Festuca arundinacea introgression forms

Accepted Manuscript Dissection of resistance to Microdochium nivale in Lolium multiflorum/Festuca arundinacea introgression forms Agnieszka Płażek, Ewa Pociecha, Adam Augustyniak, Katarzyna Masajada, Michał Dziurka, Joanna Majka, Dawid Perlikowski, Izabela Pawłowicz, Arkadiusz Kosmala PII:

S0981-9428(17)30391-1

DOI:

10.1016/j.plaphy.2017.11.022

Reference:

PLAPHY 5062

To appear in:

Plant Physiology and Biochemistry

Received Date: 15 November 2017 Revised Date:

28 November 2017

Accepted Date: 30 November 2017

Please cite this article as: A. Płażek, E. Pociecha, A. Augustyniak, K. Masajada, Michał. Dziurka, J. Majka, D. Perlikowski, I. Pawłowicz, A. Kosmala, Dissection of resistance to Microdochium nivale in Lolium multiflorum/Festuca arundinacea introgression forms, Plant Physiology et Biochemistry (2018), doi: 10.1016/j.plaphy.2017.11.022. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Dissection of resistance to Microdochium nivale in Lolium multiflorum/Festuca arundinacea introgression forms Agnieszka Płażeka#, Ewa Pociechaa#, Adam Augustyniakb, Katarzyna Masajadab, Michał Dziurkac, Joanna Majkab, Dawid Perlikowskib, Izabela Pawłowiczb, Arkadiusz

Department of Plant Physiology, University of Agriculture in Cracow, Podłużna 3, 30-239

Cracow, Poland b

Institute of Plant Genetics of the Polish Academy of Sciences, Strzeszyńska 34, 60-479

Poznań, Poland c

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a

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Kosmalab

The Franciszek Górski Institute of Plant Physiology of the Polish Academy of Sciences,

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Niezapominajek 21, 30-239 Cracow, Poland

# - these authors contributed equally to this work

Corresponding author

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Arkadiusz Kosmala e-mail: [email protected] tel. +48 61 6550 285

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Strzeszyńska 34; 60-479 Poznań, Poland

Agnieszka Płażek: [email protected] Ewa Pociecha: [email protected]

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Adam Augustyniak: [email protected] Katarzyna Masajada: [email protected] Michał Dziurka: [email protected] Joanna Majka: [email protected] Dawid Perlikowski: [email protected] Izabela Pawłowicz: [email protected]

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ACCEPTED MANUSCRIPT Abstract

The potential of resistance to Microdochium nivale is still not recognized for numerous plant species. The forage grasses of Lolium-Festuca complex are important for grass-biomass production in the temperate regions. Lolium multiflorum is a grass with a high forage quality

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and productivity but also a relatively low resistance to M. nivale. On the contrary, F. arundinacea has a higher potential of resistance but simultaneously a significantly lower

forage quality. These two species cross with each other and the intergeneric hybrids possess complementary characters of both genera. Herein, for the first time, we perform the research

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on L. multiflorum/F. arundinacea introgression forms to decipher mechanisms of resistance to M. nivale in that group of plants. Two forms with distinct levels of resistance were used as models in cytogenetic and biochemical studies. The resistant plant was shown to be a

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tetraploid with 28 L. multiflorum chromosomes, including one with three F. arundinacea introgressions. The susceptible introgression form revealed the unbalanced genomic structure and only 25 chromosomes. Twenty four chromosomes were shown to be L. multiflorum chromosomes, including one chromosome with F. arundinacea segment. One Festuca chromosome with additional two interstitial F. arundinacea segments, was also revealed in

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the susceptible form. The selected introgression forms differed in the accumulation profiles of total soluble carbohydrates, phytohormones, and phenolics in the leaf and crown tissue under the control and infection conditions. The higher amount of carbohydrates and salicylic acid in the leaves and crowns as well as a lower amount of abscisic acid in both studied organs and

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jasmonic acid in the crowns, were shown to be crucial for the expression of resistance to M. nivale in the analyzed hybrids.

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Key words: carbohydrates; forage grasses; phytohormones; GISH/FISH; phenolics; snow mould

Abbreviations: ABA, abscisic acid; DAPI, 4′,6-diamidine-2′-phenylindole dihydrochloride; DW, dry weight; Fa, Festuca arundinacea; FISH, fluorescence in situ hybrydization; H, hardening; GISH, genomic in situ hybrydization; JA, jasmonic acid; Lm, Lolium multiflorum; PH, pre-hardening; PC, phenolics; PPFD, photosynthetic photon flux density; R – resistant; S, susceptible; SA, salicylic acid; TSC, total soluble carbohydrates; UHPLC, ultra-high performance liquid chromatography

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ACCEPTED MANUSCRIPT 1. Introduction

Climate changes are the major challenge for the world economy, especially for the agricultural sector, where climate conditions strongly influence crop yield. Winter stresses are among the major environmental factors, which affect plants during their life cycle. As a

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general increase in winter temperatures under future winter conditions is expected, the extreme freezing temperatures will not be the most crucial for plant overwintering (Höglind et al. 2013). In the consequence, the traits different than frost tolerance, such as resistance to biotic stresses, including snow mould, would be more responsible for the observed differences

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in winter survival of particular plant species. Unfortunately, basic knowledge on winter

hardiness concerns mainly frost tolerance and molecular mechanisms of other overwintering traits have yet to be recognized in numerous agronomically important species. Moreover,

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expected in the future periodical warmings during winters could initiate de-hardening processes and significantly reduce plant winter survival (Rapacz et al. 2014). According to the future climate change expectations, more severe and longer periods of drought, mostly in summers, are also predicted (Chang et al. 2017). Thus, the breeding strategy for further climate changes to select plant genotypes both drought tolerant and resistant to winter

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diseases could follow the demands of sustainable agriculture to develop new cultivars of crop plants with a broad spectrum of tolerance/resistance to various environmental stresses.

Grasslands are the main survival resource for about one billion people worldwide and cover

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nearly 70% of the world’s agricultural area. In Europe, grassland accounts for 39% of this area (Marshall et al. 2016). Especially important for grass-biomass production in the temperate regions are the forage grasses of Lolium-Festuca complex. The species of both

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genera possess complementary agronomic traits. Lolium multiflorum (Italian ryegrass, Lm) is a grass species with a high forage quality and productivity but also a relatively low tolerance to abiotic (water deficit, frost, and salinity) (Humphreys and Thomas 1993) and low resistance to biotic (e.g. snow mould, caused mainly by fungi Microdochium nivale (Fr.) Samuels and Hallett) (Pociecha and Płażek 2010) environmental stresses. On the contrary, F. pratensis (meadow fescue) was proved to be one of the most winter-hardy species within the LoliumFestuca complex, mainly because of its high potential of frost tolerance (Kosmala et al. 2006a, 2009; Sandve et al. 2011) and resistance to M. nivale (Pociecha and Płażek 2010). The other Festuca species, F. arundinacea (tall fescue, Fa) has the high capacity to avoid water deficit in soil due to its deep and well-developed root system and to tolerate this stress 3

ACCEPTED MANUSCRIPT modifying its leaf metabolism (Humphreys and Pašakinskienė 1996; Wilman et al. 1998; Gibson and Newman 2001; Kosmala et al. 2012; Pawłowicz et al. 2017). This species revealed also relatively high levels of tolerance to salinity (Pawłowicz et al. 2017), frost tolerance (Kosmala et al. 2007) and resistance to M. nivale (Pociecha and Płażek 2010). In turn, these Festuca species do not match L. multiflorum for productivity, quality, and

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nutritional values in comfortable environmental conditions (Humphreys and Thomas 1993). As proved earlier, winter hardiness of Lolium-Festuca species depends mainly on their frost tolerance and resistance to M. nivale, which is a main factor of snow mould in the Middle Europe (Tronsmo et al. 2001; Prończuk et al. 2003). It was also proved that very often these

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two traits are negatively correlated in a single plant genotype (Prończuk and Zagdańska 1993; Gaudet et al. 1999; Pociecha et al. 2013). According to further climate change expectations, the resistance to M. nivale could probable become one of the most important traits required to

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survive winters in a group of Lolium-Festuca grasses. To date, F. arundinacea has attracted a relatively low scientific attention with respect to its resistance to snow mould. To the best of our knowledge, only one paper (Pociecha and Płażek 2010) has demonstrated its metabolism under infection with M. nivale, and no such papers have been published on F. arundinacea

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hybrids with L. multiflorum.

Festuca arundinacea is an allohexaploid comprising the genomes of F. pratensis (2x) and F. glaucescens (4x) (Humphreys et al. 1995). Lolium multiflorum and F. arundinacea can be crossed with each other and their chromosomes can conjugate and recombine within

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intergeneric hybrids. This gives the opportunity to transfer the agronomically important traits from one species to another during the process of crossing. One of the ways to combine the complementary traits of both species is the introgression, which involves the transfer of the

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selected genes from one species to another during backcrossing of intergeneric hybrid with one of its parental species (Kosmala et al. 2007; Perlikowski et al. 2014). One of the most important further breeding goals would be to develop L. multiflorum/F. arundinacea (Lm/Fa) introgression forms with improved tolerance to water deficit and improved resistance to M. nivale. Unfortunately, though numerous lines of such the introgression forms have been generated to date and evaluated with respect to their drought tolerance (e.g. Humphreys and Pašakinskienė 1996; Perlikowski et al. 2014, 2016 a, b), winter hardiness and frost tolerance (Kosmala et al. 2007; Augustyniak et al. 2017), no one was simultaneously analyzed to recognize their potential of resistance to M. nivale. This was the first reason to perform such experiments in the current paper. The introgression forms of L. multiflorum/F. arundinacea 4

ACCEPTED MANUSCRIPT could be also unique plant materials to recognize molecular basis of resistance to M. nivale in L. multiflorum × F. arundinacea hybrids. This resistance is a complex, polygenic trait (Gaudet at al. 1999) and in particular genotypes of introgression forms this trait may be ‘dissected’ into its different components, which are the characters specific to a particular genotype rather than to the whole population. Thus, the best way to investigate molecular

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mechanisms of resistance is the selection of two genotypes distinct in their level of resistance and differing also in their genomic structure. In other words, the unique plant materials

applied here could be used as tools in studies on molecular mechanisms involved in the

resistance to M. nivale in Lolium-Festuca species, and this was the second reason to perform

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the proposed experiments.

Resistance to M. nivale is enhanced by hardening (H) in low temperature (cold acclimation).

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During that process the plants undergo different physiological and molecular alterations in their metabolism which are essential for different components of winter survival, including resistance to M. nivale (Ergon et al. 1998; Gaudet et al. 1999, 2011; Thomashow 1999; Beck et al. 2003). Overwintering of grasses is dependent on crown survival (Hoffman et al. 2010). However, hardening process in leaves is important for winter hardiness of perennial tissues, as

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leaves are crucial machinery to produce key metabolites, e.g. sugars (Kosmala et al. 2009; Bocian et al. 2015; Augustyniak et al. 2017). Thus, the research performed on both plant organs could be important to recognize the molecular mechanisms of resistance to M. nivale. To date, different cellular compounds have been shown to be potentially associated with this

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resistance in Lolium-Festuca species, e.g. total soluble carbohydrate (TSC), abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA), and phenolic (PC) compounds (Pociecha et al.

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2009 a, b; Pociecha and Płażek 2010).

Herein, first of all, we hypothesize that within a group of closely related L. multiflorum/F. arundinacea introgression forms a wide range of diversity with respect to their resistance to M. nivale exists. Secondly, we also hypothesize that the applying into the further biochemical research the introgression forms with significantly distinct levels of resistance and different genomic structure will allow to recognize some of the crucial components of their cellular metabolism associated with resistance to M. nivale infection.

Thus, the research presented here involved: (i) selection of L. multiflorum/F. arundinacea introgression forms - highly resistant (R) and highly susceptible (S) to M. nivale, (ii) analysis 5

ACCEPTED MANUSCRIPT of the genomic structure of the selected forms by the use of genomic (GISH) and fluorescence (FISH) in situ hybridization, and (iii) biochemical analysis performed on the leaf and crown tissues of these distinct forms, including: total soluble carbohydrate, phenolic compounds, abscisic, jasmonic, and salicylic acid contents. The analyses were performed at different

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experimental time points, as indicated in the Fig. 1.

2. Materials and methods

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2.1. Plant materials

The introgression forms of L. multiflorum/F. arundinacea applied into the current research were obtained after five rounds of backcrossing of partially fertile F. arundinacea (6x) × L.

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multiflorum (4x) F1 hybrid (2n = 5x = 35) to tetraploid cultivars of L. multiflorum. Among the introgression forms of the BC4 generation (after four rounds of backcrossing) – a highly drought tolerant introgression form – no. 4/10, was selected (Perlikowski et al. 2014; 2016 a, b). This form was backcrossed to tetraploid L. multiflorum, and the forms of BC5 generation (population – no. 180/30), were obtained. The group of 140 plants were further selected with

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respect to their drought tolerance in the simulated field conditions under ‘rain-out’ shelters, according to the procedure described in details by Perlikowski et al. (2014). Twenty introgression forms with relatively high levels of drought tolerance and simultaneously without any visible symptoms of fungal diseases (April-September 2014) (Masajada et al.,

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paper in preparation), were chosen for the experiments performed in this study.

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The experimental set up was demonstrated in the Fig. 1.

2.2. Plant pre-hardening and hardening

Three well-rooted clones of each introgression form were installed in three pots containing a mixture of soil/peat/sand (2:2:1 v/v/v) at pH = 5.8 and cultured for six weeks in a greenhouse at 18 °C (day/night) in daylight (the experiment was conducted in September; 50°08’39’’ N, 19°85’37’’ E). The plants were then pre-hardened in a growth chamber for one week at 12 °C at 10h photoperiod with a light intensity of 200 µmol m-2 s-1 photosynthetic photon flux density (PPFD). Subsequently, the plants were cold acclimated (hardened) for three weeks at 4 °C at 8h photoperiod with a light intensity of 200 µmol m-2 s-1 PPFD. 6

ACCEPTED MANUSCRIPT 2.3. Artificial inoculation with M. nivale mycelium

After cold acclimation, artificial inoculation with M. nivale mycelium was performed. The M. nivale strain 2/01 was isolated from the L. perenne seeds by Prof. Maria Prończuk from the

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Plant Breeding and Acclimatization Institute in Radzikow – National Research Institute (Poland). All the inoculation experiments were done with the same isolate, which was proved to be the most aggressive among all the isolates in the collection. The inoculum was prepared by growing the fungus in soil medium (Prończuk and Prończuk 1987) at 18 °C for seven days

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in darkness. Inoculation was carried out by adding 1 g of the inoculum per plant. The

inoculated and non-inoculated (control) plants were covered with moistened blotting paper and black plastic foil to keep high humidity and darkness. Subsequently, all the plants were

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incubated for 37 days at 2 °C to imitate natural field conditions occurring in a field in winter under a snow cover. After that period, the blotting paper and foil were removed and the plants were grown for three days at 2 °C in 100 µmol m-2 s-1 PPFD and next seven days at 12 °C in the same light intensity. The resistance of the analyzed introgression forms to snow mould was evaluated on the basis of regrowth ability using the visual rating system (0-5) described by Płażek et al. (2011), where ‘5’ means a plant without any symptoms of disease and ‘0’

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means a dead plant without any signs of re-growth ability after infection. Three replicated clones of each introgression form were used to calculate means and standard errors. Two

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independent inoculation experiments were performed (n=6).

2.4. Analysis of plant genomic structure

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2.4.1. Chromosome preparations

Plant germination, chromosome accumulation and fixation, followed by chromosome preparations on slides were carried out according to Majka et al. (2017) with minor modifications. Root tips were digested by a mixture of enzymes, containing 20% (v/v) pectinase (Sigma), 1% (w/v) cellulose (Calbiochem) and 1% (w/v) cellulase ‘Onozuka R-10’ (Serva). Ten root tips were used for each introgression form to prepare 10 slides with chromosome spreads per the analyzed genotype.

2.4.2. In situ hybridization 7

ACCEPTED MANUSCRIPT Fluorescent in situ hybridization (FISH) and genomic in situ hybridization (GISH) were performed according to the protocols described by Majka et al. (2017) and Kosmala et al. (2006a,b), respectively. As a first step, FISH experiments with two types of rDNA sequences – 5S rDNA (the clone pTa79 from Triticum aestivum) (Gerlach and Dryer, 1980) and 35S

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rDNA (the coding region of 26S rDNA from Arabidopsis thaliana) (Unfried and Gruendler, 1990), were performed. 35S rDNA was labelled by nick-translation using digoxigenin-11dUTP-labelled nucleotides (Sigma-Aldrich), while 5S rDNA by PCR with tetramethyl-5dUTP-rhodamine (Sigma-Aldrich). The hybridization mixture contained 100 ng of each probe

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in 2× SSC and 50% (v/v) formamide, 10% (w/v) dextran sulfate and 0.1% (w/v) sodium dodecyl sulfate. After the denaturation of chromosomes in the hybridization mixture (80 °C, 2 min) and overnight hybridization, the slides were washed in 0.1× SSC at 42°C. The probe

isothiocyanate (FITC) (Sigma-Aldrich).

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labelled with digoxigenin was detected using anti-digoxigenin conjugated with fluorescein

Following FISH experiments, the chromosome preparations were washed off and genomic DNA probe was subjected to in situ hybridization on the same metaphase plates. Total

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genomic DNA was isolated from young leaves using C-TAB method (Doyle and Doyle, 1990). DNA was mechanically shared by boiling. DNA of L. multiflorium was labeled by nick translation method with digoxigenin-11-dUTP (Sigma-Aldrich), while DNA of F. arundinacea was used as blocking DNA. The probe to block ratio was 1:40. Observations

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were made and acquired using an Olympus XM10 CCD camera attached to an Olympus BX 61 automatic epifluorescence microscope. Image processing was carried out using the Olympus Cell-F (version 3.1; Olympus Soft Imaging Solutions GmbH: Münster, Germany)

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imaging software and Micrographx Picture Publisher software (ver. 10; Corel Corporation, Canada). Ten slides with chromosome spreads were analyzed for each introgression form.

2.5. Biochemical analysis

The leaf and crown tissue sampling for further biochemical analysis was performed at several time-points of the experiment (Fig. 1): after seven days of pre-hardening, after three weeks of hardening, on the 1st and 7th day of infection. Simultaneously, the leaf and crown tissue from the control (non-inoculated) plants, was collected.

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ACCEPTED MANUSCRIPT 2.5.1. Total soluble carbohydrate content

The estimation of total carbohydrate content was performed spectrophotometrically according to the method of Dubois et al. (1951) with the modification of Bach et al. (2015). The amount of 2 mg of lyophilized and homogenized samples were extracted with 1 cm3 of ultra pure

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water (Elga Option R) for 5 min. Then, the samples were centrifuged at 22 000 × g for 5 min. To 100 µl of supernatant, 200 µl of 5% water phenol solution and 1 cm3 of concentrated

sulphuric acid, were added. After mixing, the samples were incubated for 20 min. at 95 °C and then transferred to 96-well plates. The absorbance at 490 nm was measured with a

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microplate reader (Synergy II, Bio Tek, USA). The carbohydrate content was estimated with the use of the standard curve, prepared for glucose. The analysis was carried out in triplicate. Means and standard errors were calculated. Duncan’s Multiple Range Test was used to

2.5.2. Phenolic compounds content

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evaluate differences at a significance level of 0.05.

The total soluble phenolics content was measured spectrophotometrically according to the method of Singleton et al. (1999) with the modification of Bach et al. (2015). An aliquot of

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the extract (50 µl) was diluted in 0.5 cm3 of deionised water and 0.2 cm3 of Folina–Ciocalteu reagent, and after 10 min of incubation, 0.7 cm3 of saturated Na2CO3 was added. The samples were then mixed, incubated 2 h, centrifuged at 22 000 × g for 5 min. and transferred to 96-

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well plates. The absorbance at 765 nm was read with a microplate reader (Synergy II, Bio Tek, USA). Gallic acid was used as a standard. The analysis was carried out in five replicates. Means and standard errors were calculated. Duncan’s Multiple Range Test was used to

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evaluate differences at a significance level of 0.05.

2.5.3. Abscisic, jasmonic, and salicylic acid content

Phytohormones were measured according to Hura et al. (2017). The amount of 20 mg freezedried, pulverized samples were spiked with stable isotope labeled internal standards ([2H4]salicylic acid, and [2H6]cis,trans-abscisic acid) and extracted in 1 cm3 of methanol/water/formic acid mixture (MeOH/H2O/HCOOH 15/4/1 v/v) (Dobrev and Kaminek 2002). After triple extraction in 1 cm3 of

methanol/water/formic acid mixture

(MeOH/H2O/HCOOH 15/4/1 v/v) supernatant were combined and evaporated under N2. The 9

ACCEPTED MANUSCRIPT remnant was re-suspended in 5% MeOH in 1M HCOOH and cleaned-up on SPE cartridges (BondElut Plexa PCX, 30mg, 1mm, Agilent, USA), evaporated under N2, reconstituted in 70 µl of ACN, filtered (0.22 um nylon membrane) and used for ultra-high performance liquid chromatography (UHPLC) analyses. The system consisted of UHPLC (Agilent Infinity 1260 , Agilent, Germany) and triple quadruple mass spectrometer (Agilent 6410, Agilent, USA) with

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electro-spray ionization (ESI). Detiled measurements conditions are given by Dziurka et al. (2016). All the standards were from OlChemim (Olomouc, Czech Republik) at highest available purity. The analysis was carried out in five replicates. Means and standard errors were calculated. Duncan’s Multiple Range Test was used to evaluate differences at a

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significance level of 0.05.

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3. Results

3.1. Resistance of L. multiflorum/F. arundinacea introgression forms to M. nivale

After performing the selection on 20 introgression forms with respect to their resistance to M. nivale, a wide range of diversity was observed. However, the forms with the highest

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(maximum) level of resistance (nos 17, 19, 71, 75, and 102) and the susceptible forms (nos 84 and 138), were clearly distinguished (Fig. 2). From these two groups of plants, two introgression forms with distinct levels of resistance, were chosen for further cytogenetic and biochemical work – a highly resistant form 180/30/19 (referred here as Lm/Fa-R) and a highly

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susceptible form 180/30/138 (referred here as Lm/Fa-S).

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3.2. Genomic structure of Lm/Fa-R and Lm/Fa-S introgression forms

The genomic structure of the analyzed introgression forms differed significantly. The Lm/FaR form was a tetraploid with 28 chromosomes. Eleven loci of 35S rDNA and four of 5S rDNA were also revealed in this genotype. All the observed chromosomes were L. multiflorum chromosomes, among which one chromosome carried three F. arundinacea chromosomal segments – two interstitial and one terminal (Fig. 3A). On the other hand, the Lm/Fa-S was proved to be an aneuploid with 25 chromosomes, nine loci of 35S rDNA and three of 5S rDNA. Twenty four chromosomes were shown to be L. multiflorum chromosomes, including one chromosome with F. arundinacea interstitial segment. Additionally, one

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ACCEPTED MANUSCRIPT Festuca chromosome (with F. arundinacea centromere) was revealed in the Lm/Fa-S form. This chromosome carried two interstitial F. arundinacea segments (Fig. 3B).

3.3. Biochemical analysis

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3.3.1. Total soluble carbohydrate content

The accumulation profiles of total soluble carbohydrates were different for the Lm/Fa-R and Lm/Fa-S introgression forms both under control conditions and during infection with M.

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nivale (Fig. 4).

The observed differences were due to the amounts of accumulated sugars as well as the

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dynamics of their metabolism at different experimental time-points (Fig. 4). In the leaves, the process of plant hardening in low temperature significantly increased the carbohydrate content only in the Lm/Fa-R form. Although the sugar content in the leaves of Lm/Fa-R form on the 1st day of infection was approx. twice as higher as the content observed in the leaves of Lm/Fa-S form, in both plants a significant reduction in sugar accumulation was observed during infection progression. However, the carbohydrate utilization was clearly revealed

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already on the 1st day of infection only in the Lm/Fa-S form, while in the Lm/Fa-R form the carbohydrate content even slightly increased on this day. Finally, on the 7th day of infection these two introgression forms revealed differences in total soluble carbohydrate content in the

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leaf tissue; with its higher level present in Lm/Fa-R form (Fig. 4). In the crowns, the hardening increased sugar accumulation levels in both analyzed plants. Interestingly, this level in the Lm/Fa-S form was shown to be even significantly higher, compared to the Lm/Fa-

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R form. However, the progression of infection process revealed quite different dynamics of sugar metabolism in the crown tissue of both introgression forms. The Lm/Fa-S form significantly reduced the sugar amounts after further seven days both in the control conditions (2 °C and dark) and after infection. In the Lm/Fa-R, no changes were observed during that experimental period (Fig. 4).

3.3.2. Phenolic compounds content

The most significant differences between the analyzed plants with respect to free phenolic compound content were observed in the crown tissue (Fig. 5). The hardening process slightly 11

ACCEPTED MANUSCRIPT decreased their accumulation in the Lm/Fa-R genotype but this level increased back to the pre-hardening values after plant transferring to dark and 2 °C. In the Lm/Fa-S genotype, no significant changes in the content of phenolic compounds in that period, were observed. On the 1st day of infection, but in the control conditions, a higher amount of phenolic compounds was observed in the Lm/Fa-S plant, however this level was significantly reduced just after the

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inoculation initiation. In the consequence, on the 1st day of infection, the two inoculated plants revealed the same accumulation level of phenolic compounds in the crown tissue. This level remained unchanged in the Lm/Fa-S form (no statistically significant changes were observed), while increased significantly in the Lm/Fa-R form, during infection progression (on the 7th

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day of infection). Finally on this day, the analyzed introgression forms did not reveal any

3.3.3. Abscisic acid content

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significant differences with respect to the total phenolic compound content (Fig. 5).

In the control conditions under pre-hardening and hardening conditions, no significant differences in ABA accumulation dynamics in the leaves were observed between the analyzed introgression forms. However, after inoculation, clear differences were revealed. The Lm/Fa-

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S plant demonstrated a reduction in ABA level, both on the 1st and 7th day of infection, compared to the respective control conditions. Thus, the observed changes were, to a high probability, a result of plant infection. Simultaneously, in the Lm/Fa-R plant no significant

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differences between the respective control and infection conditions, were noticed (Fig. 6).

In the crowns, no significant differences between the analyzed introgression forms were observed during pre-hardening and hardening periods. However, further temperature decrease

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to 2 °C and simultaneous dark conditions caused a temporal quantity leap of ABA only in the Lm/Fa-S form, while in the Lm/Fa-R form its amount remained unchanged on the 1st day of new environmental conditions. Otherwise, after seven days of experiments’ duration, this amount significantly dropped in the susceptible and significantly increased in the resistant form. On the other hand, the inoculation with M. nivale, caused a significant reduction of ABA content in the Lm/Fa-S plant and a significant, temporal increase of this content in the Lm/Fa-R plant on the 1st day of infection. After next seven days, ABA accumulation level in the resistant form dropped to the values observed in the susceptible form (Fig. 6).

3.3.4. Salicylic acid content 12

ACCEPTED MANUSCRIPT The accumulation dynamics of SA during pre-hardening and hardening periods was similar in two analyzed introgression forms; the plants decreased SA content after three weeks of hardening. Simultaneously, the Lm/Fa-R plant accumulated higher amounts of this hormone in both plant organs – the leaves and crowns. Transferring the plants to dark and lower

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temperature, increased the amount of SA in the leaves of two forms and increased it in the crowns of Lm/Fa-S form. In the case of Lm/Fa-R, new experimental conditions decreased SA content in the crowns. Following next seven days at 2 °C and in dark, the amount of SA in the leaves increased in the Lm/Fa-R plant and decreased in Lm/Fa-S plant. Thus, the total content

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of this hormone in the leaf tissue at all the control experimental conditions was always higher in the resistant introgression form. In the case of crown tissue, the total content of SA was only higher on the 1st day of plant transferring to new conditions in the Lm/Fa-S plant; after

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next seven days both analyzed introgression forms revealed comparable amounts of SA. During infection, a significantly lower content of SA was observed in the leaves of both analyzed plants on the 1st and the 7th day of infection, compared to the parallel control conditions. However, a different accumulation dynamics was simultaneously observed following infection progression in the leaves – in the Lm/Fa-R plant an increase and in the

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Lm/Fa-S a decrease in the total SA content, after seven days. The similar tendency of accumulation dynamics for the analyzed here introgression forms was notice in the crowns. However, in the case of crown tissue, the content of SA under inoculation was comparable to that observed in the parallel control conditions in the Lm/Fa-R form on the 1st and on the 7th

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day after inoculation initiation and in the Lm/Fa-S form only on the 7th day. On the 1st day of inoculation, the control value was significantly higher, compared to the value of SA level in

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the inoculated Lm/Fa-S form (Fig. 7).

3.3.5. Jasmonic acid content

The accumulation dynamics of JA during pre-hardening and hardening processes in the leaves was similar in two analyzed introgression forms; the plants decreased JA content after three weeks of hardening, however in the case of Lm/Fa-R plant that decrease was not statistically significant. This dynamics was comparable to that observed for SA. In the darkness and lower temperature, the introgression form with a lower level of resistance to M. nivale revealed simultaneously a higher accumulation level of JA in the leaves. The artificial inoculation reduced significantly that level on the 1st day of infection only in the Lm/Fa-S plant (Fig. 8). 13

ACCEPTED MANUSCRIPT In the crowns, also the less resistant plant revealed higher accumulation levels of JA, compared to the resistant plant, both in the control conditions and after inoculation (Fig. 8).

4. Discussion

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4.1. Resistance of L. multiflorum/F. arundinacea introgression forms to M. nivale

Our earlier research revealed that the selected here Lm/Fa-R form possessed simultaneously no potential to tolerate frost conditions. On the other hand, the introgression form Lm/Fa-S

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was demonstrated to be highly frost tolerant (Augustyniak et al. 2017). Thus, in the case of both analyzed here plants frost tolerance and resistance to M. nivale were negatively correlated. This phenomenon was observed earlier for different species, including Lolium-

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Festuca grasses (Prończuk and Zagdańska 1993; Gaudet et al. 1999). Despite being different with respect to those components of winter hardiness, these two introgression forms revealed simultaneously relatively high levels of drought tolerance in the simulated field conditions (Masajada et al., paper in preparation).

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4.2. Genomic structure of Lm/Fa-R and Lm/Fa-S introgression forms

This was not surprising that the Lm/Fa-R plant with a balanced genomic structure and ploidy was demonstrated to be highly resistant to M. nivale. However, further research is required to

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recognize which chromosomal regions were in fact responsible for the expression of excellent resistance in this introgression form. First of all, the special attention should be paid to the identified here F. arundinacea introgressed segments. Future micro-dissection and sequencing

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of these chromosomal segments should be one of the scientific priorities. Interestingly, the unbalanced genomic structure of Lm/Fa-S form did not influence its high tolerance to frost (Augustyniak et al. 2017). The specific gene combinations, to a high probability associated with F. arundinacea chromosome segments introgressed into this form, improved its potential of abiotic stress tolerance. Further research is however required to evaluate the fertility of the Lm/Fa-S plant, and the levels of tolerance to different environmental stresses of its progeny.

4.3. Biochemical analysis

4.3.1. Total soluble carbohydrate content 14

ACCEPTED MANUSCRIPT Total soluble carbohydrates were proved earlier to be crucial to develop high levels of resistance to biotic stresses in different plant species (Ryan 1988; Pontis 1989; Ergon et al. 1998; Salzaman et al. 1998; Gaudet et al. 1999). These compounds, first of all, reduce water potential in cells and, in the consequence, hamper pathogen penetration into plant tissue

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(Gaudet et al. 1999). Furthermore, the carbohydrates synthesized in leaves are transferred to crowns, where they are the reservoir of energy both to survive winter conditions and for

further plant regeneration after winter and infection cessation (Gaudet et al. 1999; Pociecha et al. 2013). These compounds trigger many metabolic and developmental responses and

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significantly affect the expression of a large number of genes (Koch 1996; Smeekens and Rook 1997). The higher sugar abundance after hardening period in the Lm/Fa-R form was probable, at least partially, associated with a higher photosynthetic activity observed in this

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low frost tolerant genotype under cold acclimation conditions in our earlier research (Augustyniak et al. 2017). It seems that this higher accumulation of TSC under hardening in the leaves as well as their high and constant amount during infection in the crowns observed here in the case of Lm/Fa-R introgression form, could be the key component of resistance to M. nivale in the L. multiflorum/F. arundinacea introgression forms. This phenomenon was

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demonstrated earlier for winter cereals and grasses, where loss of resistance to snow mould corresponded to the depletion of carbohydrate in plant crowns during winter and early spring seasons (Gaudet et al. 1999; Płażek et al. 2011). However, on the other hand, it seems also possible that the resistant introgression form generally did not use carbohydrates as a source

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of energy to cope with the infection. In such a situation, other mechanisms to prevent a progression of infection could have been more important (e.g. Pociecha et al. 2013).

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4.3.2. Phenolic compounds content

The phenolics were shown to be important compounds to develop winter hardiness of grasses, however, their roles during cold acclimation have not been fully recognized (Pociecha et al. 2009 a, b). As it was demonstrated earlier, the phenylpropanoid pathway was activated by various stresses (Dixon and Paiva 1995; Shadle et al. 2003). During the hardening process, phenolic compounds protected plants against low temperature and pathogen infection (Peltonen et al. 1997; Hakulinen et al. 1999; Isihara et al. 1999; El Modafar et al. 2001). Phenylpropanoid alcohols were proved to be involved in the biosynthesis of lignin, accumulating in cell walls and forming a natural barrier against the propagation of pathogens 15

ACCEPTED MANUSCRIPT (Hahlbrock and Scheel 1989; Pociecha et al. 2009 a, b). Moreover, low molecular phenolic compounds, such as phytoalexins, played a very important role in defense reactions to numerous stresses, including attacks of pathogens (Ahuja et al. 2012). Here, the specific functions of free phenolics, which could have been associated with different resistance ability of the analyzed introgression forms, remain unclear. It seems that their roles in the expression

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of resistance to M. nivale, at least in the case of the analyzed genotypes, were limited. However, further research on the phenolics bound in the plant cell walls would be required to fully understand the functions of this group of compounds in the resistance of Lolium-Festuca

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grasses (Pociecha et al. 2016).

4.3.3. Abscisic acid content

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The role of ABA accumulation in the resistance to M. nivale is still not clear and disputable. Earlier work performed on androgenic lines derived from F. pratensis × L. multiflorum hybrids, revealed a rapid but short-lived increase in ABA content within a few days of cold acclimation in the high frost tolerant lines (Gąsior et al. 2003). Otherwise, no correlation between changes in ABA content and resistance to M. nivale was revealed in F. pratensis × L.

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multiflorum hybrids (Gąsior et al. 2003). The same phenomenon with respect to frost tolerance was also noticed in the current research but only in the crowns after three weeks of hardening followed by changing of the environmental conditions (2 °C and dark). We also demonstrated here the differences between the analyzed forms in their response dynamics in

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ABA content after infection. Generally, the susceptible form responded in the reduction of ABA accumulation level on the 1st day of infection both in the leaves and the crowns, compared to the parallel non-infection conditions. On the other hand, the resistant form

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revealed a stable ABA content in the leaves and an increase in the crowns, also compared to the control conditions. The differences in a total amount of ABA also existed between the analyzed plants at particular experimental time-points. For example, the Lm/Fa-R form revealed its lower values in the leaves at the control time-points after plant transferring to 2 °C and dark. Abscisic acid was shown earlier to affect plant resistance mainly by modifying transcription of the gene for glucanase (Agrawal et al. 2001; Mohr and Cahill 2001). β-1,3Glucanase is one of the pathogenesis-related proteins synthesized in response to infection (Mauch et al. 1988) and its amount could be reduced by ABA (Akiyama and Pillai 2001). A lower content of this glucanase could be, on one hand, important for a higher deposition of callose in plant cell wall and, in the consequence, a higher protection against pathogen. But, 16

ACCEPTED MANUSCRIPT on the other hand, this ABA-related reduced content of β-1,3-glucanase may simultaneously reduce plants ability to digest components of fungus cell wall (Mohr and Cahill 2001; MauchMani and Mauch 2005). Abscisic acid was also demonstrated to inhibit actions of salicylic and jasmonic acids, as well as ethylene, which play major roles in disease resistance (MauchMani and Mauch 2005). However, the quantitative relationships between ABA, JA, and SA

4.3.4. Salicylic and jasmonic acid contents

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contents were not clearly revealed in our study.

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Salicylic acid is implicated in biotic stress responses and was proved to initiate systemic

acquired resistance (SAR) and production of specific pathogenesis-related (PR) proteins (Mauch-Mani and Métraux 1998; Ganesan and Thomas 2001; Mauch-Mani and Mauch

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2005). The feeding style of M. nivale suggests to include it to the hemibiotrophic fungi (Żur et al. 2011). This type of host tissue penetration induces plant defense reactions resulted in the synthesis of both salicylic (typical for biotrophic fungi) and jasmonic (typical for necrotrophic fungi) acids (Mendgen and Hahn 2002; Ding et al. 2011). The observed in the current study pattern of SA level changes during infection suggests that on the first days M. nivale feeding

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style had a biotrophic character. The importance of SA in the resistance level was noticed here in its generally higher amount in the leaves of the resistant introgression form, and in the increasing accumulation level of this hormone during infection progression in both the leaf

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and crown tissue of this form.

It seems that the important phenomenon, which could be responsible, at least partially, for the reduced ability of the Lm/Fa-S introgression form to cope with the infection progression, was

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the reduction of JA amount during infection, compared to the parallel control conditions. A higher JA amount determined seven days after inoculation, suggests a necrotrophic feeding style of M. nivale. In our opinion, the patterns of SA and JA content changes in the leaves and crowns confirmed our earlier suggestion that this pathogen should be included to the group of hemibiotrophic fungi (Żur et al. 2011).

5. Conclusions

The results presented here concern the first research on potential mechanisms of resistance to M. nivale in L. multiflorum × F. arundinacea hybrids. These plant materials belong to the 17

ACCEPTED MANUSCRIPT Lolium-Festuca complex, comprising species, intergeneric hybrids and their introgression derivatives, which are crucial for grass-biomass production in the temperate regions. It was possible to select here the L. multiflorum/F. arundinacea introgression forms with a different genomic structure, significantly distinct with respect to their resistance to M. nivale. Furthermore, it was noticed also that the resistance to M. nivale was negatively correlated

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with the tolerance to frost in the selected forms. However, the molecular basis underlying that lack of positive correlation between both key components of winter hardiness, has not been explained in the current research. The higher total soluble carbohydrates and SA contents as well as a lower level of ABA and JA, in the leaf and crown tissue under the applied

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experimental conditions, were suggested to be crucial for the resistance to M. nivale in the L. multiflorum/F. arundinacea introgression forms. However, it seems that with respect to that trait the accumulation dynamics of these compounds is even more important. The role of the

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phenolics bound in the plant cell walls cannot be also excluded here. Furthermore, the accumulation profiles of SA and JA in the leaves and crowns confirmed our earlier suggestion that M. nivale should be included to the group of hemibiotrophic fungi.

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Contributions

Agnieszka Płażek coordinated and interpreted phytopathological studies. Ewa Pociecha prepared fungal cultures, inoculated plants, and estimated the levels of plant resistance. Adam Augustyniak and Izabela Pawłowicz developed and selected plants for the studies. Katarzyna

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Masajada and Joanna Majka performed cytogenetic analysis. Michał Dziurka performed HPLC analysis of hormones’ contents in the leaves and crowns. Dawid Perlikowski performed statistical analysis. Arkadiusz Kosmala coordinated the whole project, prepared

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final diagrams, and wrote the first version of the manuscript All the authors participated in the preparation of the final version of this manuscript.

Acknowledgments. The research was funded by the Polish Ministry of Agriculture and Rural Development (2014-2020); project: “Identification of genes associated with the expression of winter-hardiness and tolerance to drought in Lolium multiflorum/Festuca arundinacea introgression forms”.

Figure legends 18

ACCEPTED MANUSCRIPT Fig. 1. The general scheme of the experimental set up. In the parentheses the days after artificial inoculation, were indicated. Fig. 2. Level of resistance to M. nivale of 20 L. multiflorum/F. arundinacea introgression forms. The evaluation scale was [0-5], where ‘5’ means a plant without any symptoms of

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disease and ‘0’ means a dead plant without any signs of re-growth ability after infection. Results are shown as means (n=6) of two independent inoculation experiments and standard error bars.

Fig. 3. Genomic structure of the L. multiflorum/F. arundinacea introgression forms – Lm/Fa-

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R (resistant) (A) and Lm/Fa-S (susceptible) (B). Upper part: FISH with rDNA probes – 5S rDNA (pink), 35S rDNA (green); chromosomes counterstained with DAPI (blue). Middle part: GISH with the total genomic DNA of L. multiflorum used as a probe (yellow);

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chromosomes counterstained with propidium iodide (orange). Chromosomes with F. arundinacea chromatin (orange) are indicated with red arrows. Lower part: enlarged chromosomes with F. arundinacea chromatin.

Fig. 4. Accumulation of total soluble carbohydrates (TSC) in the leaves and crowns under control and infection conditions in the Lm/Fa-R (resistant) and Lm/Fa-S (susceptible)

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introgression forms. Means (n=3) and standard error bars, are indicated. Values marked with the same letter did not differ at a significance level of 0.05 (Duncan’s Multiple Range Test). Abbreviations: pre-H – pre-hardening, H – hardening, DW – dry weight. Time-points of tissue sampling were described in the Fig. 1.

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Fig. 5. Accumulation of phenolic (PC) compounds in the leaves and crowns under control and infection conditions in the Lm/Fa-R (resistant) and Lm/Fa-S (susceptible) introgression forms. Means (n=5) and standard error bars, are indicated. Values marked with the same letter did

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not differ at a significance level of 0.05 (Duncan’s Multiple Range Test). Abbreviations: preH – pre-hardening, H – hardening, DW – dry weight. Time-points of tissue sampling were described in the Fig. 1.

Fig. 6. Accumulation of abscisic acid (ABA) in the leaves and crowns under control and infection conditions in the Lm/Fa-R (resistant) and Lm/Fa-S (susceptible) introgression forms. Means (n=5) and standard error bars, are indicated. Values marked with the same letter did not differ at a significance level of 0.05 (Duncan’s Multiple Range Test). Abbreviations: preH – pre-hardening, H – hardening, DW – dry weight. Time-points of tissue sampling were described in the Fig. 1.

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ACCEPTED MANUSCRIPT Fig. 7. Accumulation of salicylic acid (SA) in the leaves and crowns under control and infection conditions in the Lm/Fa-R (resistant) and Lm/Fa-S (susceptible) introgression forms. Means (n=5) and standard error bars, are indicated. Values marked with the same letter did not differ at a significance level of 0.05 (Duncan’s Multiple Range Test). Abbreviations: preH – pre-hardening, H – hardening, DW – dry weight. Time-points of tissue sampling were

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described in the Fig. 1. Fig. 8. Accumulation of jasmonic acid (JA) in the leaves and crowns under control and

infection conditions in the Lm/Fa-R (resistant) and Lm/Fa-S (susceptible) introgression forms. Means (n=5) and standard error bars, are indicated. Values marked with the same letter did

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not differ at a significance level of 0.05 (Duncan’s Multiple Range Test). Abbreviations: preH – pre-hardening, H – hardening, DW – dry weight. Time-points of tissue sampling were

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described in the Fig. 1.

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ACCEPTED MANUSCRIPT Highlights •

Two Lolium multiflorum/Festuca arundinacea introgression forms with distinct levels of resistance to Microdochium nivale were selected. The selected plants revealed a different genomic structure.

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The resistant introgression form was characterized by a higher amount of soluble

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carbohydrates and salicylic acid, and a lower amount of abscisic acid in the leaf and crown tissues. •

The susceptible introgression form demonstrated a higher level of jasmonic acid in the

The patterns of salicylic and jasmonic acids accumulation in the analyzed plants

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confirmed suggestions that M. nivale should be regarded as a hemibiotrophic fungus.

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crowns.

ACCEPTED MANUSCRIPT Contributions. Agnieszka Płażek coordinated and interpreted phytopathological studies. Ewa Pociecha prepared fungal cultures, inoculated plants, and estimated the levels of plant resistance. Adam Augustyniak and Izabela Pawłowicz developed and selected plants for the studies. Katarzyna Masajada and Joanna Majka performed cytogenetic analysis. Michał Dziurka performed

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HPLC analysis of hormones’ contents in the leaves and crowns. Dawid Perlikowski performed statistical analysis. Arkadiusz Kosmala coordinated the whole project, prepared final diagrams, and wrote the first version of the manuscript All the authors participated in

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