Evaluation of Trichoderma atroviride endophytes with growth-promoting activities on tomato plants and antagonistic action on Fusarium oxysporum

Rodrigo Pawloski Schoffen, Amanda da Silva Ribeiro, Verci Alves de Oliveira-Junior, Julio Cesar Polonio, Andressa Domingos Polli, Ravely Casarotti Orlandelli, Marcos Alessandro dos Santos Ribeiro, João Alencar Pamphile, João Lúcio Azevedo


In Brazil, tomato is one of the most consumed vegetables and the fungus Fusarium oxysporum is one of the most important phytopathogen of tomato plants (Lycopersicon esculentum Mill.). Thus, the search of beneficial microorganisms with growth-promoting and/or biological control properties represent an important tool for agricultural biotechnology. Herein, two Trichoderma endophytes (strains 36b and 164b) associated with Coffea arabica were investigated on their growth-promoting activities on plants and their antagonist effects and interactions against F. oxysporum. Molecular multigene (ITS- TEF-TUB-CAL) identification and phylogenetic analysis allowed the identification of these endophytes as belonging to Trichoderma atroviride species. When inoculated with the endophytic strain 36b, tomato plants reached the highest speed of seedling emergence (83.3%), but both endophytes increased the number of leaves, root length and dry biomass of treated plants. Regarding the in vitro antagonism assay, reduced phytopathogen growth by approximately 70 (strain 36b) and 52% (strain 164b) which indicates a partial replacement of endophytes after initial deadlock with mycelial contact. Scanning electron microscopy allowed to observe the presence of Fusarium macroconidia between endophytic hyphae and conidia, with the helicoidization of endophytic hyphae, which wrapped around the pathogen hyphae, suggesting a mechanical inhibition by strangulation.


Molecular multigene identification; Growth-promoting endophytes; Scanning electron microscopy.

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AZEVEDO JL, MACCHERONI W, PEREIRA JO, ARAÚJO WL. Endophytic microorganisms: a review on insect control and recent advances on tropical plants. Electron. J. Biotechnol. 2000;3(1)1;40-65. https://doi.org/10.2225/vol3-issue1-fulltext-4

AZEVEDO JL, MACCHERONI JÚNIOR W, ARAÚJO WL, PEREIRA JO. Microrganismos endofíticos e seu papel em plantas tropicais. Biotecnol. avanços na Agric. e na agroindústria. Caxias do Sul: EDUCS; 2002.

BADALYAN SM, INNOCENTI G, GARIBYAN NG. Antagonistic activity of xylotrophic mushrooms against pathogenic fungi of cereals in dual culture. Phytopathol. Mediterr. 2002;41:200-25. https://doi.org/http://dx.doi.org/10.14601/Phytopathol_Mediterr-1668

BADER NA, SALERNO GL, COVACEVICH F, CONSOLO VF. Native Trichoderma harzianum strains from Argentina produce indole-3 acetic acid and phosphorus solubilization, promote growth and control wilt disease on tomato (Solanum lycopersicum L.) J. King Saud Univ. Sci. [internet]. Forthcoming. [cited 2019 sept 09]. Author’s manuscript available at: https://doi.org/10.1016/j.jksus.2019.04.002

BERGER G, CZARNOCKA K, COCHARD B, OSZAKO T, LEFORT F. Biocontrol endotherapy with Trichoderma spp. and Bacillus amyloliquefaciens against Phytophthora spp.: a comparative study with phosphite treatment on Quercus robur and Fagus sylvatica. J. Agric. Sci. Technol. A 2015;5:428-39. https://doi.org/10.17265/2161-6256/2015.06.005

BONGIORNO VA, RHODEN SA, GARCIA A, POLONIO JC, AZEVEDO JL, PEREIRA JO et al. Genetic diversity of endophytic fungi from Coffea arabica cv. IAPAR-59 in organic crops. Ann. Microbiol. 2016;66:855-65. https://doi.org/10.1007/s13213-015-1168-0

BRASIL. Ministério da Agricultura, Pecuária e Abastecimento. Regras para análise de sementes. Brasília: Mapa/ACS; 2009.

BUSBY PE, PEAY KG, NEWCOMBE G. Common foliar fungi of Populus trichocarpa modify Melampsora rust disease severity. New Phytol. 2016;209(4):1681-92. https://doi.org/10.1111/nph.13742

CAMPANILE G, RUSCELLI A, LUISI N. Antagonistic activity of endophytic fungi towards Diplodia corticola assessed by in vitro and in planta tests. Eur. J. Plant Pathol. 2007;117:237-46. https://doi.org/10.1007/s10658-006-9089-1

CARBONE I, KOHN LM. A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia 1999;91:553-56. https://doi.org/10.2307/3761358

CHAKRABORTYA S, TIEDEMANN V, TENG S. Climate change: potential impact on plant diseases. Environ. Pollut. 2000;108:317-26. https://doi.org/10.1016/S0269-7491(99)00210-9

CHOU CH. Role of allelopathy in sustainable agriculture: use of allelochemicals as naturally occurring bio-agrochemicals. Allelopathy J. 2010;25(10):3-16

DRUZHININA IS, SEIDL-SEIBOTH V, HERRERA-ESTRELLA A, HORWITZ BA, KENERLEY CM, MONTE E et al. Trichoderma: The genomics of opportunistic success. Nat. Rev. Microbiol. 2011;9:749-59. https://doi.org/10.1038/nrmicro2637

EZIASHI EI, OMAMOR IB, ODIGIE EE. Antagonism of Trichoderma viride and effects of extracted watersoluble compounds from Trichoderma species and benlate solution on Ceratocystis paradoxa. Afr. J. Biotechnol. 2007;6(4):388-92

FOOD AND AGRICULTURE ORGANIZATION. [internet]. FAOSTAT: Statistics division, 2018 [cited 2019 sept 09]. Available from http://faostat3.fao.org/ browse/Q/Q C/E

FELBER AC, POLONIO JC, ORLANDELLI RC, COSTA AT, AMBROSIO-ALBUQUERQUE, EP, CASTRO RA, QUECINE-VERDI MC et al. Agrobacterium-mediated transformation of Diaporthe schini endophytes associated with Vitis labrusca L. and its antagonistic activity against grapevine phytopathogens. Indian J. Microbiol. 2019;59(2):217-24. https://doi.org/10.1007/s12088-019-00787-0

FERREIRA DF. Sisvar: a computer statistical analysis system. Ciênc. Agrotec. 2011;35(6):1039-42. http://dx.doi.org/10.1590/S1413-70542011000600001

GHINI R, HAMADA E, BETTIOL W. Climate change and plant diseases. Sci. Agric. 2088;65:98-107. http://dx.doi.org/10.1590/S0103-90162008000700015

GLASS NL, DONALDSON GC. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Appl. Environ. Microbiol. 1995;61(4):1323-30

GRAVEL V, ANTOUN H, TWEDDELL RJ. Growth stimulation and fruit yield improvement of greenhouse tomato plants by inoculation with Pseudomonas putida or Trichoderma atroviride: possible role of indole acetic acid (IAA). Soil Biol. Biochem. 2007;39:1968-77. https://doi.org/10.1016/j.soilbio.2007.02.015

GUPTA S, MAHAJAN S, GUPTA M, SHARMA D. Screening of native isolates of Trichoderma spp. of Jammu for their biocontrol potential through hydrolytic enzyme activities. Indian Phytopathol. 2016;69(2):173-80.

HARMAN GE, HOWELL CR, VITERBO A, CHET I, LORITO M. Trichoderma species - opportunistic, avirulent plant symbionts. Nat. Rev. Microbiol. 2004;2,43-56. https://doi.org/10.1038/nrmicro797

HASAN, S., GUPTA, G., ANAND, S., CHATURVEDI, A., KAUR, H. Biopotential of microbial antagonists against soilborne fungal plant pathogens. Int. J. Agric. Food Sci. Technol. 2013;42(2):37-9. https://doi.org/10.1007/s11104-010-0615-8.5

HYDE KD, CAI L, CANNON PF, CROUCH JA, CROUS PW, DAMM U et al. Colletotrichum – names in current use. Fungal Divers. 2009;39:147-82

JULIATTI FC, REZENDE AA, JULIATTI BCM, MORAIS TP. Trichoderma as a biocontrol agent against Sclerotinia stem rot or white mold on soybeans in Brazil: usage and Technology. In: SHAH MM, organizer. Trichoderma - the most widely used fungicide. 1 ed. Zagreb: IntechOpen; 2019. p. 1-24.

KATOH K, TOH H. Recent developments in the MAFFT multiple sequence alignment program. Brief. Bioinform. 2008;9(4):286-98. https://doi.org/10.1093/bib/bbn013

LO PRESTI L, LIANG L, TANAKA S, SCHWEIZER G, TOLLOT M, ZUCCARO A. et al. Fungal effectors and plant susceptibility. Annu. Rev. Plant Biol. 2015;66:513-45. https://doi.org/10.1146/annurev-arplant-043014-114623

LOUZADA GAS, CARVALHO DDC, MELLO SCM, LOBO JÚNIOR M, MARTINS I, BRAÚNA LM. Potencial antagônico de Trichoderma spp. originários de diferentes agroecossistemas contra Sclerotinia sclerotiorum e Fusarium solani. Biota Neotrop. 2009;9(3):145-9. http://dx.doi.org/10.1590/S1676-06032009000300014

MAGUIRE JD. Speed of germination-aid selection and evaluation for seedling emergence and vigor. Crop Sci. 1962;2: 176-7. http://dx.doi.org/10.2135/cropsci1962.0011183X000200020033x

MAHARACHCHIKUMBURA SSN, HYDE KD, GROENEWALD JZ, XU J, CROUS PW. Pestalotiopsis revisited. Stud. Mycol. 2014;79:121-86. https://doi.org/10.1016/j.simyco.2014.09.005

MANAMGODA DS, CAI L, BAHKALI AH, CHUKEATIROTE E, HYDE KD. Cochliobolus: an overview and current status of species. Fungal Divers. 2011;51:3-42. https://doi.org/10.1007/s13225-011-0139-4

MARTÍNEZ-MEDINA A, DEL MAR ALGUACIL M, PASCUAL JA, VAN WEES SCM. Phytohormone profiles induced by Trichoderma isolates correspond with their biocontrol and plant growth-promoting activity on melon plants. J. Chem. Ecol. 2014;40:804-15. https://doi.org/10.1007/s10886-014-0478-1

NIETO-JACOBO MF, STEYAERT JM, SALAZAR-BADILLO FB, NGUYEN DV, ROSTÁS M, BRAITHWAITE M. Environmental growth conditions of Trichoderma spp. affects indole acetic acid derivatives, volatile organic compounds, and plant growth promotion. Front. Plant Sci. 2017;98:102. https://doi.org/10.3389/fpls.2017.00102

NYLANDER JAA. MrModeltest v2. Uppsala: Evolutionary Biology Centre; 2004.

O'DONNELL K, CIGELNIK E. Two divergent intragenomic rDNA ITS2 types within a monophyletic lineage of the fungus Fusarium are non-orthologous. Mol. Phylogenet. Evol. 1997;7:103-16. https://doi.org/10.1007/BF02464387

ORLANDELLI RC, ALMEIDA TT, ALBERTO RN, POLONIO JC, AZEVEDO JL, PAMPHILE JA. Antifungal and proteolytic activities of endophytic fungi isolated from Piper hispidum Sw. Braz. J. Microbiol. 2015;46(2):359-66. http://dx.doi.org/10.1590/S1517-838246220131042

PAMPHILE JA, GAI CS, PILEGGI M, ROCHA CLMSC, PILEGGI SAV. Plant-microbe interactions between host and endophytes observed by Scanning Electron Microscopy (SEM). In: SORVARI S, PIRTTILÄ AM, editors. Prospects and applications for plant-associated microbes. A Laboratory Manual, Part A: Bacteria. Finland: BioBien Innovations); 2008. p. 184-9

PATTERN CL, GLICK BR. Role of Pseudomonas putida indo lactic acid in development of the host plant root system. App. Environ. Microbiol. 2002;68:3795-801. https://doi.org/10.1128/aem.68.8.3795-3801.2002

POLONIO JC, ALMEIDA TT, GARCIA A, MARIUCCI GEG, AZEVEDO JL, RHODEN SA et al. Biotechnological prospecting of foliar endophytic fungi of guaco (Mikania glomerata Spreng.) with antibacterial and antagonistic activity against phytopathogens. Genet. Mol. Res. 2015;14:7297-309. https://doi.org/10.4238/2015.July.3.5

RAMBAUT A. FigTree v1. 3.1: Tree figure drawing tool. [cited 2019 sept 09]. Available from: http://tree.bio.ed.ac. uk/software/figtree.

RIBEIRO A S, POLONIO JC, COSTA AT, SANTOS CM, RHODEN SA, AZEVEDO JL et al. Bioprospection of culturable endophytic fungi associated with the ornamental plant Pachystachys lutea. Curr. Microbiol. 2018;75:588-96. https://doi.org/10.1007/s00284-017-1421-9

RONQUIST F, VAN DER MARK E, TESLENKO M, AYRES DL, DARLING A, HÖHNA S, LARGET B et al. Mrbayes 3.2: Efficient bayesian phylogenetic inference and model choice across a large model space. Syst. Biol. 2012;61(3):539-42. https://doi.org/10.1093/sysbio/sys029

SOARES DA, OLIVEIRA DP, SANTOS TT, MARSON PG, PIMENTA RS. Multiloci identification of Diaporthe fungi isolated from the medicinal plant Costus spiralis (Jacq.) Roscoe (Costaceae). J. Appl. Microbiol. 2018;125(1):172-80. https://doi.org/10.1111/jam.13769

SONKAR P. Determination of interaction between Trichoderma asperellum and Fusarium oxysporum sp. by digital light microscopy and confocal microscopy. J. Microb. Biochem.Technol. 2019;11(1):1-4. https://doi.org/10.4172/1948-5948.1000407

STRANGE RN, SCOTT PR. Plant disease: a threat to global food security. Annu Rev Phytopathol. 2005;43:83-116. https://doi.org/10.1146/annurev.phyto.43.113004.133839

UDAYANGA D, ROSSMAN AY, CHUKEATIROTE E, HYDE KD, CASTLEBURY LA. Insights into the genus Diaporthe: phylogenetic species delimitation in the D. eres species complex. Fungal Divers. 2014;67:203-29. https://doi.org/10.1007/s13225-014-0297-2

VAIDYA G, LOHMAN DJ, MEIER R. SequenceMatrix: concatenation software for the fast assembly of multi-gene datasets with character set and codon information. Cladistics 2011;27:171-80. https://doi.org/10.1111/j.1096-0031.2010.00329.x

VILLAMIZAR-GALLARDO RA, ORTÍZ-RODRIGUEZ OO, ESCOBAR JW. Symbiotic and endophytic fungi as biocontrols against cocoa (Theobroma cacao L.) phytopathogens. Summa Phytopathol. 2017;43(2):87-93. http://dx.doi.org/10.1590/0100-5405/2175

VINALE F, LORITO M, MARRA R, WOO SL, SIVASITHAMPARAM K, GHISALBERTI EL. Trichoderma–plant–pathogen interactions. Soil Biol. Biochem. 2008;40:1-10. https://doi.org/10.1016/j.soilbio.2007.07.002

VOS CM, YANG Y, CONINCK B, CAMMUE BPA. Fungal (-like) biocontrol organisms in tomato disease control. Biol. Control 20104;74:65-81. https://doi.org/10.1016/j.biocontrol.2014.04.004

WHITE TJ, BRUNS T, LEE S, TAYLOR J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: INNIS MA, GELFAND DH, SNINSKY JJ, editors. PCR protocols: a guide to methods and applications. New York: Academic Press; 1990. p. 315-322

ZHANG M, XU JH, LIU G, YAO XF, LI PF, YANG XP. Characterization of the watermelon seedling infection process by Fusarium oxysporum f. sp. niveum. Plant Pathol. 2015;64:1076-84. https://doi.org/10.1111/ppa.12355

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