Can We Define an Experimental Framework to Approach the Genetic Basis of Root Colonization?

  • Ariella Alperovitch-Lavy
  • Benjamin A. HorwitzEmail author
Part of the Rhizosphere Biology book series (RHBIO)


The contributions of Trichoderma to the biocontrol of soilborne disease and plant growth promotion depend on the interaction between the biocontrol fungus, the plant, fungal pathogens, and other organisms in the rhizosphere. In some interactions, Trichoderma colonizes the root surface, epidermis, and outer cortex layers. In contrast to arbuscular or even ectomycorrhizae, the fungal hyphae are not precisely localized to a structure, making it somewhat of a challenge to define and quantify colonization. Following ingress into the root, hyphae grow in the apoplast in a parallel pattern defined by the root tissue, much like pathogens such as Fusarium oxysporum. Trichoderma, however, does not penetrate to inner cortex layers or the vascular tissues. To elucidate the genetic basis of root colonization, one would ideally isolate mutants defective in the process, and this needs a good definition of colonization phenotypes. Here, we compare different methods to follow and quantify Trichoderma entry and proliferation in roots and discuss data obtained for mutants.


Colonization Root cortex Rhizosphere Fungal-plant interaction Symbiont Apoplast 



We thank Rinat Zaid for providing us with unpublished data and Dr. Nitsan Dahan at the Life Sciences & Engineering Center, Technion, for expert assistance with confocal microscopy. Studies of Trichoderma in the Horwitz Lab, including unpublished data shown here, were supported by the US-Israel Agricultural Research and Development Fund (BARD) and the US-Israel Binational Science Foundation (BSF), together with Charles M. Kenerley, Texas A&M University, USA.


  1. Alonso-Ramírez A, Poveda J, Martín I, Hermosa R, Monte E, Nicolás C (2014) Salicylic acid prevents Trichoderma harzianum from entering the vascular system of roots. Mol Plant Pathol 15:823–831. Scholar
  2. Bi F, Barad S, Ment D, Luria N, Dubey A, Casado V, Glam N, Mínguez JD, Espeso EA, Fluhr R, Prusky D (2016) Carbon regulation of environmental pH by secreted small molecules that modulate pathogenicity in phytopathogenic fungi. Mol Plant Pathol 17:1178–1195. Scholar
  3. Białas A, Zess EK, De la Concepcion JC, Franceschetti M, Pennington HG, Yoshida K, Upson JL, Chanclud E, Wu C-H, Langner T, Maqbool A, Varden FA, Derevnina L, Belhaj K, Fujisaki K, Saitoh H, Terauchi R, Banfield MJ, Kamoun S (2018) Lessons in effector and NLR biology of plant-microbe systems. Mol Plant Microbe Interact 31:34–45. Scholar
  4. Bonfante P, Genre A (2010) Mechanisms underlying beneficial plant–fungus interactions in mycorrhizal symbiosis. Nat Commun 1:1–11. Scholar
  5. Brotman Y, Briff E, Viterbo A, Chet I (2008) Role of swollenin, an expansin-like protein from Trichoderma, in plant root colonization. Plant Physiol 147:779–789. Scholar
  6. 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 10:19–27PubMedPubMedCentralGoogle Scholar
  7. Contreras-Cornejo HA, Macias-Rodriguez L, Cortes-Penagos C, Lopez-Bucio J (2009) Trichoderma virens, a plant beneficial fungus, enhances biomass production and promotes lateral root growth through an auxin-dependent mechanism in arabidopsis. Plant Physiol 149:1579–1592. Scholar
  8. Crutcher FK, Moran-Diez ME, Ding S, Liu J, Horwitz BA, Mukherjee PK, Kenerley CM (2015) A paralog of the proteinaceous elicitor SM1 is involved in colonization of maize roots by Trichoderma virens. Fungal Biol 119:476–486. Scholar
  9. Djonovic S, Vargas WA, Kolomiets MV, Horndeski M, Wiest A, Kenerley CM (2007) A proteinaceous elicitor Sm1 from the beneficial fungus trichoderma virens is required for induced systemic resistance in maize. Plant Physiol 145:875–889. Scholar
  10. van der Does HC, Duyvesteijn RGE, Goltstein PM, van Schie CN, Manders EMM, Cornelissen BJC, Rep M (2008) Expression of effector gene SIX1 of Fusarium oxysporum requires living plant cells. Fungal Genet Biol 45:1257–1264. Scholar
  11. Dotson BR, Soltan D, Schmidt J, Areskoug M, Rabe K, Swart C, Widell S, Rasmusson AG (2018) The antibiotic peptaibol alamethicin from Trichoderma permeabilises Arabidopsis root apical meristem and epidermis but is antagonised by cellulase-induced resistance to alamethicin. BMC Plant Biol 18:165. Scholar
  12. 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 9:749–759. Scholar
  13. Elad Y (2003) Biocontrol of foliar pathogens: mechanisms and application. Commun Agric Appl Biol Sci 68:17–24PubMedGoogle Scholar
  14. Engelberth J, Koch T, Schüler G, Bachmann N, Rechtenbach J, Boland W (2001) Ion channel-forming alamethicin is a potent elicitor of volatile biosynthesis and tendril coiling Cross talk between jasmonate and salicylate signaling in lima bean. Plant Physiol 125:369–377CrossRefGoogle Scholar
  15. Franceschetti M, Maqbool A, Jiménez-Dalmaroni MJ, Pennington HG, Kamoun S, Banfield MJ (2017) Effectors of filamentous plant pathogens: commonalities amid diversity. Microbiol Mol Biol Rev 81:e00066-16. Scholar
  16. González-Pérez E, Ortega-Amaro MA, Salazar-Badillo FB, Bautista E, Douterlungne D, Jiménez-Bremont JF (2018) The Arabidopsis-Trichoderma interaction reveals that the fungal growth medium is an important factor in plant growth induction. Sci Rep 8:16427. Scholar
  17. Guzmán-Guzmán P, Alemán-Duarte MI, Delaye L, Herrera-Estrella A, Olmedo-Monfil V (2017) Identification of effector-like proteins in Trichoderma spp and role of a hydrophobin in the plant-fungus interaction and mycoparasitism. BMC Genet 18:16. Scholar
  18. Harman GE, Howell CR, Viterbo A, Chet I, Lorito M (2004) Trichoderma species — opportunistic, avirulent plant symbionts. Nat Rev Microbiol 2:43–56. Scholar
  19. Karlsson M, Atanasova L, Jensen DF, Zeilinger S (2017a) Necrotrophic mycoparasites and their genomes. Microbiol Spectrosc 5:1005–1026. Scholar
  20. Karlsson M, Atanasova L, Jensen DF, Zeilinger S (2017b) Necrotrophic mycoparasites and their genomes. In: The fungal kingdom. American Society of Microbiology, Washington, DC, pp 1005–1026. Scholar
  21. Kloppholz S, Kuhn H, Requena N (2011) A secreted fungal effector of Glomus intraradices promotes symbiotic biotrophy. Curr Biol 21:1204–1209. Scholar
  22. Kubicek CP, Herrera-Estrella A, Seidl-Seiboth V, Martinez DA, Druzhinina IS, Thon M, Zeilinger S, Casas-Flores S, Horwitz BA, Mukherjee PK, Mukherjee M, Kredics L, Alcaraz LD, Aerts A, Antal Z, Atanasova L, Cervantes-Badillo MG, Challacombe J, Chertkov O, McCluskey K, Coulpier F, Deshpande N, von Döhren H, Ebbol DJ, Esquivel-Naranjo EU, Fekete E, Flipphi M, Glaser F, Gómez-Rodríguez EY, Gruber S, Han C, Henrissat B, Hermosa R, Hernández-Oñate M, Karaffa L, Kosti I, Le Crom S, Lindquist E, Lucas S, Lübeck M, Lübeck PS, Margeot A, Metz B, Misra M, Nevalainen H, Omann M, Packer N, Perrone G, Uresti-Rivera EE, Salamov A, Schmoll M, Seiboth B, Shapiro H, Sukno S, Tamayo-Ramos JA, Tisch D, Wiest A, Wilkinson HH, Zhang M, Coutinho PM, Kenerley CM, Monte E, Bake SE, Grigoriev IV (2011) Comparative genome sequence analysis underscores mycoparasitism as the ancestral life style of Trichoderma. Genome Biol 12:R40. Scholar
  23. Lahrmann U, Ding Y, Banhara A, Rath M, Hajirezaei MR, Döhlemann S, von Wirén N, Parniske M, Zuccaro A (2013) Host-related metabolic cues affect colonization strategies of a root endophyte. Proc Natl Acad Sci 110:13965–13970. Scholar
  24. Lamdan N-L, Shalaby S, Ziv T, Kenerley CM, Horwitz BA (2015) Secretome of trichoderma interacting with maize roots: role in induced systemic resistance. Mol Cell Proteomics 14:1054–1063. Scholar
  25. Lorito M, Woo SL, Harman GE, Monte E (2010) Translational research on Trichoderma: from ’omics to the field. Annu Rev Phytopathol 48:395–417. Scholar
  26. Lu Z, 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 70:3073–3081CrossRefGoogle Scholar
  27. Luo Y, Zhang D-D, Dong X-W, Zhao P-B, Chen L-L, Song X-Y, Wang X-J, Chen XL, Shi M, Zhang Y-Z (2010) Antimicrobial peptaibols induce defense responses and systemic resistance in tobacco against tobacco mosaic virus. FEMS Microbiol Lett 313:120–126. Scholar
  28. Martinez C, Blanc F, Le Clair E, Besnard O, Nicole M, Baccou JC (2001) Salicylic acid and ethylene pathways are differentially activated in melon cotyledons by active or heat-denatured cellulase from Trichoderma longibrachiatum. Plant Physiol 127:334–344CrossRefGoogle Scholar
  29. Martínez-Medina A, Appels FW, van Wees SCM (2017) Impact of salicylic acid- and jasmonic acid-regulated defences on root colonization by Trichoderma harzianum T-78. Plant Signal Behav 12:e1345404. Scholar
  30. Masachis S, Segorbe D, Turrà D, Leon-Ruiz M, Fürst U, El Ghalid M, Leonard G, López-Berges MS, Richards TA, Felix G, Di Pietro A (2016) A fungal pathogen secretes plant alkalinizing peptides to increase infection. Nat Microbiol 1:16043. Scholar
  31. Mathys J, De Cremer K, Timmermans P, Van Kerckhove S, Lievens B, Vanhaecke M, Cammue BA, De Coninck B (2012) Genome-wide characterization of ISR induced in Arabidopsis thaliana by Trichoderma hamatum T382 against Botrytis cinerea infection. Front Plant Sci 3:108. Scholar
  32. Minker KR, Biedrzycki ML, Kolagunda A, Rhein S, Perina FJ, Jacobs SS, Moore M, Jamann TM, Yang Q, Nelson R, Balint-Kurti P, Kambhamettu C, Wisser RJ, Caplan JL (2018) Semiautomated confocal imaging of fungal pathogenesis on plants: microscopic analysis of macroscopic specimens. Microsc Res Tech 81:141–152. Scholar
  33. Morán-Diez E, Hermosa R, Ambrosino P, Cardoza RE, Gutiérrez S, Lorito M, Monte E (2009) The ThPG1 endopolygalacturonase is required for the Trichoderma harzianum – plant beneficial interaction. Mol Plant Microbe Interact 22:1021–1031. Scholar
  34. Morán-Diez ME, Trushina N, Lamdan N, Rosenfelder L, Mukherjee PK, Kenerley CM, Horwitz BA (2015) Host-specific transcriptomic pattern of Trichoderma virens during interaction with maize or tomato roots. BMC Genomics 16:8. Scholar
  35. Nieto-Jacobo MF, Steyaert JM, Salazar-Badillo FB, Nguyen DV, Rostás M, Braithwaite M, De Souza JT, Jimenez-Bremont JF, Ohkura M, Stewart A, Mendoza-Mendoza A (2017) Environmental growth conditions of trichoderma spp. affects indole acetic acid derivatives, volatile organic compounds, and plant growth promotion. Front Plant Sci 8:102. Scholar
  36. Nogueira-Lopez G, Greenwood DR, Middleditch M, Winefield C, Eaton C, Steyaert JM, Mendoza-Mendoza A (2018) The apoplastic secretome of Trichoderma virens during interaction with maize roots shows an inhibition of plant defence and scavenging oxidative stress secreted proteins. Front Plant Sci 9:409. Scholar
  37. Oldroyd GED, Harrison MJ, Paszkowski U (2009) Reprogramming plant cells for endosymbiosis. Science 324:753–754. Scholar
  38. Parke JL (1991) Root colonization by indigenus and introduced microorganisms. In: The rhizosphere and plant growth. Springer, Dordrecht, pp 33–42. Scholar
  39. Pelagio-Flores R, Esparza-Reynoso S, Garnica-Vergara A, López-Bucio J, Herrera-Estrella A (2017) Trichoderma-induced acidification is an early trigger for changes in arabidopsis root growth and determines fungal phytostimulation. Front Plant Sci 8:822. Scholar
  40. Peñalva MA, Tilburn J, Bignell E, Arst HN (2008) Ambient pH gene regulation in fungi: making connections. Trends Microbiol 16:291–300. Scholar
  41. Plett J, Martin F (2011) Blurred boundaries: lifestyle lessons from ectomycorrhizal fungal genomes. Trends Genet 27:14–22. Scholar
  42. Plett JM, Kemppainen M, Kale SD, Kohler A, Legué V, Brun A, Tyler BM, Pardo AG, Martin F (2011) A secreted effector protein of laccaria bicolor is required for symbiosis development. Curr Biol 21:1197–1203. Scholar
  43. Plett JM, Daguerre Y, Wittulsky S, Vayssieres A, Deveau A, Melton SJ, Kohler A, Morrell-Falvey JL, Brun A, Veneault-Fourrey C, Martin F (2014) Effector MiSSP7 of the mutualistic fungus Laccaria bicolor stabilizes the Populus JAZ6 protein and represses jasmonic acid (JA) responsive genes. Proc Natl Acad Sci 111:8299–8304. Scholar
  44. Rippa S, Eid M, Formaggio F, Toniolo C, Béven L (2010) Hypersensitive-like response to the pore-former peptaibol alamethicin in Arabidopsis thaliana. Chembiochem 11:2042–2049. Scholar
  45. Rodriguez RJ, White JF Jr, Arnold AE, Redman RS (2009) Fungal endophytes: diversity and functional roles. New Phytol 182:314–330. Scholar
  46. Rotblat B, Enshell-Seijffers D, Gershoni JM, Schuster S, Avni A (2002) Identification of an essential component of the elicitation active site of the EIX protein elicitor. Plant J 32:1049–1055CrossRefGoogle Scholar
  47. Rubio MB, Dominguez S, Monte E, Hermosa R (2012) Comparative study of Trichoderma gene expression in interactions with tomato plants using high-density oligonucleotide microarrays. Microbiology 158:119–128. Scholar
  48. Ryder LS, Harris BD, Soanes DM, Kershaw MJ, Talbot NJ, Thornton CR (2012) Saprotrophic competitiveness and biocontrol fitness of a genetically modified strain of the plant-growth-promoting fungus Trichoderma hamatum GD12. Microbiology 158:84–97. Scholar
  49. Salas-Marina MA, Isordia-Jasso MI, Islas-Osun MA, Delgado-Sánchez P, Jiménez-Bremont JF, Rodríguez-Kessler M, Rosales-Saavedra MT, Herrera-Estrella A, Casas-Flores S (2015) The Epl1 and Sm1 proteins from Trichoderma atroviride and Trichoderma virens differentially modulate systemic disease resistance against different life style pathogens in Solanum lycopersicum. Front Plant Sci 6:77. Scholar
  50. Saloheimo M, Paloheimo M, Hakola S, Pere J, Swanson B, Nyyssönen E, Bhatia A, Ward M, Penttilä M (2002) Swollenin, a Trichoderma reesei protein with sequence similarity to the plant expansins, exhibits disruption activity on cellulosic materials. Eur J Biochem 269:4202–4211CrossRefGoogle Scholar
  51. Saravanakumar K, Fan L, Fu K, Yu C, Wang M, Xia H, Sun J, Li Y, Chen J (2016) Cellulase from Trichoderma harzianum interacts with roots and triggers induced systemic resistance to foliar disease in maize. Sci Rep 6:35543. Scholar
  52. Sarrocco S, Matarese F, Baroncelli R, Vannacci G, Seidl-Seiboth V, Kubicek CP, Vergara M (2017) The constitutive endopolygalacturonase TvPG2 regulates the induction of plant systemic resistance by Trichoderma virens. Phytopathology 107:537–544. Scholar
  53. Schmoll M, Dattenböck C, Carreras-Villaseñor N, Mendoza-Mendoza A, Tisch D, Alemán MI, Baker SE, Brown C, Cervantes-Badillo MG, Cetz-Chel J, Cristobal-Mondragon GR, Delay L, Esquivel-Naranjo EU, Frischmann A, Gallardo-Negrete JJ, García-Esquivel M, Gomez-Rodriguez EY, Greenwood DR, Hernández-Oñate M, Kruszewska JS, Lawry R, Mora-Montes HM, Muñoz-Centeno T, Nieto-Jacobo MF, Nogueira Lopez G, Olmedo-Monfil V, Osorio-Concepcion M, Piłsyk S, Pomraning K, Rodriguez-Iglesias A, Rosales-Saavedra MT, Sánchez-Arreguín JA, Seidl-Seiboth V, Stewart A, Uresti-Rivera EE, Wang C-L, Wang T-F, Zeilinger S, Casas-Flores S, Herrera-Estrella A (2016) The genomes of three uneven siblings: footprints of the lifestyles of three trichoderma species. Microbiol Mol Biol Rev 80:205–327. Scholar
  54. Shoresh M, Harman GE, Mastouri F (2010) Induced systemic resistance and plant responses to fungal biocontrol agents. Annu Rev Phytopathol 48:21–43. Scholar
  55. Thürich J, Meichsner D, Furch ACU, Pfalz J, Krüger T, Kniemeyer O, Brakhage A, Oelmüller R (2018) Arabidopsis thaliana responds to colonisation of Piriformospora indica by secretion of symbiosis-specific proteins. PLoS One 13:e0209658. Scholar
  56. Tisserant E, Malbreil M, Kuo A, Kohler A, Symeonidi A, Balestrin R, Charron P, Duensing N, Frei dit Frey N, Gianinazzi-Pearson V, Gilbert LB, Handa Y, Herr JR, Hijri M, Koul R, Kawaguchi M, Krajinski F, Lammers PJ, Masclaux FG, Murat C, Morin E, Ndikumana S, Pagni M, Petitpierre D, Requena N, Rosikiewicz P, Riley R, Saito K, San Clemente H, Shapiro H, van Tuinen D, Becard G, Bonfante P, Paszkowski U, Shachar-Hill YY, Tuskan GA, Young JPW, Sanders IR, Henrissat B, Rensing SA, Grigoriev IV, Corradi N, Roux C, Martin F (2013) Genome of an arbuscular mycorrhizal fungus provides insight into the oldest plant symbiosis. Proc Natl Acad Sci 110:20117–20122. Scholar
  57. Trushina N, Levin M, Mukherjee PK, Horwitz BA (2013) PacC and pH–dependent transcriptome of the mycotrophic fungus Trichoderma virens. BMC Genomics 14:138. Scholar
  58. Viterbo A, Chet I (2006) TasHyd1, a new hydrophobin gene from the biocontrol agent Trichoderma asperellum, is involved in plant root colonization. Mol Plant Pathol 7:249–258. Scholar
  59. Viterbo A, Wiest A, Brotman Y, Chet I, Kenerley C (2007) The 18mer peptaibols from Trichoderma virens elicit plant defence responses. Mol Plant Pathol 8:737–746. Scholar
  60. Weaver MA, Kenerley CM (2008) Competitiveness of a genetically engineered strain of Trichoderma virens. Mycopathologia 166:51–59. Scholar
  61. Yedidia II, Benhamou N, Chet II (1999) Induction of defense responses in cucumber plants (Cucumis sativus L.) By the biocontrol agent Trichoderma harzianum. Appl Environ Microbiol 65:1061–1070CrossRefGoogle Scholar
  62. Yu C, Dou K, Wang S, Wu Q, Ni M, Zhang T, Lu Z, Tang J, Chen J (2019) Elicitor hydrophobin Hyd1 interacts with Ubiquilin1-like to induce maize systemic resistance. J Integr Plant Biol:12796.

Copyright information

© Springer Nature Singapore Pte Ltd. 2020

Authors and Affiliations

  • Ariella Alperovitch-Lavy
    • 1
  • Benjamin A. Horwitz
    • 1
    Email author
  1. 1.Faculty of BiologyTechnion – Israel Institute of TechnologyHaifaIsrael

Personalised recommendations