Interactions between Trichoderma harzianum and

Interactions between Trichoderma harzianum and defoliating Verticillium dahliae in

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resistant and susceptible wild olive clones

I. Carrero-Carrónab, M. B. Rubioa, J. Niño-Sáncheza, J. A. Navas-Cortésc, R. M. Jiménez-Díazbc, E. Montea and R. Hermosaa*

a

Spanish-Portuguese Institute for Agricultural Research (CIALE), Department of

Microbiology and Genetics, University of Salamanca, Río Duero 12, Campus de Villamayor, 37185 Salamanca, Spain b

Departamento de Agronomía, College of Agriculture and Forestry (ETSIAM), Universidad

de Córdoba, Alameda del Obispo s/n, 14080 Córdoba, Spain c

Instituto de Agricultura Sostenible (IAS), Consejo Superior de Investigaciones Científicas

(CSIC), Avda. Menéndez Pidal s/n, 14080 Córdoba, Spain * Corresponding author: Rosa Hermosa; Telephone: +34 923 294500 (ext. 5116); Fax: + 34 923 294399; E-mail: [email protected]

Abstract Verticillium wilt (VW) in olive is best managed by an integrated disease management strategy, of which use of host resistance is a key element. The widespread occurrence of a highly virulent defoliating (D) Verticillium dahliae pathotype has jeopardized the use of commercial olive cultivars lacking sufficient resistance to this pathogen. However, the This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/ppa.12879 This article is protected by copyright. All rights reserved.

combined use of resistant wild olive rootstocks and Trichoderma spp. effective in the

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biocontrol of VW can improve the management of VW in olive. In vivo interactions between D V. dahliae and Trichoderma harzianum were studied in olive and wild olive plants displaying different degrees of resistance against this pathogen using confocal microscopy. This multitrophic system included: wild olive clones ‘Ac-4’ and ‘Ac-15’, and olive cv. Picual, and the fungal fluorescent transformants T. harzianum GFP22 and V. dahliae V138IYFP, this latter being obtained herein. In planta observations indicated that V138I-YFP colonizes the roots and stems of those olive and wild olive genotypes, and that GFP22 grows endophytically within the roots all of them. YFP fluorescence signal quantifications showed that: i) the degree of root and stem colonization by the pathogen varied depending upon the susceptibility of the tested wild olive genotype, being that higher in ‘Ac-15’ than in ‘Ac-4’ plants; and ii) treatment with T. harzianum GFP22 reduced the extent of pathogen growth in both clones. Moreover, root colonization by strain GFP22 reduced the percentage of pathogen colonies recovered from stems of olive and wild olive plants.

Keywords: Verticillium wilt; Olea europaea; biological control agents; confocal microscopy

Introduction

Verticillium dahliae Kleb. is a vascular-colonizing, soilborne, mitosporic fungus that can survive in the soil by means of melanized microsclerotia without a host for at least 14 years and cause vascular diseases known as Verticillium wilts (VW) in a wide range of economically important crops (Klosterman et al., 2009). This pathogen has a clonal population structure in which nine clonal lineages have been identified that correlate with vegetative compatibility groups (VCGs), most of which show strong evidence of having

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arisen by recombination (Milgroom et al., 2014). Populations of V. dahliae also comprise two

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types of pathogenic variation, namely defoliating (D) and non-defoliating (ND) pathotypes (symptom types), and pathogenic races 1 and 2 that are correlated with VCGs and clonal lineages (Korolev et al., 2008; Milgroom et al., 2016; Jiménez-Díaz et al., 2017). Isolates of the D pathotype cause defoliation of cotton, olive and okra only and are exclusively in VCG1A and lineage 1A, whereas isolates of the ND pathotype do not defoliate those plant species and are in any of all other VCGs and lineages (Collado-Romero et al., 2006; Korolev et al., 2008; Milgroom et al., 2016; Jiménez-Díaz et al., 2017). Similarly, isolates of race 1, which is avirulent on tomato plants with resistance gene Ve1 (de Jonge et al., 2012), are found mostly in lineage 2A and belong to the ND pathotype, whereas the Ve1-resistance breaking isolates of race 2 are in all other lineages and the D and ND pathotypes (JiménezDíaz et al., 2017). All together makes VWs amongst the most devastating and challenging diseases to

manage in agricultural production worldwide, and determines the need of integrated disease management strategies for its efficient control (Inderbitzin & Subbarao, 2014). One of best examples of such a need is VW in olive (Olea europaea L. subsp. europaea var. europaea). This disease has become a major threat to olive production in Spain and elsewhere because of the widespread occurrence of the D pathotype that is highly virulent on the most widely grown olive cultivars, such as ‘Arbequina’ and ‘Picual’ (Jiménez-Díaz et al., 2011; 2012; Milgroom et al., 2016). A suitable strategy for the integrated VW management in susceptible olive cultivars would be the use of selected wild olive (O. europaea L. subsp. europaea var. sylvestris) rootstocks that have shown a high degree of resistance to D V. dahliae (JiménezFernández et al., 2016; Palomares-Rius et al., 2016; Jiménez-Díaz et al., 2017), together with root treatments with biological control agents (BCAs) (Tjamos et al., 2004; Jiménez-Díaz et al., 2012).


Strains of Trichoderma spp. offer a good choice for that integrated management

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strategy of VW in olive because the biocontrol activity of most strains is well understood (Lorito et al., 2010), including a beneficial growth promotion and induction of systemic defense in the host plant (Shoresh et al., 2010; Hermosa et al., 2012). For example, root treatments with a commercial Trichoderma formulation reduced severity of infections by D V. dahliae by over 30% in ‘Picual’ olives under conditions conducive for severe disease in field microplots (Jiménez-Díaz et al., 2009). Different mechanisms regarding the direct inhibition of V. dahliae by Trichoderma strains have been reported recently, which are directly related to the reduction of VW symptoms severity and progress in susceptible ‘Picual’ olive (Carrero-Carrón et al., 2016; Ruano-Rosa et al., 2016). Moreover, an olive growth promotion effect was observed in plants treated with each of two Trichoderma asperellum strains tested, and isolations from disinfested ‘Picual’ roots pieces indicated that they were colonized endophytically (Carrero-Carrón et al., 2016). Also, T. harzianum strain CECT 2413 was shown to bear biocontrol potential against D V. dahliae in ‘Picual’ plants (Ruano-Rosa et al., 2016), but authors failed to observe endophytic colonization of olive roots by the fluorescently-tagged derivative of this strain (GFP22) and claimed that biocontrol capacity was associated with colonization of their rhizoplane. Confocal laser scanning microscopy (CLSM) has allowed studying the interactions of

D V. dahliae and/or Pseudomonas fluorescens PICF7 in olive plants (Prieto & MercadoBlanco, 2008; Prieto et al., 2009). Studies also showed that P. fluorescens PICF7 is endophytic and triggers systemic defense responses in olive cultivars that differ in susceptibility to D V. dahliae (Gómez-Lama Cabanás et al., 2014, 2015), which may contribute to its previously reported biocontrol potential against VW in olive (MercadoBlanco et al., 2004).


In this present study, using confocal microscopy, we have comparatively investigated

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the colonization by D V. dahliae of wild olive clones ‘Ac-4’ and ‘Ac-15’, which are resistant and susceptible to this pathogen respectively (Palomares-Rius et al., 2016; Jiménez-Díaz et al., 2017), and the role of T. harzianum hindering this process. Neither the endophytic root colonization of wild olive plants by Trichoderma nor its biocontrol potential against D V. dahliae in resistant and susceptible wild olive genotypes were previously studied. A better understanding of these features would be of help for increased efficiency in the integrated management of VW of olive.

Materials and Methods

Fungal strains Trichoderma harzianum GFP22, a GFP-marked strain derived from the wild type strain T. harzianum CECT 2413 (Spanish Type Culture Collection, Burjassot, Spain), kindly provided by Dra. Ana Rincón (University of Seville, Spain), was used as antagonistic fungus. Strains CECT 2413 (referred to as T34 strain) and GFP22 are commonly used in basic biocontrol (Lorito et al., 2010) and confocal (Samolski et al., 2012; Alonso-Ramírez et al., 2014; Ruano-Rosa et al., 2016) studies, respectively. Monoconidial V. dahliae V-138I isolate (Department of Crop Protection culture collection, Instituto de Agricultura Sostenible, CSIC, Córdoba, Spain) (referred to as V-138I strain), previously typed to linage 1A, D pathotype and race 2 (Jiménez-Díaz et al., 2017), was used as a host in the transformation experiments to express YFP in V. dahliae. Fungal strains were routinely grown on potato dextrose agar (PDA, Difco-Becton Dickinson, Sparks, MD) on Petri dishes at 25 ºC in the dark. Conidia of T. harzianum strains were stored in test tubes at -80 ºC in 30% glycerol. V. dahliae cultures


on plum-lactose-yeast extract agar (PLYA) in test tubes covered with liquid paraffin were

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stored at 4 ºC in the dark (Bejarano-Alcázar et al., 1996). Active cultures of V. dahliae were obtained by placing small agar plugs from stock cultures on chlortetracycline-amended water agar (1 L distilled water, 20 g agar, 30 mg chlortetracycline) and further subculturing on PDA.

Plant material Four-month-old, 40-45 cm height plants of wild olive ‘Ac-4’ and ‘Ac-15’, and of ‘Picual’ olive, certified free from V. dahliae, were used in this work. These plants were propagated by Plantas Continental, S.A. (Posadas, Córdoba, Spain) as previously described (Carrero-Carrón et al., 2016). ‘Ac-4’ is resistant (Palomares-Rius et al., 2016; Jiménez-Díaz et al., 2017), and ‘Ac-15’ and ‘Picual’ are highly susceptible, to D V. dahliae (Jiménez-Díaz et al., 2012).

Development of YFP-expressing strains of Verticillium dahliae Plasmid pRF-HUE-YFP was constructed to express YFP in V. dahliae under the control of the glyceraldehyde 3-phosphate dehydrogenase constitutive promoter of Aspergillus nidulans.

The

pair

of

(GGACTTAAUATGGTGAGCAAGGGCGAGGAG)

primers

YFP-UserF

and

YFP-UserR

(GGGTTTAAUTTACTTGTACAGCTCGTCCATGCCG) was used to amplify a 720- bp DNA fragment containing the coding region of eYFP using as template the plasmid pFLG (Gong et al., 2015). Both primers included the sequences (underlined letters) needed to ligate the PCR product to plasmid pRF-HUE digested with PacI and Nt.BbvCI USERTM enzymes (Frandsen et al., 2008). Plasmid pRF-HUE-YFP was used to genetically transform V. dahliae by means of the Agrobacterium tumefaciens-mediated transformation procedure as previously described Mullins et al. (2001). Transformants were selected on PDA medium


containing 100 and 300 g of hygromycin B and of cefotaxime mL-1, respectively. Selection

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of transformants was done after four alternative rounds of culturing on selective (PDA containing 100 g of hygromycin B mL-1) and non-selective medium (PDA). The acquired fluorescence was visualized in hyphae of four transformant strains grown in PDB medium at 25 ºC and 125 rpm in the dark for 4 days, using confocal microscopy. In vitro assays were performed to compare mycelial growth rate of V138I-YFP and its

response to antagonistic T. harzianum strains with those of the wild-type V. dahliae parent strain V-138I. For comparison of mycelia growth, agar plugs cut from growing colonies of V. dahliae strains were placed at the center of Petri dishes with PDA or PLYA media, incubated at 25 °C in the dark, and examined daily for 12 days. To comparatively test for inhibition of hyphal growth of both V. dahliae strains by T. harzianum strains T34 and GFP22, in vitro dual culture assays on PDA and PLYA were performed using V. dahliae strains V-138I and V138I-YFP as targets. These assays were performed as previously described (Carrero-Carrón et al., 2016), except for data of radial growth data which were recorded at 7 and 3 days after plating the strains of V. dahliae and T. harzianum, respectively. Transformant V138I-YFP was tested for pathogenicity on olive cv. Picual as

previously described (Carrero-Carrón et al., 2016), using infested cornmeal-sand mixture (CMS; sand: cornmeal: deionized water, 9:1:2, w/w) and six replicated plants for each of V138I-YFP-infested and -uninfested treatments. The experiment lasted for 7 weeks. The disease reaction in the plants was assessed by the severity of foliar symptoms assessed on individual plants on a 0 to 4 rating scale according to the percentage of affected leaves and twigs at weekly intervals, as previously described (Mercado-Blanco et al., 2004).


In vivo assay of plants colonization by Verticillium dahliae and Trichoderma harzianum

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Four-month-old plants of ‘Picual’, ‘Ac-15’ and ‘Ac-4’ were used. The experiment consisted of four treatments: i) uninoculated control (T1), ii) inoculation with T. harzianum GFP22 (T2), iii) inoculation with V. dahliae V138I-YFP (T3), and iv) inoculation with T. harzianum GFP22 and V. dahliae V138I-YFP (T4). For joint inoculations in T4, plants were first inoculated with strain GFP22, incubated for 12 days (see below), and then inoculated with strain V138I-YFP. Inocula consisted of conidia of T. harzianum GFP22 and V. dahliae V138I-YFP produced on PDA at 25 ºC for 7 and 12 days, respectively. Conidia from PDA cultures were harvested by adding 5 mL of sterile water to the plates and scraping the colonies surface with a rubber spatula. Conidia suspensions were filtered through eight layers of sterile cheesecloth and inoculum concentrations were adjusted to 1 x 107 and 3 x 107 conidia mL-1 for GFP22 and V138I-YFP, respectively, with sterile water using a haemocytometer. Viability of conidia in the inoculum suspensions was determined by dilution plating on PDA and incubating at 25 ºC in the dark for 1 and 3 days for GFP22 and V138I-YFP, respectively. Plants were grown in a pasteurized soil mixture (clay loam: peat, 2:1, v/v; pH 8.4, 24% water holding capacity) in 0.9 L (9 x 9 x 11 cm) disinfested plastic pots (one plant per pot). Plants in pots were first inoculated with T. harzianum GFP22 by drenching the soil mixture around a plant with 60 mL of a suspension of 1 x 107 conidia mL-1, followed by additional 20 mL of sterile water to allow for conidia of GFP22 strain being washed down the soil profile. Control plants were treated similarly except for the absence of T. harzianum GFP22 inoculum. T. harzianum GFP22-inoculated and control plants were incubated in a growth chamber adjusted to 22 ± 2 ºC, 60 to 80% relative humidity and a 14-h photoperiod of fluorescent light of 360 μmol m–2 s–1 for 12 days. Thereafter, plants were carefully uprooted to retain most of rhizosphere soil, and transplanted to 1.5-L (11 × 11 × 13 cm) disinfested plastic pots (one plant per pot) filled with the pasteurized soil mixture of


above infested with 100 mL of a 3 x 107 conidia mL-1 of V. dahliae V138I-YFP to reach an

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inoculum concentration of 2 x 106 conidia mL-1. Control plants were treated similarly except for the absence of V. dahliae V138I-YFP inoculum. A total of 56 plants of each of ‘Picual’, ‘Ac-15’ and ‘Ac-4’ (14 replicated plants per treatment combination) were included in this bioassay. Plants were incubated for 62 days in the growth chamber at same conditions of above. Plants were watered as needed and fertilized every 2 weeks with 50 mL Hoagland´s nutrient solution. Plants were sampled in a destructive manner and a time-course after inoculation, and

pieces of their roots and stem were used for CLSM examination (Fig. 1) or isolation of V138I-YFP (only stems). Root and stem pieces from ‘Ac-4’, ‘Ac-15’ and ‘Picual’ plants inoculated with T. harzianum GFP22 (treatment T2) were examined at weekly intervals from 7 to 62 days after inoculation (dait). Also, noninoculated plants (T1) were examined for absence of fluorescence at weekly intervals. ‘Picual’ plants singly (treatment T3) or double inoculated (treatment T4) were sampled in the following weeks after inoculation with V138IYFP (daiv) (Fig. 1): one (7 daiv), two (11 daiv) and three (15 and 20 daiv) for root examination; and four (28 daiv), six (36, 37 and 39 daiv), seven (43 daiv) and eight (50 daiv) for stem examination. Roots and stem pieces from one ‘Picual’ plant per sample time were used for CLSM examination, and stem pieces from four ‘Picual’ plants sampled at 43 daiv were used for isolation of V138I-YFP. ‘Ac-4’ and ‘Ac-15’ plants from T3 and T4 treatments were sampled (Fig. 1) in weeks two (11 daiv) and three (15 and 20 daiv) for root examination, and in weeks six (36, 37 and 39 daiv), seven (43 daiv) and eight (50 daiv) for stem examination. One plant of each of ‘Ac-15’ and ‘Ac-4’ was used for each sampling time, with the exception that three, four and two plants were used at 20, 43 and 50 daiv time points, respectively.


Confocal laser microscopy of olive and wild olive plants

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Plant roots and stems from the bioassay, sampled as described above, were cross or longitudinally sectioned using a hand microtome model 501 (Nahita, Leica Biosystems, Wetzlar, Germany) and a vibratome Series 1000plus (TAAB Laboratories Equipment, Aldermarston, UK), respectively. For every sampled plant, 30 sections from root and/or stem tissue were obtained and placed on microscope slides, submerged in glycerol 50% (v/v) and gently squashed with a cover glass. Microscopic examination was performed using a laser scanning spectral confocal microscope (TCS2-SP2, Leica Biosystems). Excitation was provided by an argon laser (488 nm). Fluorescence of GFP and YFP was detected at 495-520 and 530-620 nm, respectively. Images were acquired with Leica LSM Image Browser software (Leica Biosystems) and mounted with Leica Application Suite Advanced Fluorescence Lite 1.8.2 (Leica Biosystems). The extent of colonization of wild olive ‘Ac-4’ and ‘Ac-15’ plants by V138I-YFP was

quantified with the help of ImageJ (US National Institutes of Health, Bethesda, MA, USA). Data were obtained for each of wild olive clones inoculated with V138I-YFP alone (treatment T3) or GFP22 + V138I-YFP (treatment T4) sampled at 20 (three plants) and 43 daiv (four plants) for examinations of root and stem tissues. At least four images per plant tissue showing hyphal growth of V138I-YFP were used. Images of root and stem tissues were compared and YFP cell fluorescence was used to calculate the fungal colonization ratio of similar areas in root or stem for both wild olive clones.

Isolation of Verticillium dahliae from inoculated plants The extent of colonization of ‘Ac-4’, ‘Ac-15’ and ‘Picual’ plants by V. dahliae V138I-YFP was determined by isolations on PDA from stem pieces of four plants of each of the olive genotypes and treatment combination sampled at 43 daiv. This sampling time was chosen on


the basis of the high disease severity (3.5 on a 0-4 rating scale) reached on ‘Picual’ plants.

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The main stem of plants deprived of leaves and lateral shoots was equally divided into three zones (upper, medium and lower). Forty eight 1-cm-long pieces from each stem zone were thoroughly washed under running tap water, the bark aseptically removed, and the pieces were surface disinfested as previously described (Carrero-Carrón et al., 2016), plated onto PDA (six pieces per plate) and incubated at 25 ºC in the dark for 7 days. Colonies of V. dahliae were identified by microscopic observation of verticillate conidiophores (JiménezDíaz et al., 2012). Results were expressed as the percentage of positive isolations.

Data analysis Data on mycelia radial growth of V. dahliae growing in single or dual cultures with T. harzianum were subjected to standard analysis of variance (ANOVA) with the GLM procedure in SAS (v. 9.4, SAS Institute Inc.) to determine whether radial growth of V. dahliae was significantly affected (P < 0.05) by T. harzianum. A single factor experimental design was used. Separate analyses were performed for each growth media. Linear singledegree-of-freedom contrasts were computed to test the effect of selected experimental condition combinations at P < 0.05. Similarly, ANOVA analyses were used to assess the effects of plant genotype, plant tissue and inoculation treatment on the YFP fluorescence signal intensity. For each plant tissue (i.e., root and stem) a factorial experimental design was used with plant genotype, plant tissue and inoculation treatment as factors. Linear singledegree-of-freedom contrasts were computed to test the effect of selected experimental treatment combinations at P < 0.05. Data on frequency of stem colonization by V. dahliae V138I-YFP were analyzed with

the GENMOD procedure using the binomial distribution and the logit as link function in SAS. A factorial experimental design was used with plant genotype, stem zone and


inoculation treatment as factors. A likelihood ratio test was used to determine whether the

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plant genotype, plant stem zone or inoculation treatment significantly affected (P < 0.05) plant colonization by V138I-YFP. The statistical significance (P < 0.05) of the likelihood ratio was determined by a chi-square test and the least squares means statement was used to determine significant differences (P < 0.05) among treatments (Agresti, 2007).

Results

Development and characterization of Verticillium dahliae V138I-YFP transformant Yellow fluorescent protein (YFP) transformants were generated to monitor the colonization of plants of wild olives ‘Ac-4’ and ‘Ac-15’, and ‘Picual’ olive by D V. dahliae. Eighteen transformants with hygromycin B resistance were obtained, of which only four kept the antibiotic resistance phenotype after four consecutive rounds of culturing for selection. Strain V138I-YFP of V. dahliae was chosen among these four stable transformants because it showed strong and uniform yellow fluorescence when hyphae from PDB cultures were checked by confocal microscopy. V. dahliae V138I-YFP was further characterized to check for possible phenotypic alterations compared with its parental strain V. dahliae V-138I. Radial mycelial growths of colonies of transformant and parental strains did not differ significantly (P ≥ 0.05) neither after 7 days of incubation on PDA and PLYA media (Table 1), nor after 12 days of incubation in these two media (data not shown). Compared with single cultures, growth of both V. dahliae strains in dual culture with either T. harzianum strain T34 or GFP22 on the same media resulted in a significant (P < 0.05) reduction of V. dahliae colony growth, with no significant differences (P ≥ 0.05) in growth reduction between V138-I and V138I-YFP (Table 1). Also, both V. dahliae strains showed similar


behavior as overgrown targets for the biocontrol activity of the two T. harzianum strains

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tested (data not shown). Finally, V138I-YFP proved highly virulent when tested for pathogenicity on susceptible olive cv. Picual, inoculated plants showing defoliation and a mean severity of symptoms of 3.3 (within a 0-4 rating scale) by 5 weeks (39 daiv) after inoculation, 3.5 in the week six (43 daiv) and all plants were dead (disease severity = 4) by 7 weeks after inoculation.

Effect of treatment with Trichoderma harzianum and olive genotype on colonization of plants by defoliating Verticillium dahliae The pattern of colonization of wild olive clones ‘Ac-4’ and ‘Ac-15’ and olive cv. Picual by D V. dahliae V138I-YFP in the presence or absence of T. harzianum GFP22 was comparatively assessed by means of CLSM examinations. To that aim, ‘Ac-4’, ‘Ac-15’ and ‘Picual’ plants were inoculated with the fluorescent transformants T. harzianum GFP22 (green) (treatment T2) or (treatment T3)/and (treatment T4) V. dahliae V138I-YFP (yellow), or nonninoculated (treatment T1). ‘Picual’ plants from the four treatments (T1 to T4) were used for periodical searching

of fluorescence signals to set sampling times of ‘Ac-4’ and ‘Ac-15’ plants. As expected, roots and stem samples from uninoculated control plants of ‘Ac-4’, ‘Ac-15’ and ‘Picual’ (treatment T1) did not show any fluorescence at any time point (data not shown). For treatment T2, strain GFP22 was not seen in tissues of any of the three genotypes sampled 7 dait; however the fungus was found on root hairs and within, epidermis and cortex of ‘Picual’ plants sampled 11 dait, and in those of ‘Ac-4’ and ‘Ac-15’ plants sampled 20 dait. Subsequently, GFP22 was detected in the root cortex of plants of the three genotypes sampled at weekly intervals until the end of the bioassay, 62 dait (Fig. 2); however, the fungus was found neither within the root vascular tissues nor in the stem of any of those samples. These observations


are indicative of endophytic colonization of both olive and wild olive roots by T. harzianum

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GFP22. Root and stem of ‘Picual’ plants singly inoculated with V. dahliae V138I-YFP

(treatment T3) were colonized by the pathogen (Fig. 3). This fungus was detected in the root vascular tissues of ‘Picual’ plants in treatment T3 sampled 11 daiv (Fig. 1) but it was not observed in same tissues of wild olive ‘Ac-4’ and ‘Ac-15’ sampled at this time point. Contrary to what was expected, V138I-YFP was not observed in tissues of ‘Ac-4’ and ‘Ac15’ plants in treatment T3 sampled 15 daiv, but it was present in root tissues of ‘Ac-15’ plants jointly inoculated with V138I-YFP and GPF22 (treatment T4) sampled 15 daiv. Examination of root tissues sampled 20 daiv indicated that V138I-YFP infected the two wild olive genotypes, the yellow signal being found in plants of treatments T3 and T4 (Fig. 4). Moreover, observations in cross sections of roots sampled 20 daiv indicated that V138I-YFP had reached the vascular bundles of ‘Ac-15’ plants, whereas in similarly inoculated ‘Ac-4’ plants the pathogen was found only in the root cortex and epidermis (data not shown). Intensity of fluorescence signal was significantly influenced (P < 0.05) by host genotype, plant tissue and treatment but not (P ≥ 0.05) by their interactions. As shown in Table 2, the mean values of fluorescence signal intensity recorded in root of wild olive plants sampled 20 daiv were significantly higher (P < 0.05) in plants singly inoculated with V. dahliae V138IYFP (treatment T3) than those jointly inoculated with V138I-YFP and GPF22 (treatment T4), as well as in plants of ‘Ac-15’ compared to that in ‘Ac-4’ plants (P < 0.05) in either of the two treatments. Sampling of plant stems in treatments T3 and T4 started 4 weeks after inoculation (28

daiv) for ‘Picual’ olive and 2 weeks later (36 daiv) for ‘Ac-4’ and ‘Ac-15’ wild olives (Fig. 1). At this last sampling time, V138I-YFP was found in the vascular bundles of pieces of the upper stem zone of ‘Picual’ and ‘Ac-15’ plants. Conversely, V138I-YFP was not found in


any of stem zones of ‘Ac-4’ plants sampled at 36, 37 and 39 daiv, but it was detected in

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sections of the upper stem zone sampled 43 daiv (Fig. 4). While ‘Picual’ and ‘Ac-15’ plants showed severe defoliation by 39 daiv, little defoliation developed in plants of ‘Ac-4’ and it did not occur until 50 daiv in the two remaining plants of treatment T3. The extent of pathogen colonization was comparatively quantified in stems of ‘Ac-4’ and ‘Ac-15’ wild olives sampled 43 daiv (Table 2). Mean values of fluorescence signal were significantly higher (P < 0.05) in plants singly inoculated with V138I-YFP (treatment T3) compared with those assessed in plants inoculated with GFP22 and V138I-YFP (treatment T4), and such values were significant lower (P < 0.05) in stems than in roots for both wild olive genotypes, being significantly lower (P < 0.05) for ‘Ac-4’ plants and both plant tissues (Table 2). To confirm the differences observed among the four treatments assayed in ‘Ac-4’, ‘Ac-15’ and ‘Picual’ during CLSM examinations, we alternatively quantified the pathogen stem colonization by means of isolations of V. dahliae V138I-YFP on PDA from the lower, medium and upper stem zones of the same four plants sampled at 43-daiv for YFP cell fluorescence quantifications (Table 3). V. dahliae V138I-YFP was recovered neither from stems of plants inoculated only with T. harzianum GFP22 (treatment T2) nor from noninoculated controls (treatment T1). Also, V138I-YFP was not isolated from the upper and medium stem zones of ‘Ac-4’ plants jointly inoculated with GFP22 and V138I-YFP (treatment T4). The percentages of positive isolation differed (P < 0.05) among host genotypes, treatments and sampled stem zones in the three olive genotypes, with a significant interaction (P < 0.05) between genotype and treatment. Overall, stem colonization was highest in ‘Picual’ decreasing significantly in ‘Ac-15’ and ‘Ac-4’, in that order. Stem colonization tended to decrease significantly along the stem axis, being lowest (P < 0.05) in the upper zone in all three genotypes. Strain GFP22 significantly reduced (P < 0.05) the stem colonization by V138I-YFP in ‘Picual’ (upper zone) and ‘Ac-4’ (upper, medium and lower


zones). Although lower percentages of V138I-YFP isolation were obtained in ‘Ac-15’ plants

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(upper and medium zones) inoculated with GFP22 plusV138I-YFP (treatment T4) compared with those got in ‘Ac-15’ plants inoculated with V138I-YFP alone (treatment T3), the differences were not significant (P ≥ 0.05).

Discussion

Host resistance is the single most practical, sustainable and cost-efficient method for management of VW in olive (Jiménez-Díaz et al., 2012). However, most of commercial olive cultivars lack suitable resistance to the highly virulent D pathotype of V. dahliae that has become widespread in Spain and that also occurs elsewhere (Jiménez-Díaz et al., 2012; Trapero et al., 2013). In previous studies, we identified three wild olive clones that proved resistant to D V. dahliae, whose use as a rootstock for grafting D V. dahliae-susceptible olive cultivars is now under commercial production (Jiménez-Fernández et al., 2016; PalomaresRius et al., 2016). Here, we have investigated the potential of combining T. harzianum with use of a resistant wild olive rootstock for enhancing the integrated management of D V. dahliae-induced VW in olive. Previous studies have demonstrated that CLSM is of much help for monitoring infection and colonization of herbaceous and woody hosts by V. dahliae (Prieto et al., 2009; Zhang et al., 2013), as well as for simultaneous visualization of this pathogen and the biocontrol strains P. fluorescens PICF7 and T. harzianum GFP22 (Prieto et al., 2009; Ruano-Rosa et al., 2016). The use of the fluorescent transformant D V. dahliae V138I-YFP, obtained in the present study, and of the commonly employed strain T. harzianum GFP22 (Chacón et al., 2007; Samolski et al., 2012; Alonso-Ramírez et al., 2014; Ruano-Rosa et al., 2016), allowed us to simultaneously examine growth of D V. dahliae and


T. harzianum in olive and wild olive plants by means of CLSM. The newly developed V.

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dahliae YFP transformant was proved suitable for the study because it did not show any difference compared with the wild type strain V. dahliae V-138I regarding morphology and growth rate on different culture media (Table 1). The transformant strain retained the parental virulence phenotype, as shown by the severe defoliation and plant death (mean symptoms severity = 4 on a 0-4 rating scale) induced on susceptible ‘Picual’ olive by 7 weeks after inoculation, a disease reaction similar to that found for the parental isolate on ‘Picual’ olive in previous work (Jiménez-Díaz et al., 2009; Jiménez-Fernández et al., 2016). Also, we found that there were not significant differences (P ≥ 0.05) in the antagonistic ability of T. harzianum GFP22 against V. dahliae V-138I and V. dahliae V138I-YFP (Table 1). The effectiveness of bacterial and fungal BCAs against VWs was related with their

populations in the plant host rhizosphere (Angelopoulou et al., 2014). Also, it has been described that efficient root colonization is needed for the well-known beneficial effect of Trichoderma spp. to host plants (Lorito et al., 2010). Endophytic lifestyle has been shown for several Trichoderma spp. (Bae et al., 2009), including the strain GFP22 of T. harzianum in tomato roots (Chacón et al., 2007). Endophytic infections by T. asperellum strains Bt3 and T25 have also been reported in ‘Picual’ olive plants (Carrero-Carrón et al., 2016). Previous CLSM studies have demonstrated that T. harzianum GFP22 is efficient in rhizosphere colonization of Arabidopsis thaliana and cucumber plants (Samolski et al., 2012; AlonsoRamírez et al., 2014). Similarly, Ruano-Rosa et al. (2016) reported efficient colonization of ‘Picual’ olive roots by GPF22 and related that to its biocontrol activity against VW in olive because they did not observe inner colonization of the roots by the antagonist. Contrary to that, the CLSM examination that we carried out in roots of ‘Picual’, ‘Ac-15’, and ‘Ac-4’ plants from treatments T2 and T4 clearly demonstrate that T. harzianum GFP22 grows within the root epidermis and upper cortex of olive and wild olive plants without reaching the


vascular system (Fig. 2 and 4). These results are in agreement with previous studies (Chacón

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et al., 2007; Samolski et al., 2012; Alonso-Ramírez et al., 2014) and indicate that the endophytic lifestyle of T. harzianum develops in herbaceous and woody plants. In the present study, we found that hyphae of V. dahliae V138I-YFP grew on root

hairs and within epidermal cells along roots of resistant (‘Ac-4’) and susceptible (‘Ac-15’) wild olives, as well as of susceptible ‘Picual’ olive (Fig. 3 and 4). It has been suggested that V. dahliae may gain ingress in olive roots through micro- or macro-breakages that can occur when olive plants are managed in the process of artificial inoculation and/or during root growth (Prieto et al., 2009). Although we intentionally avoided injuring the plants while transplanting them into infested soil, occurrence of wound that might have facilitated a passive entrance of the pathogen into roots cannot be disregarded. In any case, colonization of the root cortex of host plants by Verticillium spp. can occur in different manners (Eynck et al., 2007; Klosterman et al., 2009). It has been reported for V. dahliae a massive production of conidia and microsclerotia outside the root tissue of unsuitable host plants (Eynck et al., 2007). Although V138I-YFP retained a capacity for microsclerotia production on the infested CMS mixed with the pasteurized potting soil, neither conidia nor microsclerotia or appressoria of the transformant strain were observed within the root cell junctions of ‘Picual’, ‘Ac-15’ and ‘Ac-4’ plants under confocal microscopy. Our results show that V. dahliae V138I-YFP reached the stem of the three olive

genotypes assayed (Fig. 3 and 4). However, plant colonization by V138I-YFP differed among host genotypes as observed by confocal microscopy in the sampling made over time (Fig. 1), as well as by quantification of the fluorescence signal made on ‘Ac-4’ and ‘Ac-15’ plants (Table 2). Differences detected between ‘Ac-15’ and ‘Ac-4’ wild olive clones indicate that the latter one has valuable resistance and potential as a rootstock for the integrated management of VW in susceptible olive cultivars, which can be further enhanced with root


treatment with T. harzianum CECT 2413. Thus, in this present work we demonstrate that T.

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harzianum GFP22 reduces the level of colonization of resistant (‘Ac-4’) and susceptible (‘Ac-15’, ‘Picual’) genotypes by V. dahliae V138I-YFP (Tables 2 and 3). In fact, prior infection by GFP22 slowed down colonization of the root vascular bundles in ‘Picual’ plants, and reduced the mean fluorescence signal of V138I-YFP as well as the percentage of pathogen isolation from stems in plants of ‘Ac-15’ and ‘Ac-4’, compared with those in same plant genotypes singly inoculated with V138I-YFP. Prieto & Mercado-Blanco (2008) claimed that early and localized colonization of the rhizoplane and inner root by P. fluorescens PICF7 is needed to impair full progress of D V. dahliae in olive plants. We did not observe high fluorescence of GFP22 on the root surface of olive and wild olive plants; however, substantial endophytic growth of this strain took place in plants inoculated either singly with the antagonist or jointly with the antagonist and the pathogen (Fig. 2 and 4), although the extent of endophytic colonization did not avoid that the pathogen could gain access to vascular tissues. In summary, we conclude that ‘Ac-4’ wild olive bears a degree of resistance against D

V. dahliae that has a potential for use as a rootstock for the management of D V. dahliaeinduced VW in susceptible olive cultivars. Moreover, we conclude that use of that resistant rootstock can be combined with root treatment with endophytic strains of Trichoderma spp. that are able to reduce the extent of plant colonization by the pathogen. Since the D pathotype belongs to race 2 of the pathogen that lacks the effector gene Ave1 (Jiménez-Díaz et al., 2017) and therefore is potentially virulent on plants carrying the resistance gene Ve1, further studies are needed to unravel the genetic basis of resistance to D V. dahliae observed in selected clones of wild olive.


Acknowledgments

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This work was supported by projects from the Spanish Government MINECO (AGL201240041-C02 and AGL2015-70671-C2) and the “Consejería de Innovación, Ciencia y Empresa (CICE)”, Regional Government of Andalusia (P10-AGR 6082). I. Carrero-Carrón was under pre- and postdoctoral fellowships of CICE.

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Figure legends

Fig. 1 Schematic representation of sampling time points of roots and stems of olive cv. Picual and wild olive clones ‘Ac-15’ and ‘Ac-4’ plants from treatments T3 (Verticillium dahliae V138I-YFP alone) and T4 (inoculation with Trichoderma harzianum GFP22 followed by that with V138I-YFP 12 days latter), with indication of negative (empty circle) or positive (black circle) observation of V138I-YFP fluorescence under confocal microscopy in both treatments. Time points are referred to days after inoculation with V138I-YFP (daiv). Circles represent the number of plants sampled. Grey circle indicates, positive observation in T3 and negative in T4. Crossed grey circle indicates, negative observation in T3 and positive in T4. Framed plants were also used for fluorescence signal quantification. Asterisk indicates that the four plants sampled at 43 daiv were used to isolate the pathogen on agar plates.


Fig. 2 Trichoderma harzianum GFP22 (green fluorescence) growing in the root epidermis

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and cortex of ‘Ac-15’, ‘Ac-4’ and ‘Picual’ plants. Confocal images correspond to longitudinal root sections at 20 days after inoculation with GFP22 (dait) in ‘Ac-15’, 29 dait in ‘Ac-4’ and 62 dait in ‘Picual’.

Fig. 3 Colonization of plants of olive cv. Picual by the V138I-YFP transformant of defoliating Verticillium dahliae V-138I (yellow fluorescence). A) Longitudinal root section at 15 days after inoculation with V138I-YFP (daiv) showing the pathogen in cortex and vessels. B) Cross root section at 39 daiv showing the pathogen in cortex and vessels. C) Cross stem section at 43 daiv showing the pathogen in cambium layer (CL) and xylem vessels (XV). D) Cross stem section of a dead plant at 50 daiv showing extensive colonization by the pathogen in CL and XV.

Fig. 4 Extent of colonization by the V138I-YFP transformant of defoliating Verticillium dahliae V-138I in ‘Ac-15’ (A, C, E and G) and ‘Ac-4’ (B, D, F and H) wild olive clones singly inoculated with V138I-YFP (A, B, E and F) or inoculated with the pathogen 12 days after prior inoculation with Trichoderma harzianum GFP22 (C, D, G and H). Times refer to days after inoculation with V138I-YFP (daiv). A-D) Longitudinal root sections at 20 daiv. EH) Cross sections of upper stem at 43-daiv. Yellow arrows indicate strain V138I-YFP (yellow fluorescence) and the green ones strain GFP22 (green fluorescence).


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Table 1 Radial mycelial growth of Verticillium dahliae isolates V-138I (wild type) and V138I-YFP (YFP-marked) in single and dual cultures with Trichoderma harzianum isolates T34 (wild type) and GFP22 (GFP-marked) on two growth media. Data were recorded after 7 days. For dual cultures, V. dahliae was allowed to grow for 4 days before plating T. harzianum Growth medium PDA

PLYA

V-138I

2.53 ± 0.12*

2.40 ± 0.00*

V138I-YFP

2.46 ± 0.12*

2.40 ± 0.00*

V-138I vs T34

1.43 ± 0.12

1.63 ± 0.12

V138I-YFP vs T34

1.30 ± 0.00

1.63 ± 0.12

V-138I vs GFP22

1.40 ± 0.10

1.60 ± 0.10

V138I-YFP vs GFP22

1.27 ± 0.06

1.60 ± 0.10

Single culture

Dual culture

Data are means of 3 replications. Means with an asterisk indicate that radial mycelial growth in single culture was significantly higher (P < 0.05) compared by single degree of freedom contrasts with this same V. dahliae isolate growing in dual culture with T. harzianum isolates. No significant differences existed (P ≥ 0.05) between radial mycelial growth of V. dahliae wild type (V-138I) and YFP-marked (V138I-YFP) growing in single culture.


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Table 2 Effects of plant genotype, prior inoculation with Trichoderma harzianum GFP22 and plant tissue sampled (root and stem) on the extent of colonization of ‘Ac-15’ and ‘Ac-4’ wild olives by Verticillium dahliae V138I-YFP ‘Ac-15’

‘Ac-4’

Treatment

Root

Stem

Root

Stem

V138I-YFP

53.97 ± 20.57a*

5.60 ± 2.23b*

4.06 ± 2.04a*

0.31 ± 0.10b*

GFP22 + V138I-YFP

22.85 ± 9.16a

0.78 ± 0.54b

1.94 ± 1.94a

0.04 ± 0.04b

Data are mean values of YFP cell fluorescence signal for each genotype, treatment and plant tissue from at least four images. Plants were inoculated singly with V138I-YFP or 12 days after prior inoculation with GFP22. Root data were from three plants sampled at 20 days after inoculation with V138I-YFP (daiv) and stem data were from four plants sampled at 43 daiv. For each genotype and treatment, means followed by different superscript letters represent significant differences between plant zones as determined by Least Squares means’s test at P < 0.05. For each genotype and plant zone, values followed by asterisk represent significant differences between V138I-YFP treatment compared to that for GFP22 + V138I-YFP treatment as determined by Least Squares means’s test at P < 0.05.


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This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/ppa.12879 This article is protected by copyright. All rights reserved.

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Table 3 Effects of plant genotype, prior inoculation with Trichoderma harzianum GFP22 and stem zone sampled [Upper (U), Medium (M) and Lower (L)] on the extent of stem colonization of ‘Picual’ olive, and ‘Ac-15’ and ‘Ac-4’ wild olives by Verticillium dahliae V138I-YFP, calculated by means of isolations on pure cultureª ‘Picual’

‘Ac-15’

‘Ac-4’

Treatment

U

M

L

U

M

L

U

M

L

Untreated

0

0

0

0

0

0

0

0

0

GFP22

0

0

0

0

0

0

0

0

0

V138I-YFP

96.7 ± 3.3b*

100a

100a

60.0 ± 11.5c

92.5 ± 3.8b

100a

16.7 ± 9.6c* 29.2 ± 7.7b* 37.4 ± 11.3a*

GFP22 + V138I-YFP

73.6 ± 9.5b

100a

100a

38.9 ± 16.6c

87.8 ± 6.2b

100a

0b

0b

4.8 ± 4.8a

ªValues are expressed as percentage of recovered pathogen colonies, calculated from 48 stem pieces of one-cm-long (12 pieces per zone and plant, from four plants), for each genotype, treatment and stem zone. Samples were taken 43 days after V138I-YFP inoculation or not in plants previously (12 days before) inoculated with GFP22 or not. For each genotype and treatment, values followed by different superscript letters indicate significant differences as determined by Least Squares means’s test (P < 0.05). Values followed by an asterisk indicate that percentage of stem colonization in the V138I-YFP inoculated plants was significantly higher (P < 0.05) compared to that in the GFP22 + V138I-YFP treatment as determined by Least Squares means’s test (P < 0.05).


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This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/ppa.12879 This article is protected by copyright. All rights reserved.

Accepted Article This article is protected by copyright. All rights reserved.

