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
1
, Pilar Prieto
2
, Ana María Rincón
3
, María Victoria Gómez-
Rodríguez
4
, Raquel Valderrama
4
, Juan Bautista Barroso
4
, Jesús Mercado-Blanco
1*
Departments of
1
Crop Protection and
2
Plant Breeding, Institute for Sustainable
Agriculture, Agencia Estatal Consejo Superior de Investigaciones Científicas (CSIC),
Córdoba, Spain.
3
Department of Genetics, Faculty of Biology, University of Seville, Spain.
4
Group of Biochemistry and Cell Signalling in Nitric Oxide, Department of
Experimental Biology, Center for Advanced Studies in Olive Grove and Olive Oils,
University of Jaén, Jaén, Spain.
1
Dr. Jesús Mercado-Blanco
Institute for Sustainable Agriculture (CSIC); Department of Crop Protection;
Laboratory of Plant-Microorganism Interaction; C/ Alameda del Obispo s/n
CP 14080; Córdoba, Spain
jesus.mercado@ias.csic.es
author(s) details
David Ruano-Rosa is a postdoctoral researcher skilled on integrated control of soil-
borne pathogens of woody plants, on the selection and characterization of biological
control agents with special interest in the genera T richoderma and Pseudomonas, and
on the study of phytopathogen-biological control agent interactions.
Pilar Prieto is interested in the analysis of genome organization in cereals, particularly
rice and wheat, combining confocal microscopy with genomic and proteomic
approaches. The study of chromosome associations during meiosis is a relevant research
topic. She aims to transfer agronomic traits into wheat by manipulating chromosome
associations to promote inter-specific recombination between wheat and related species
such as wild and cultivated barley. The study of the colonization processes of cereals
and olive tissues by microorganisms using confocal microscopy is another research
area.
Ana Rincón is researcher and lecturer at the University of Seville, Spain. Her work has
been focused on molecular genetics and improvement of strains of Trichoderma spp. as
biological control agents since 1997. She has taken part in numerous projects to develop
Trichoderma-based formulations for crop protection.
María Victoria Gómez-Rodríguez is lecturer in plant physiology at the Universidad de
Jaén, Spain, since 23 years ago. Her main research interests are plant-pathogen
interaction and plant induced resistance mechanisms against oomycetes."
Raquel Valderrama is professor in Biochemistry and Molecular Biology and her
investigation is focused on the study of the response of antioxidant systems and several
reactive oxygen and nitrogen species (ROS and RNS) in plants during development and
biotic /abiotic stress conditions.
Juan B. Barroso is full professor of Biochemistry and Molecular Biology and his
research is focused on the study of the metabolism of several reactive oxygen and
nitrogen species (ROS and RNS) and their implications on the plant defense
mechanisms against different biotic and abiotic stress conditions.
Jesús Mercado-Blanco is tenured scientist in the Institute for Sustainable Agriculture
(Spanish National Research Council, CSIC). His main research interests focus on
agricultural microbiology and biotechnology and the development of control tools
within integrated disease management strategies, using Verticillium wilt of olive as
study model. Specific research topics are the bases underlying plant-microbe
interactions by molecular and ‘-omic’ approaches and the identification,
characterization and use of microbiological control agents, with emphasis on bacterial
endophytes.
author(s) biography
1
Fate of Trichoderma harzianum in the olive rhizosphere: time course of the root
1
colonization process and interaction with the fungal pathogen Verticillium dahliae
2
3
Abstract
4
Trichoderma harzianum Rifai is a well-known biological control agent (BCA) effective
5
against a wide range of phytopathogens. Since colonization and persistence in the target
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niche is crucial for biocontrol effectiveness we aimed to: (i) shed light on the olive roots
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colonization process by T. harzianum CECT 2413, (ii) unravel the fate of its biomass
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upon application; and (iii) study the in planta interaction with the soil-borne pathogen
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Verticillium dahliae Kleb. Fluorescently-tagged derivatives of CECT 2413 and V.
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dahliae and confocal laser scanning microscopy were used. In vitro assays showed for
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the first time mycoparasitism of V. dahliae by T. harzianum, evidenced by events such
12
as hyphal coiling. In planta assays revealed that CECT 2413 profusely colonized the
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rhizoplane of olive roots. Interestingly, biomass of the BCA was visualized mainly as
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chlamydospores. This observation was independent on the presence or absence of the
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pathogen. Evidence of inner colonization of olive roots by CECT 2413 was not
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obtained. These results suggest that CECT 2413 is not able to persist in a metabolically-
17
active form when applied as a spore suspension. This may have strong implications in
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the way this BCA should be introduced and/or formulated to be effective against
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Verticillium wilt of olive.
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21
Keywords
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Chlamydospores, Confocal laser scanning microscopy, Mycoparasitism, Olea europaea
23
L., Trichoderma harzianum Rifai, Root colonization, Verticillium dahliae Kleb.,
24
Verticillium wilt.
25
Revised (blind) manuscript indicating where changes were made
2
Introduction
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A growing social demand for agricultural products free of harmful compounds for both
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human and animal health and for the environment has encouraged the research on
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biological control agents (BCA) as an alternative to chemical-based products for
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effective crop diseases management. Among microorganisms studied and employed as
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BCA, species of the filamentous fungal genus Trichoderma arise as one of the most
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outstanding. Trichoderma spp. are thus considered the BCA par excellence among fungi
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due to: i) their well-documented antimicrobial activity, consequence of mechanisms
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(not mutually exclusive) such as antibiosis, mycoparasitism and/or competition
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(Harman and Kubicek 1998; Verma et al. 2007); ii) their ability to induce systemic
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resistance (Harman et al. 2004; Contreras-Cornejo et al. 2013); and/or iii) their positive
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effects on seed germination and plant growth (e.g. Hermosa et al. 2012). Moreover,
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Trichoderma spp. are highly versatile and show cosmopolitan distribution (Druzhinina
38
et al. 2011; Kredick et al. 2014). Taking into account all these characteristics,
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Trichoderma spp. are widely selected as BCA against soil-borne pathogens (e.g. Ruano-
40
Rosa et al. 2010; 2014), and constitute the base of many registered bioformulations
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worldwide (Verma et al. 2007; Lorito and Woo 2015). Even though our knowledge on
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mechanisms underlying biocontrol exerted by Trichoderma spp. is abundant, there is
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still an important lack of information on how Trichoderma spp. interact with the host
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plant and the target phytopathogen in a scenario such as the rhizosphere, where multiple
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trophic interactions take place (Raaijmakers et al. 2009). Research pursuing this aim is
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scant and therefore needed to better understand the fate of this BCA once applied to
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roots or soil, especially in the case of woody plants with large root systems.
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Trichoderma harzianum Rifai is employed as BCA against a wide range of plant
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pathogens, such as Fusarium oxysporum f. sp. phaseoli (Carvalho et al. 2014).
50
3
Trichoderma harzianum CECT 2413 is a well-documented isolate of this genus due to
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its demonstrated mycoparasitic activity against different pathogens, for instance
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Rhizoctonia meloni and Phytophthora citrophthora (Moreno-Mateos et al. 2007), and
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its plant growth promotion capability (Chacón et al. 2007). Related to root colonization,
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CECT 2413 has mostly been studied on non-woody plants like tomato (Solanum
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lycopersicum L.) (Chacón et al. 2007), cucumber (Cucumis sativus L.) (Samolski et al.
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2012) or Arabidopsis thaliana L. (Alonso-Ramírez et al. 2014).
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Verticillium wilt of olive (Olea europaea L.) (VWO) is caused by the soil-borne
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fungus Verticillium dahliae Kleb. This disease is considered one of the most important
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biotic constraints for olive cultivation in many regions, particularly in the
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Mediterranean Basin. Unfortunately, VWO is very difficult to control and must be
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confronted by means of an integrated disease management (IDM) strategy (López-
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Escudero and Mercado-Blanco 2011). An interesting approach to control VWO in a
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sustainable, environmentally-friendly way and within IDM frameworks is by using
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BCA, particularly at the nursery production stage (Tjamos 1993). So far, however, only
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a few reports have demonstrated the effectiveness of BCA against VWO (Mercado-
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Blanco et al. 2004; Prieto et al. 2009), identified a number of taxa with potential to
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control V. dahliae (Papasotiriou et al. 2013), or used promising combinations of BCA
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and organic amendments (Vitullo et al. 2013). Trichoderma spp. have also been
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investigated either on their potential to antagonize V. dahliae in nurseries potting mixes
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(Aleandri et al. 2015), or as bioformulations against VWO caused by the defoliating (D)
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pathotype of V. dahliae (Jiménez-Díaz et al. 2009). Recently, the use of a formulation
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based on T. harzianum CECT 2413 to control VWO has been patented and licensed
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(Spanish patent number ES 2393728 A1, Barroso et al. 2014). Isolate CECT 2413 has
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been demonstrated to be an effective in vitro antagonist against different isolates of V.
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