Biological control effect of Trichoderma harzianum and Bacillus amyloliquefaciens on Neopestalotiopsis rosae under controlled conditions

Maria Magdalena Cervantes Zúñiga, Kenzy Ivvet Peña Carrillo, Agustín Hernádez Juárez, Gabriel Gallegos Morales, Juan Carlos Delgado Ortiz, Epifanio Castro del Ángel

Abstract


Background: The strawberry crop (Fragaria x ananassa) faces a wide range of fungal phytopathogens that cause various diseases during its phenological cycle, with Neopestalotiopsis rosae having a significant impact. Objective: To evaluate the biocontrol in-vitro of N. rosae-induced crown rot by using Trichoderma harzianum and Bacillus amyloliquefaciens with potential applications in strawberry crops. Methodology: The pathogen was isolated from infected plants of the Frontera, Festival and Albion varieties in the state of Michoacán, followed by its purification and morphological and molecular identification. Under laboratory conditions, a strain of T. harzianum provided by a phytopathology laboratory was used to perform dual-culture inhibition assays on potato dextrose agar. Results: The results showed a percentage of N. rosae growth inhibition ranging from 58.94% to 81.63%. Furthermore, in bioassays carried out with B. amyloliquefaciens, inhibition percentages were observed, which ranged between 62.50% and 72.87, highlights the potential of both biological agents in the management of this disease. Implications: These findings suggest that Trichoderma harzianum and Bacillus amyloliquefaciens could represent promising components of integrated disease management strategies. Their significant antagonistic activity indicates potential for reducing reliance on synthetic fungicides and supports further investigation under greenhouse and field conditions to validate their efficacy against crown rot caused by Neopestalotiopsis rosae. Conclusion: The strains of Trichoderma harzianum and Bacillus amyloliquefaciens demonstrated strong antagonistic activity against Neopestalotiopsis rosae, indicating their potential as effective biocontrol agents for managing crown rot in strawberry crops.  

Keywords


Biocontrol; inhibition; crown rot.

Full Text:

PDF

References


Acosta-González, U., García-García, G., Silva-Rojas, H.V., Peres, N.A., Marin, M.V., Rebello, C.S., Fuentes-Aragón, D., Leyva-Mir, S.G. and Rebollar-Alviter, Á., 2025. Molecular, cultural and pathogenic characterization of Neopestalotiopsis rosae from strawberry in Mexico. https://doi.org/10.31219/osf.io/wz7ms_v1

Ahmad, H., Sajjid, M., Hayat, S., Ullah, R., Ali, M., Jamal, A. and Ali, J., 2017. Growth, yield and fruit quality of strawberry (Fragaria ananassa Duch.) under different phosphorus levels. Research in Agriculture, 2(2), pp. 19–26. http://dx.doi.org/10.22158/ra.v2n2p19

Álvarez-Medina, A., Silva-Rojas, H. V., Leyva-Mir, S. G., Marbán-Mendoza, N. and Rebollar-Alviter, Á., 2017. Resistencia de Botrytis cinerea de strawberry (Fragaria × ananassa Duch.) a fungicidas en Michoacán, México. Agrociencia, 51(7), pp. 783–798.

Arroyo, C.A.J. and Hernández, F.A.A., 2021. Competitiveness of the Mexican strawberry in the US market from 1992 to 2017. Ciencia y Tecnología Agropecuaria, 22(1). https://doi.org/10.21930/rcta.vol22

Baggio, J.S., Forcelini, B.B., Wang, N.Y., Ruschel, R.G., Mertely, J.C. and Peres, N.A., 2021. Outbreak of leaf spot and fruit rot in Florida strawberry caused by Neopestalotiopsis spp. Plant Disease, 105(2), pp. 305–315. https://doi.org/10.1094/PDIS-06-20-1290-RE

Bell, D.K., Wells, H.D. and Markham, C.R., 1982. In vitro antagonism of Trichoderma species against six fungal plant pathogens. Phytopathology, 72(4), pp. 379–382. https://doi.org/10.1094/Phyto-72-379

Carbone, I. and Kohn, L.M., 1999. A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia, 91(3), pp. 553–556. https://doi.org/10.2307/3761358

Castillo-Reyes, F., Hernández-Castillo, F.D., Gallegos-Morales, G., Flores-Olivas, A., Rodríguez-Herrera, R. and Aguilar, C. N. (2015). Efectividad in vitro de Bacillus y polifenoles de plantas nativas de México sobre Rhizoctonia solani. Revista Mexicana de Ciencias Agrícolas, 6(3), pp. 549-562.

Chen, S., Daly, P., Anjago, W.M., Wang, R., Zhao, Y., Wen, X., Zhou, D., Deng, S., Lin, X., Voglmeir, J., Cai, F., Shen, Q., Druzhinina, I.S. and Wei, L., 2024. Genus-wide analysis of Trichoderma antagonism toward Pythium and Globisporangium plant pathogens and the contribution of cellulases to the antagonism. Applied and Environmental Microbiology, 90(9), e00681-24. https://doi.org/10.1128/aem.00681-24

Chen, X., Lu, Y., Liu, X., Gu, Y. and Li, F., 2025. Trichoderma: dual roles in biocontrol and plant growth promotion. Microorganisms, 13(8), 1840. https://doi.org/10.3390/microorganisms13081840

Chun, J., Yang, H. E. and Kim, D. H., 2018. Identification of a novel partitivirus of Trichoderma harzianum NFCF319 and evidence for the related antifungal activity. Frontiers in Plant Science, 9, 1699. https://doi.org/10.3389/fpls.2018.01699

Darapanit, A., Boonyuen, N., Leesutthiphonchai, W., Nuankaew, S. and Piasai, O., 2021. Identification, pathogenicity and effects of plant extracts on Neopestalotiopsis and Pseudopestalotiopsis causing fruit diseases. Scientific Reports, 11(1), 22606. https://doi.org/10.1038/s41598-021-02113-5

Ding, N., Dong, H. and Ongena, M., 2025. Bacterial cyclic lipopeptides as triggers of plant immunity and systemic resistance against pathogens. Plants, 14(17), 2644. https://doi.org/10.3390/plants14172644

Essa, T.A., Kamel, S.M., Ismail, A.M. and El-Ganainy, S., 2018. Characterization and chemical control of Neopestalotiopsis rosae, the causal agent of strawberry root and crown rot in Egypt. Egyptian Journal of Phytopathology, 46(1), pp. 1–19. https://doi.org/10.21608/ejp.2018.87411

Gharsallah, H., Cheffi, M., Mallek, R., Massoudi, A., Omri, N., Triki, M.A., Öztop, M.H. and Zarai, Z., 2025. Antifungal potential of Bacillus strains: implications for biocontrol strategies in food safety and sustainable agriculture. Frontiers in Microbiology, 16, 1615252. https://doi.org/10.3389/fmicb.2025.1615252

Güçlü, T. and Özer, N., 2022. Trichoderma harzianum antagonistic activity and competition for seed colonization against seedborne pathogenic fungi of sunflower. Letters in Applied Microbiology, 74(6), pp. 1027–1035. https://doi.org/10.1111/lam.13698

Guindon, S., Dufayard, J.F., Lefort, V., Anisimova, M., Hordijk, W. and Gascuel, O., 2010. New algorithms and methods to estimate maximum-likelihood phylogenies: Assessing the performance of PhyML 3.0, Systematic Biology, 59(3), pp. 307–321. https://doi.org/10.1093/sysbio/syq010

Guzmán-Guzmán, P., Etesami, H. and Santoyo, G., 2025. Trichoderma: a multifunctional agent in plant health and microbiome interactions. BMC Microbiology, 25(1), 434. https://doi.org/10.1186/s12866-025-04158-2

Hanif, A., Zhang, F., Li, P., Li, C., Xu, Y., Zubair, M., Zhang, M., Jia, D., Zhao, X., Liang, J., Majid, T., Yan, J., Farzand, A., Wu, H., Gu, Q. and Gao, X., 2019. Fengycin produced by Bacillus amyloliquefaciens FZB42 inhibits Fusarium graminearum growth and mycotoxin biosynthesis. Toxins, 11(5), 295. https://doi.org/10.3390/toxins11050295

Hernández-Rodríguez, M., Jasso-de Rodríguez, D., Hernández-Castillo, F.D., Moggio, I., Arias, E., Valenzuela-Soto, J.H. and Flores-Olivas, A., 2024. The rhizobacterium Bacillus amyloliquefaciens MHR24 has biocontrol ability against fungal phytopathogens and promotes growth in Arabidopsis thaliana. Microorganisms, 12(11), 2380. https://doi.org/10.3390/microorganisms12112380

Hoang, D.T., Chernomor, O., von Haeseler, A., Minh, B.Q. and Vinh, L.S., 2018. UFBoot2: improving the ultrafast bootstrap approximation. Molecular Biology and Evolution, 35(2), pp. 518–522. https://doi.org/10.1093/molbev/msx281

Infante, D., Martínez, B., González, N. and Reyes, Y., 2009. Mecanismos de acción de Trichoderma frente a hongos fitopatógenos. Revista de Protección Vegetal, 24(1), pp. 14–21.

Kaur, H., Gelain, J., Marin, M.V., Peres, N.A. and Schnabel, G., 2022. Development of a molecular tool for identification of a new Neopestalotiopsis species associated with disease outbreaks on strawberry. Plant Disease, 107(5), pp. 1544–1549. https://doi.org/10.1094/PDIS-09-22-2117-RE

Kazerooni, E.A., Maharachchikumbura, S.S., Al-Sadi, A. M., Kang, S. M., Yun, B.W. and Lee, I.J., 2021. Biocontrol potential of Bacillus amyloliquefaciens against Botrytis pelargonii and Alternaria alternata on Capsicum annuum. Journal of Fungi, 7(6), 472. https://doi.org/10.3390/jof7060472

Kalyaanamoorthy, S., Minh, B.Q., Wong, T.K.F., von Haeseler, A. and Jermiin, L.S., 2017. ModelFinder: fast model selection for accurate phylogenetic estimates. Nature Methods, 14, pp. 587–589. https://doi.org/10.1038/nmeth.4285

Kredics, L., Büchner, R., Balázs, D., Allaga, H., Kedves, O., Raci?, G., Varga, A., Nagy, V.D., Vágvölgyi, C. and Sipos, G., 2024. Recent advances in the use of Trichoderma-containing multicomponent microbial inoculants for pathogen control and plant growth promotion. World Journal of Microbiology and Biotechnology, 40(5), 162. https://doi.org/10.1007/s11274-024-03965-5

Kumar, S., Stecher, G., Suleski, M., Sanderford, M., Sharma, S. and Tamura, K., 2024. Molecular evolutionary genetics analysis version 12 for adaptive and green computing. Molecular Biology and Evolution, 41(12), pp. 1–9. https://doi.org/10.1093/molbev/msae263

Lee, T., Park, D., Kim, K., Lim, S.M., Yu, N.H., Kim, S., Kim, H., Jung, K.S., Jang, J.Y., Park, J., Ham, H., Lee, S., Hong, S.K. and Kim, J.C., 2017. Characterization of Bacillus amyloliquefaciens DA12 showing potent antifungal activity against mycotoxigenic Fusarium species. The Plant Pathology Journal, 33(5), pp. 499–507. https://doi.org/10.5423/PPJ.FT.06.2017.0126

Maharachchikumbura, S.S., Hyde, K.D., Groenewald, J.Z., Xu, J. and Crous, P.W., 2014. Pestalotiopsis revisited. Studies in Mycology, 79(1), pp. 121–186. https://doi.org/10.1016/j.simyco.2014.09.005

Marnani, M.A., Madani, M. and Shakib, P., 2026. Extraction and characterization of iturin A as a key factor in the antagonism of Bacillus amyloliquefaciens M13RW01 toward pathogenic fungi. Recent Patents on Biotechnology, 20(1), pp. 155–165. https://doi.org/10.2174/0118722083352297250326041149

Minh, B.Q., Schmidt, H.A., Chernomor, O., Schrempf, D., Woodhams, M.D., von Haeseler, A. and Lanfear, R., 2020. IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era. Molecular Biology and Evolution, 37(5), pp. 1530–1534. https://doi.org/10.1093/molbev/msaa015

Mitra, B., Chowdhury, A.R., Dey, P., Hazra, K.K., Sinha, A.K., Hossain, A. and Meena, R.S., 2022. Use of agrochemicals in agriculture: alarming issues and solutions. In: Input Use Efficiency for Food and Environmental Security. Springer Nature Singapore, pp. 85–122. https://doi.org/10.1007/978-981-16-5199-1_4

Nguyen, L.T., Schmidt, H.A., von Haeseler, A. and Minh, B.Q., 2015. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Molecular Biology and Evolution, 32(1), pp. 268–274. https://doi.org/10.1093/molbev/msu300

Saiyam, D., Dubey, A., Malla, M. A. and Kumar, A., 2024. Lipopeptides from Bacillus: unveiling biotechnological prospects—sources, properties, and diverse applications. Brazilian Journal of Microbiology, 55(1), pp. 281–295. https://doi.org/10.1007/s42770-023-01228-3

Saldaña Jaramillo, J., 2021. Control de hongos patógenos con Bacillus subtilis y Trichoderma koningiopsis en un cultivo de strawberry en condiciones de campo. Tesis de maestría. Instituto Politécnico Nacional, México.

Servicio de Información Agroalimentaria y Pesquera., 2024. Producción agrícola por cultivo: fresa (Fragaria × ananassa). Secretaría de Agricultura y Desarrollo Rural, Ciudad de México, México. SIAP. Disponible en: https://www.gob.mx/siap

Stange, P., Seidl, S., Karl, T. and Benz, J.P., 2023. Evaluation of Trichoderma isolates as biocontrol measure against Claviceps purpurea. European Journal of Plant Pathology, 167(4), pp. 651–675. https://doi.org/10.1007/s10658-023-02716-w

Stenberg, J.A., Sundh, I., Becher, P.G., Björkman, C., Dubey, M., Egan, P.A., Friberg, H., Gil, J.F., Jensen, D.F., Jonsson, M., Karlsson, M., Khalil, S., Ninkovic, V., Rehermann, G., Vetukuri, R. R. and Viketoft, M., 2021. When is it biological control? A framework of definitions, mechanisms, and classifications. Journal of Pest Science, 94(3), pp. 665–676. https://doi.org/10.1007/s10340-021-01354-7

Sun, Q., Harishchandra, D., Jia, J., Zuo, Q., Zhang, G., Wang, Q., Yan, J., Zhang, W. and Li, X., 2021. Role of Neopestalotiopsis rosae in causing root rot of strawberry in Beijing, China. Crop Protection, 147, 105710. https://doi.org/10.1016/j.cropro.2021.105710

Shi, J., Li, B., Wang, S., Zhang, W., Shang, M., Wang, Y. and Liu, B., 2024. Occurrence of Neopestalotiopsis clavispora causing apple leaf spot in China. Agronomy, 14(8), 1658. https://doi.org/10.3390/agronomy14081658

Wang, X.Y., Xu, T.T., Sun, L.J., Cen, R.H., Su, S., Yang, X.Q., Yang, Y. and Ding, Z.T., 2021. The chemical diversity, the attractant, anti-acetylcholinesterase, and antifungal activities of metabolites from biocontrol Trichoderma harzianum uncovered by OSMAC strategy. Bioorganic Chemistry, 114, 105148. https://doi.org/10.1016/j.bioorg.2021.105148

White, T.J., Bruns, T., Lee, S. and Taylor, J., 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR Protocols: A Guide to Methods and Applications. Academic Press, pp. 315–322.

Yan, F., Li, C., Ye, X., Lian, Y., Wu, Y. and Wang, X., 2020. Antifungal activity of lipopeptides from Bacillus amyloliquefaciens MG3 against Colletotrichum gloeosporioides in loquat fruits. Biological Control, 146, 104281. https://doi.org/10.1016/j.biocontrol.2020.104281

Zalila-Kolsi, I., Kessentini, S., Tounsi, S. and Jamoussi, K., 2022. Optimization of Bacillus amyloliquefaciens BLB369 culture medium by response surface methodology for low cost production of antifungal activity. Microorganisms, 10(4), 830. https://doi.org/10.3390/microorganisms10040830

Zhang, R., Yu, J., Yang, L., Qiao, J., Qi, Z., Yu, M., Du, Y., Song, T., Cao, H., Pan, X., Liu, Y. and Liu, Y., 2024. Surfactins and iturins produced by Bacillus velezensis Jt84 strain synergistically contribute to the biocontrol of rice false smut. Agronomy, 14(10), 2204. https://doi.org/10.3390/agronomy14102204




URN: http://www.revista.ccba.uady.mx/urn:ISSN:1870-0462-tsaes.v29i1.67095

DOI: http://dx.doi.org/10.56369/tsaes.6709

Bookmark and Share


Copyright (c) 2026 Epifanio Castro del Ángel, Maria Magdalena Cervantes Zúñiga, Kenzy Ivvet Peña Carrillo, Agustín Hernádez Juárez, Gabriel Gallegos Morales, Juan Carlos Delgado Ortiz

Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 International License.