L, Miranda T, Basso C, Arcia M (2020). Efecto de la aplicación de bioestimulantes y Trichoderma sobre el crecimiento en plántulas de maracuyá (Passiflora edulis Sims) en vivero. Bioagro 32(3):195-204.

Duarte-Leal Y, Lamz-Piedra A, Martínez-Coca B (2017). Antagonismo in vitro de aislamientos de Trichoderma asperellum Samuels, Lieckfeldt y Nirenberg frente a Sclerotium rolfsii Sacc [In vitro antagonism of Trichoderma asperellum Samuels, Lieckfeldt and Nirenberg isolates against Sclerotium rolfsii Sacc]. Revista de Protección Vegetal 32(3):1-11.

Esmail SM, Draz IS, Saleem MH. Hamzah MS, Mumtaz S, Mohamed ME (2022). Penicillium simplicissimum and Trichoderma asperellum counteract the challenge of Puccinia striiformis f. sp. tritici in wheat plants. Egyptian Journal of Biological Pest Control 32:116.

Filajdic N, Sutton TB (1992). Influence of temperature and wetness duration on infection of apple leaves and virulence of different isolates of Alternaria mali. Phytopathology 82:1279-1283.

Garganese F, Schena L, Siciliano I, Prigigallo MI, Spadaro D, De Grassi A, … Sanzani SM (2016). Characterization of citrus-associated Alternaria species in Mediterranean areas. PLoS One 16:e0163255.

Halifu S, Deng X, Song X, Song R (2019). Effects of two Trichoderma strains on plant growth, rhizosphere soil nutrients and fungalcommunity of Pinus sylvestris var. mongolica annual seedlings. Forests 10:758.

Hall TA (1999). BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT (ver. 2007). Nucleic Acids Symposium Series 41:95-98.

Hao D, Lang B, Wang Y, Wang X, Liu T, Chen J (2022). Designing synthetic consortia of Trichoderma strains that improve antagonistic activities against pathogens and cucumber seedling growth. Microbial Cell Factories 21(2022): 234.

Hastings WK (1970). Monte Carlo sampling methods using Markov chains and their applications. Biometria 57:97-109.

Hermosa R, Viterbo A, Chet I, Monte E (2012). Plant-beneficial effects of Trichoderma and of its genes. Microbiology 158:17-25.

IBM Corp (2017). IBM SPSS Statistics para Windows. In: (Versión 25.0) IBM Corp.

Iftikhar S, Anwar W, Akhter A, Ali S, Azhar H, Khurshid M, Saleem HM (2021). Genetic analysis and pathogenic characterization of Alternaria tenuissima induced fruit rot of bitter gourd. Biodiversitas 22(2):615-623.

Ikram M, Ali N, Jan G, Iqbal A, Hamayun M, Jan FG, Hussain A, Lee IJ (2019). Trichoderma reesei improved the nutrition status of wheat crop under salt stress. Journal of Plant Interactions 14(1):590-602.

Jaroszuk-Ściseł J, Tyśkiewicz R, Nowak A, Ozimek E, Majewska M, Hanaka A, … Janusz G (2019). Phyto hormones (auxin, gibberellin) and ACC deaminase in vitro synthesized by the mycoparasitic Trichoderma DEMTkZ3A0 strain and changes in the level of auxin and plant resistance markers in wheat seedlings inoculated with this strain conidia. International Journal of Molecular Sciences 20:4923.

Jasnić SM, Marjanović ZS, Vidić MB, Bagi F, Budakov DB, Pavlović SD, Stojšin VB (2011). Pathogenic, morphological and molecular characteristics of Alternaria tenuissima from soybean. Zbornik Matice srpske za prirodne nauke 120:181-194.

Ketta HA, Hewedy OAER (2021). Biological control of Phaseolus vulgaris and Pisum sativum root rot disease using Trichoderma species. Egyptian Journal of Biological Pest Control 31:1-9.

Kuliešis A, Saladis J, Kuliešis AA (2010). Development and productivity of young Scots pine stands by regulating density. Baltic Forestry 16(2):235-246.

Kumar NV, Rajam KS, Rani ME (2017). Plant growth promotion efficacy of indole acetic acid (IAA) produced by a mangrove associated fungi Trichoderma viride VKF3. International Journal of Current Microbiology and Applied Sciences 6(11):2692-2701.

Lin S, Hand PF (2019). Determining the sources of primary and secondary inoculum and seasonal inoculum dynamics of fungal pathogens causing fruit rot of deciduous holly. Plant Diseases 103:951-958.

López-Bucioa J, Pelagio-Floresa R, Herrera-Estrella A (2015). Trichoderma as biostimulant: exploiting the multilevel properties of a plant beneficial fungus. Scientia Horticulturae 196:109-123.

López-Valenzuela BE, Tzintzun-Camacho O, Armenta-Bojórquez AD, Valenzuela-Escoboza FA, Lizárraga-Sánchez GJ, Ruelas- Islas JR, González-Mendoza D (2022). Microorganismos del género Trichoderma productores de fitohormonas y antagonistas de fitopatógenos [Microorganisms of the genus Trichoderma that produce phytohormones and antagonists of phytopathogens]. Bioagro 34(2):163-172.

Matas-Baca MA, Urías-García C, Pérez-Álvarez S, Flores-Córdova MA, Escobedo-Bonilla CM, Magallanes-Tapia MA, Sánchez-Chávez E (2022). Morphological and molecular characterization of a new autochthonous Trichoderma sp. isolate and its biocontrol efficacy against Alternaria sp. Saudi Journal of Biological Sciences 29(2022):2620-2625.

Mohammadi A, Bahramikia S (2019). Molecular identification and genetic variation of Alternaria species isolated from tomatoes using ITS1 sequencing and inter simple sequence repeat methods. Current Medical Mycology 5:1-8.

Mohammedi A, Trachi S, Ayad D, Messgo-Moumene S, Boureghda H, Bouznad Z (2022). Antagonistic potential of Trichoderma spp. evaluated under in vitro and in vivo conditions against Alternaria spp. responsible for early blight of tomato in Algeria. Bulgarian Journal of Agricultural Science 28 (4):626-635.

Morales-Mora LA, Andrade-Hoyos P, Valencia-de Ita MA, Romero-Arenas O, Silva-Rojas HV, Contreras-Paredes CA (2020). Characterization of strawberry associated fungi and in vitro antagonistic effect of Trichoderma harzianum. Revista Mexicana de Fitopatología 38(3):434-449

Promwee A, Intana W (2022). Trichoderma asperellum (NST-009): A potential native antagonistic fungus to control Cercospora leaf spot and promote the growth of ‘Green Oak’ lettuce (Lactuca sativa L.) cultivated in the commercial NFT hydroponic system. Plant Protection Science 58(2):139-149.

Rambaut A (2018). FigTree v 1.4.3. Retrieved 2023 February 09 from

Ramírez-Legarreta MR, Ruiz-Corral JA, Medina-García G, Jacobo-Cuéllar JL, Parra-Quezada RA, Ávila-Marioni MR, Armando-Álvarez JP (2011). Perspectivas del sistema de producción de manzano en Chihuahua, ante el cambio climático [Perspectives of the apple production system in Chihuahua, in the face of climate change]. Revista Mexicana de Ciencias Agrícolas 2:265-279.

Rodarte-Díaz YM, Guzmán-Moreno J, Ramírez-Santoyo RM, Reveles-Hernández RG, Vidales-Rodríguez LE (2020). Aislamiento e identificación morfológica y molecular de hongos asociados a plantas de zanahoria enfermas [Isolation and morphological and molecular identification of fungi associated with diseased carrot plants]. Investigación Científica 14(2):14-19.

Rodríguez-Roa JH, Cárdenas ME, Jiménez P (2013). Especies de Alternaria de la sabana de Bogotá, Colombia. Facultad de Ciencias Básicas 9(2):228-247.

Ronnie-Gakegne E, Martínez-Coca B (2018). Antibiosis y efecto de pH-temperatura sobre el antagonismo de cepas de Trichoderma asperellum frente a Alternaria solani [Antibiosis and effect of pH-temperature on the antagonism of Trichoderma asperellum strains against Alternaria solani]. Revista de Protección Vegetal 33:1-9.

Ronquist F, Teslenko M, Van Der Mark P, Ayres D, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012). MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61:539-542.

Rubio-Tinajero S, Osorio-Hernández E, Estrada-Drouaillet B, Silva-Espinosa T, Espinoza-Ahumada C, Delgado-Martínez R, … Ríos-Velasco C (2022). In vitro biocontrol of Fusarium oxysporum with antagonistic microorganisms and in vivo effect on Solanum lycopersicum L. Journal of Environmental Science and Health, Part B 57(8):625-635.

Sabre WI, Ghoneem KM, Rashad YM, Al-Askar AA (2017). Trichoderma harzianum WKY1: an indole acetic acid producer for growth improvement and anthracnose disease control in sorghum. Biocontrol Science and Technology 27(5):654-676.

Saleem A, El-Shahir AA (2022). Morphological and molecular characterization of some Alternaria species isolated from tomato fruits concerning mycotoxin production and polyketide synthase genes. Plants 11:1168.

Sharma A, Salwan R, Kaur R, Sharma R, Sharma V (2022). Characterization and evaluation of bioformulation from antagonistic and flower inducing Trichoderma asperellum isolate UCRD5. Biocatalysis and Agricultural Biotechnology 43:102437.

SIAP (Servicio de Información Agroalimentaria y Pesquera) (2023). Anuario Estadístico de la Producción Agrícola [Statistical Yearbook of Agricultural Production]. Retrieved 2023 February 03 from

Simmons EG (1993). Alternaria themes and variations (54-62). Mycotaxon 46:171-199.

Simmons EG (1999). Alternaria themes and variations (236-243): Host-specific toxin producers. Mycotaxon 70:325-370.

Singh A, Shukla N, Kabadwal BC, Tewari AK, Kumar J (2018). Review on plant-Trichoderma-pathogen interaction. International Journal of Current Microbiology and Applied Sciences 7(2):2382-2397.

Studholme DJ, Harris B, Le Cocq K, Winsbury R, Perera V, Ryder L, … Grant M (2013). Investigating the beneficial traits of Trichoderma hamatum GD12 for sustainable agriculture–insights from genomics. Frontiers in Plant Science 4:1-13.

Tamandegani PR, Marik T, Zafari D, Balázs D, Vágvölgyi C, Szekeres A, Kredics L (2020). Changes in peptaibol production of Trichoderma species during in vitro antagonistic interactions with fungal plant pathogens. Biomolecules 10:730.

Thambugala KM, Daranagama DA, Phillips AJL, Kannangara SD, Promputtha I (2020). Fungi vs. Fungi in biocontrol: An overview of fungal antagonists applied against fungal plant pathogens. Frontiers in Cellular and Infection Microbiology 10:604923. 0.3389/fcimb.2020.604923

Thompson JD, Higgins DG, Gibson TJ (1994). ClustalW: improving the sensivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Research 22:673-4680.

Turaeva B, Soliev A, Eshboev F, Kamolov L, Azimova N, Karimov H, ... Khamidova K (2020). The use of three fungal strains in producing of indole-3-acetic acid and gibberellic acid. Plant Cell Biotechnology and Molecular Biology 21(35-36):32-43.

Verma M, Brar SK, Tyagi RD, Surampalli RY, Valéro JR (2007). Antagonistic fungi, Trichoderma spp.: panoply of biological control. Biochemical Engineering Journal 37:1-20.

Wang H, Zhang R, Duan Y, Jiang W, Chen X, Shen X, Yin C, Mao Z (2021). The endophytic strain Trichoderma asperellum 6S-2: An efficient biocontrol agent against apple replant disease in China and a potential plant-growth-promoting fungus. Journal of Fungi 7:1050.

Wang H, Zhang R, Mao Y, Jiang W, Chen X, Shen X, Yin C, Mao Z (2022). Effects of Trichoderma asperellum 6S-2 on apple tree growth and replanted soil microbial environment. Journal of Fungi (Basel) 7(1):63.

White TJ, Bruns TD, Lee S, Taylor J (1990). Amplification and direct sequencing of fungal ribosomal genes form phylogenetics. In: Innis MA, Gelfrand DH, Sninsky JJ, White TJ (Eds). PCR Protocols. Academic Press; San Diego, California, USA.

Zhang S, Xu B, Zhang J, Gan Y (2018). Identification of the antifungal activity of Trichoderma longibrachiatum T6 and assessment of bioactive substances in controlling phytopathgens. Pesticide Biochemistry and Physiology 147:59-66.

Zhang Y, Uyemoto JK, Kirkpatrick BC (1998). A small-scale procedure for extracting nucleic acids from woody plants infected with various phytopathogens for PCR assay. Journal of Virological Methods 71:45-50.



How to Cite

MADRID-MOLINA, M., PÉREZ-ÁLVAREZ, S., ESCOBEDO-BONILLA, C. M., & URÍAS-GARCÍA, C. (2023). Application of Trichoderma asperellum in apple trees as a growth regulator and antagonist for the control of Alternaria sp . Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 51(1), 13108.



Research Articles
DOI: 10.15835/nbha51113108

Most read articles by the same author(s)