, Volume 61, Issue 3, pp 269–282 | Cite as

Fate of Trichoderma harzianum in the olive rhizosphere: time course of the root colonization process and interaction with the fungal pathogen Verticillium dahliae

  • David Ruano-Rosa
  • Pilar Prieto
  • Ana María Rincón
  • María Victoria Gómez-Rodríguez
  • Raquel Valderrama
  • Juan Bautista Barroso
  • Jesús Mercado-Blanco


Trichoderma harzianum Rifai is a well-known biological control agent (BCA) effective against a wide range of phytopathogens. Since colonization and persistence in the target niche is crucial for biocontrol effectiveness we aimed to: (i) shed light on the olive roots colonization process by T. harzianum CECT 2413, (ii) unravel the fate of its biomass upon application, and (iii) study the in planta interaction with the soil-borne pathogen Verticillium dahliae Kleb. Fluorescently-tagged derivatives of CECT 2413 and V. dahliae and confocal laser scanning microscopy were used. In vitro assays showed for the first time mycoparasitism of V. dahliae by T. harzianum, evidenced by events such as hyphal coiling. In planta assays revealed that CECT 2413 profusely colonized the rhizoplane of olive roots. Interestingly, biomass of the BCA was visualized mainly as chlamydospores. This observation was independent on the presence or absence of the pathogen. Evidence of inner colonization of olive roots by CECT 2413 was not obtained. These results suggest that CECT 2413 is not able to persist in a metabolically-active form when applied as a spore suspension. This may have strong implications in the way this BCA should be introduced and/or formulated to be effective against Verticillium wilt of olive.


Chlamydospores Confocal laser scanning microscopy Mycoparasitism Olea europaea L. Trichoderma harzianum Rifai Root colonization Verticillium dahliae Kleb. Verticillium wilt 



We are very grateful to M. N. Casa Adán for the help with CLSM at the University of Jaén facilities, and to M. Maldonado-González and A. Valverde-Corredor for their technical assistance. We appreciate the collaboration of NUTESCA SL in this project. This work was supported by European Regional Development Fund-cofinanced grants from the Spanish Ministry of Economy and Competitiveness [Project number BIO2012-33904] and ‘Junta de Andalucía’ [Project number AGR-6038].


  1. Aleandri MP, Chilosi G, Bruni N, Tomassini A, Vettraino AM, Vannini A (2015) Use of nursery potting mixes amended with local Trichoderma strains with multiple complementary mechanisms to control soil-borne diseases. Crop Prot 67:269–278CrossRefGoogle Scholar
  2. Alonso-Ramirez A, Poveda J, Martin I, Hermosa R, Monte E, Nicolas C (2014) Salicylic acid prevents Trichoderma harzianum from entering the vascular system of roots. Mol Plant Pathol 15(8):823–831CrossRefPubMedGoogle Scholar
  3. Askew DJ, Laing MD (1993) An adapted selective medium for the quantitative isolation of Trichoderma species. Plant Pathol 42(5):686–690CrossRefGoogle Scholar
  4. Bae YS, Knudsen GR (2000) Cotransformation of Trichoderma harzianum with beta-glucuronidase and green fluorescent protein genes provides a useful tool for monitoring fungal growth and activity in natural soils. Appl Environ Microbiol 66(2):810–815CrossRefPubMedPubMedCentralGoogle Scholar
  5. Barroso Albarracín JB, Carreras Egaña A, Valderrama Rodríguez R, Chaki M, Begara Morales J, Mercado-Blanco J, Pérez Artés E, Rincón Romero A, Carballo Codón A, Benítez Fernández T, Valverde Corredor A, Guevara Pezoa F, Rodríguez Palero MJ, Dueñas Sánchez R, Fierro Risco J, López García A (2014) Cepa de Trichoderma útil para el tratamiento y/o prevención de infecciones provocadas por hongos pertenecientes al género Verticilium. Spanish Patent ES2393728Google Scholar
  6. Benítez T, Rincón AM, Limón MC, Codón AC (2004) Biocontrol mechanisms of Trichoderma strains. Int Microbiol 7:249–260PubMedGoogle Scholar
  7. Brotman Y, Kapuganti JG, Viterbo A (2010) Trichoderma. Curr Biol 20(9):R390–R391CrossRefPubMedGoogle Scholar
  8. Carvalho DDC, Lobo M, Martins I, Inglis PW, Mello SCM (2014) Biological control of Fusarium oxysporum f. sp phaseoli by Trichoderma harzianum and its use for common bean seed treatment. Trop Plant Pathol 39(5):384–391CrossRefGoogle Scholar
  9. Chacón MR, Rodríguez-Galán O, Benítez T, Sousa S, Rey M, Llobell A, Delgado-Jarana J (2007) Microscopic and transcriptome analyses of early colonization of tomato roots by Trichoderma harzianum. Int Microbiol 10(1):19–27PubMedGoogle Scholar
  10. Chet I, Benhamou N, Haran S (1998) Mycoparasitism and lytic enzymes. In: Harman GE, Kubicek CP (eds) Trichoderma & Gliocladium. Enzymes, biological control and commercial applications, vol 2. Taylor and Francis Ltd, London, UK, pp 153–172Google Scholar
  11. Cohen SD, Lewis JA, Papavizas GC, Bean GA (1983) Chlamydospore formation by Trichoderma spp. in organic-matter amended soil. Phytopathology 73(5):820Google Scholar
  12. Contreras-Cornejo HA, Ortiz-Castro R, López-Bucio J (2013) Promotion of plant growth and the induction of systemic defence by Trichoderma: physiology, genetics and gene expression. In: Mukherjee PK, Horwitz BA, Singh US, Mukherjee M, Schmoll M (eds) Trichoderma: biology and applications. CABI, Walingford, UK, pp 173–194CrossRefGoogle Scholar
  13. Druzhinina IS, Seidl-Seiboth V, Herrera-Estrella A, Horwitz BA, Kenerley CM, Monte E, Mukherjee PK, Zeilinger S, Grigoriev IV, Kubicek CP (2011) Trichoderma: the genomics of opportunistic success. Nat Rev Microbiol 9(10):749–759CrossRefPubMedGoogle Scholar
  14. El-Hassan SA, Gowen SR (2006) Formulation and delivery of the bacterial antagonist Bacillus subtilis for management of lentil vascular wilt caused by Fusarium oxysporum f. sp lentis. J Phytopathol 154(3):148–155CrossRefGoogle Scholar
  15. Ghanbarzadeh B, Safaie N, Goltapeh EM (2014) Antagonistic activity and hyphal interactions of Trichoderma spp. against Fusarium proliferatum and F. oxysporum in vitro. Arch Phytopathol Plant Prot 47(16):1979–1987CrossRefGoogle Scholar
  16. Harman GE, Kubicek CP (1998) Trichoderma & Gliocladium: enzymes, biological control and commercial applications, vol 2. Taylor & Francis Ltd, London, UKGoogle Scholar
  17. Harman GE, Howell CR, Viterbo A, Chet I, Lorito M (2004) Trichoderma species: opportunistic, avirulent plant symbionts. Nat Rev Microbiol 2(1):43–56CrossRefPubMedGoogle Scholar
  18. Hermosa R, Viterbo A, Chet I, Monte E (2012) Plant-beneficial effects of Trichoderma and of its genes. Microbiology 158:17–25CrossRefPubMedGoogle Scholar
  19. Hohmann P, Jones EE, Hill RA, Stewart A (2011) Understanding Trichoderma in the root system of Pinus radiata: associations between rhizosphere colonisation and growth promotion for commercially grown seedlings. Fungal Biol 115(8):759–767CrossRefPubMedGoogle Scholar
  20. Hohmann P, Jones EE, Hill RA, Stewart A (2012) Ecological studies of the bio-inoculant Trichoderma hamatum LU592 in the root system of Pinus radiata. FEMS Microbiol Ecol 80(3):709–721CrossRefPubMedGoogle Scholar
  21. Jiménez-Díaz RM, Trapero-Casas JL, Boned J, Landa del Castillo BB, Navas-Cortés JA (2009) Uso de Bioten para la protección biológica de plantones de olivo contra la Verticilosis causada por el patotipo defoliante de Verticillium dahliae. Bol San Veg. Plagas, 35:595–615 (with an abstract in English)Google Scholar
  22. Kato A, Miyake T, Nishigata K, Tateishi H, Teraoka T, Arie T (2012) Use of fluorescent proteins to visualize interactions between the Bakanae disease pathogen Gibberella fujikuroi and the biocontrol agent Talaromyces sp KNB-422. J Gen Plant Pathol 78(1):54–61CrossRefGoogle Scholar
  23. Knudsen GR, Eschen DJ, Dandurand LM, Bin L (1991) Potential for biocontrol of Sclerotinia sclerotiorum through colonization of sclerotia by Trichoderma harzianum. Plant Dis 75:466–470CrossRefGoogle Scholar
  24. Kredics L, Hatvani L, Naeimi S, Körmöczi P, Manczinger L, Vágvölgyi C, Druzhinina I (2014) Chapter 1: biodiversity of the genus Hypocrea/Trichoderma in different habitats. In: Gupta VK, Schmoll M, Herrera-Estrella A, Upadhyay RS, Druzhinina I, Tuohy M (eds) Biotechnology and biology of Trichoderma. Elsevier, Amsterdam, The Netherlands, pp 3–24CrossRefGoogle Scholar
  25. Lace B, Genre A, Woo S, Faccio A, Lorito M, Bonfante P (2015) Gate crashing arbuscular mycorrhizas: in vivo imaging shows the extensive colonization of both symbionts by Trichoderma atroviride. Env Microbiol Rep 7(1):64–77CrossRefGoogle Scholar
  26. Lewis JA, Papavizas GC (1983) Production of chlamydospores and conidia by Trichoderma spp in liquid and solid growth media. Soil Biol Biochem 15(3):351–357CrossRefGoogle Scholar
  27. López-Escudero FJ, Mercado-Blanco J (2011) Verticillium wilt of olive: a case study to implement an integrated strategy to control a soil-borne pathogen. Plant Soil 344(1):1–50CrossRefGoogle Scholar
  28. López-Escudero FJ, del Río C, Caballero JM, Blanco-López MA (2004) Evaluation of olive cultivars for resistance to Verticillium dahliae. Eur J Plant Pathol 110(1):79–85CrossRefGoogle Scholar
  29. Lorito M, Woo S (2015) Trichoderma: A multi-purpose tool for integrated pest management. In: Lugtenberg B (ed) Principles of plan-microbe interactions. Microbes Sustain Agric, Springer, USA, pp 345–353Google Scholar
  30. Lu ZX, Tombolini R, Woo S, Zeilinger S, Lorito M, Jansson JK (2004) In vivo study of Trichoderma-pathogen-plant interactions, using constitutive and inducible green fluorescent protein reporter systems. Appl Environ Microbiol 70(5):3073–3081CrossRefPubMedPubMedCentralGoogle Scholar
  31. Mercado-Blanco J, Rodríguez-Jurado D, Hervás A, Jiménez-Diaz RM (2004) Suppression of verticillium wilt in olive planting stocks by root-associated fluorescent Pseudomonas spp. Biol Control 30(2):474–486CrossRefGoogle Scholar
  32. Moreno-Mateos MA, Delgado-Jarana J, Codón AC, Benítez T (2007) pH and Pac1 control development and antifungal activity in Trichoderma harzianum. Fungal Genet Biol 44(12):1355–1367CrossRefPubMedGoogle Scholar
  33. Papasotiriou FG, Varypatakis KG, Christofi N, Tjamos SE, Paplomatas EJ (2013) Olive mill wastes: a source of resistance for plants against Verticillium dahliae and a reservoir of biocontrol agents. Biol Control 67(1):51–60CrossRefGoogle Scholar
  34. Papavizas GC (1985) Trichoderma and Gliocladium- biology, ecology, and potential for biocontrol. Annu Rev Phytopathol 23:23–54CrossRefGoogle Scholar
  35. Park D (1954) Chlamydospores and survival in soil fungi. Nature 173:454–455CrossRefPubMedGoogle Scholar
  36. Pegg GF, Brady BL (2002) Verticillium wilts. CAB International, Wallingford, UKCrossRefGoogle Scholar
  37. Penttilä M, Nevalainen H, Rättö M, Salminen E, Knowles J (1987) A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei. Gene 61(2):155–164CrossRefPubMedGoogle Scholar
  38. Pertot I, Gobbin D, De Luca F, Prodorutti D (2008) Methods of assessing the incidence of Armillaria root rot across viticultural areas and the pathogen’s genetic diversity and spatial–temporal pattern in northern Italy. Crop Prot 27(7):1061–1070CrossRefGoogle Scholar
  39. Prieto P, Navarro-Raya C, Valverde-Corredor A, Amyotte SG, Dobinson KF, Mercado-Blanco J (2009) Colonization process of olive tissues by Verticillium dahliae and its in planta interaction with the biocontrol root endophyte Pseudomonas fluorescens PICF7. Microb Biotechnol 2(4):499–511CrossRefPubMedPubMedCentralGoogle Scholar
  40. Raaijmakers JM, Paulitz TC, Steinberg C, Alabouvette C, Moenne-Loccoz Y (2009) The rhizosphere: a playground and battlefield for soilborne pathogens and beneficial microorganisms. Plant Soil 321(1–2):341–361CrossRefGoogle Scholar
  41. Rincón AM, Chaki M, Fierro-Risco J, Valverde-Corredor A, Carreras A, Pérez-Artés E, Begara JC, Valderrama R, Barroso JB, Mercado-Blanco J (2014) Biological control of Verticillium wilt of olive by Trichoderma harzianum. In: XII Meeting of the IOBC/WPRS Working Group, Biological control of fungi and bacterial plant pathogens. Upsala, Sweden, p 78Google Scholar
  42. Ruano-Rosa D, del Moral-Navarrete L, Lopez-Herrera CJ (2010) Selection of Trichoderma spp. isolates antagonistic to Rosellinia necatrix. Span J Agric Res 8(4):1084–1097CrossRefGoogle Scholar
  43. Ruano-Rosa D, Cazorla FM, Bonilla N, Martín-Pérez R, De Vicente A, López-Herrera CJ (2014) Biological control of avocado white root rot with combined applications of Trichoderma spp. and rhizobacteria. Eur J Plant Pathol 138(4):751–762CrossRefGoogle Scholar
  44. Samolski I, Rincón AM, Pinzón LM, Viterbo A, Monte E (2012) The qid74 gene from Trichoderma harzianum has a role in root architecture and plant biofertilization. Microbiol-SGM 158(1):129–138CrossRefGoogle Scholar
  45. Tjamos EC (1993) Prospects and strategies in controlling Verticillium wilt of olive. EPPO Bull 23(3):505–512CrossRefGoogle Scholar
  46. Verma M, Brar SK, Tyagi RD, Surampalli RY, Valéro JR (2007) Antagonistic fungi, Trichoderma spp.: panoply of biological control. Biochem Eng J 37(1):1–20CrossRefGoogle Scholar
  47. Vitullo D, Altieri R, Esposito A, Nigro F, Ferrara M, Alfano G, Ranalli G, De Cicco V, Lima G (2013) Suppressive biomasses and antagonist bacteria for an eco-compatible control of Verticillium dahliae on nursery-grown olive plants. Int J Environ Sci Technol 10(2):209–220CrossRefGoogle Scholar
  48. Yang XM, Chen LH, Yong XY, Shen QR (2011) Formulations can affect rhizosphere colonization and biocontrol efficiency of Trichoderma harzianum SQR-T037 against Fusarium wilt of cucumbers. Biol Fertil Soils 47(3):239–248CrossRefGoogle Scholar
  49. Yedidia I, Benhamou N, Kapulnik Y, Chet I (2000) Induction and accumulation of PR proteins activity during early stages of root colonization by the mycoparasite Trichoderma harzianum strain T-203. Plant Physiol Biochem 38(11):863–873CrossRefGoogle Scholar
  50. Zachow C, Fatehi J, Cardinale M, Tilcher R, Berg G (2010) Strain-specific colonization pattern of Rhizoctonia antagonists in the root system of sugar beet. FEMS Microbiol Ecol 74(1):124–135CrossRefPubMedGoogle Scholar

Copyright information

© International Organization for Biological Control (IOBC) 2015

Authors and Affiliations

  1. 1.Laboratory of Plant-Microorganism Interaction, Department of Crop Protection, Institute for Sustainable AgricultureAgencia Estatal Consejo Superior de Investigaciones Científicas (CSIC)CórdobaSpain
  2. 2.Department Plant Breeding, Institute for Sustainable AgricultureAgencia Estatal Consejo Superior de Investigaciones Científicas (CSIC)CórdobaSpain
  3. 3.Department of Genetics, Faculty of BiologyUniversity of SevilleSevilleSpain
  4. 4.Group of Biochemistry and Cell Signalling in Nitric Oxide, Department of Experimental Biology, Center for Advanced Studies in Olive Grove and Olive OilsUniversity of JaénJaénSpain

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