Jukuri, open repository of the Natural Resources Institute Finland (Luke) All material supplied via Jukuri is protected by copyright and other intellectual property rights. Duplication or sale, in electronic or print form, of any part of the repository collections is prohibited. Making electronic or print copies of the material is permitted only for your own personal use or for educational purposes. For other purposes, this article may be used in accordance with the publisher’s terms. There may be differences between this version and the publisher’s version. You are advised to cite the publisher’s version. This is an electronic reprint of the original article. This reprint may differ from the original in pagination and typographic detail. Author(s): S. M. Velmala, T. Rajala, A. Smolander, R.-L. Petäistö, A. Lilja & T. Pennanen Title: Infection with foliar pathogenic fungi does not alter the receptivity of Norway spruce seedlings to ectomycorrhizal fungi Year: 2014 Version: Accepted manuscript Copyright: The Authors 2014 Rights: This Item is protected by copyright and/or related rights. You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights- holder(s) Rights url: https://rightsstatements.org/page/InC/1.0/?language=en Please cite the original version: Velmala, S. M., Rajala, T., Smolander, A., Petäistö, R.-L., Lilja, A., & Pennanen, T. (2014). Infection with foliar pathogenic fungi does not alter the receptivity of Norway spruce seedlings to ectomycorrhizal fungi. Plant and Soil, 385(1–2), 329–342. https://doi.org/10.1007/s11104-014- 2238-y 1 Infection with foliar pathogenic fungi does not alter the receptivity of 1 Norway spruce seedlings to ectomycorrhizal fungi 2 Velmala1 SM, Rajala1 T, Smolander1 A, Petäistö2 R-L, Lilja1 A & Pennanen1 T. 3 1Finnish Forest Research Institute Metla, PO Box 18, 01301 Vantaa, Finland; 4 sannakajsa.velmala@metla.fi, tiina.rajala@metla.fi, aino.smolander@metla.fi, arja.lilja@metla.fi, 5 taina.pennanen@metla.fi; 2Finnish Forest Research Institute Metla, Suonenjoki, Finland, 6 raij.petaisto@gmail.com. 7 Author for correspondence: 8 Sannakajsa M. Velmala, Finnish Forest Research Institute, PO Box 18, 01301 Vantaa, Tel: +35829 9 5322580, Email: sannakajsa.velmala@metla.fi, Fax: +35829 5322103 10 11 2 Abstract 12 Aims: We studied whether the induction of defence against foliar pathogens affects the interaction 13 of Norway spruce (Picea abies) with ectomycorrhizal fungi (EMF) and whether the response differs 14 between seedlings originating from families showing variable growth performance in long-term 15 trials. 16 Methods: The shoots were inoculated with Botrytis cinerea and Gibberella avenacea. The roots 17 were simultaneously inoculated with sieved humus to provide the EMF inoculum. The severity of 18 the pathogenic infection was based on the amount of damage and induced production of condensed 19 tannins in the needles. 20 Results: EMF richness and colonisation were not affected by the pathogens and were also identical 21 between the fast- and slow-growing seedlings. The fast-growing seedlings were more vulnerable to 22 the pathogens; however, the constitutive level of condensed tannins in the needles did not correlate 23 with their susceptibility to either the pathogenic or symbiotic fungi. G. avenacea induced a 24 marginally greater production of condensed tannins in the slow-growing seedlings, which was 25 linked to a slight reduction in EMF richness and less needle damage after wintering. 26 Conclusions: Our results suggest that there are differences in resource allocation strategies between 27 the fast- and slow-growing spruce families, which may indicate the presence of underlying host 28 effects that regulate interactions with associated fungi.29 3 Introduction30 As sessile organisms, coniferous trees rely on both constitutive and induced defence mechanisms 31 against phytopathogenic microbes (Bonello et al. 2006). Many necrotising fungal pathogens infect 32 container seedlings of Norway spruce (Picea abies (L.) Karst.), and a primary risk both during the 33 growing period and after winter storage in frost is the common spruce pathogen Botrytis cinerea 34 Pers. (teleomorph Botryotinia fuckeliana (de Bary) Whetzel), which is the cause of grey mould 35 disease (Petäistö et al. 2004; Petäistö 2006). Fusarium (anamorphic Gibberella) species are also 36 widely distributed and cause damping-off and root rot diseases in nurseries (Hansen and Hamm 37 1988; Lilja et al. 1992) and infect the foliage of spruce saplings, resulting in needle death (Petäistö 38 et al. 2012). 39 Damage by necrotising microbial attackers can lead to unspecific and comprehensive systemic 40 responses distant to the actual location of damage and further primes defence reactions upon a 41 second challenge (Becker and Conrath 2007). Systemic signals may be bidirectional as reported for 42 the Austrian pine (Pinus nigra Arnold) (Blodgett et al. 2007) and asymmetric in time (Eyles et al. 43 2010). Moreover, the microbe induced systemic resistance may interfere with the mutualistic plant-44 microbe interactions in distal organs (Román et al. 2011; van Dam and Heil 2011) and may render 45 the host even more vulnerable to other enemies (Heil and Baldwin 2002). 46 Both local and induced resistance have been reported in Norway spruce after infection with 47 pathogenic fungi (Christiansen et al. 1999; Krokene et al. 2001; Swedjemark et al. 2007; Fossdal et 48 al. 2012b; Nagy and Fossdal 2013) and beneficial microbes, such as ectomycorrhizal fungi (EMF) 49 and mycorrhiza helper bacteria (Sampangi et al. 1986; Lehr et al. 2007; Likar and Regvar 2008; 50 Nagy and Fossdal 2013) since also infection with mutualistic fungi suppresses the immune system 51 of the host (Adomas et al. 2008; Heller et al. 2008; Nagy and Fossdal 2013). The resistance 52 activation caused by pathogens may cause conflicting effects on fungal symbiosis with plants (van 53 Dam and Heil 2011) depending on the symbiotic tissue (Blodgett et al. 2007) and the defence 54 pathway it activates (de Román et al. 2011; Pfabel et al. 2012 etc.). Infecting fungi can produce 55 phytohormones or induce the hormone production of the host plant (Robert-Seilaniantz et al. 2011). 56 Infection with a necrotising fungus may activate the jasmonic acid and ethylene-based broad-57 spectrum nonspecific resistance, which may have positive effects on biotrophic associations such as 58 the mycorrhizal colonization because jasmonic acid may suppress the endogenous salicylic acid -59 mediated biotroph-specific defence (van Dam and Heil 2011). Moreover, salicylic acid seems to 60 support the production of cytokinins and gibberellins, and to antagonize auxin biosynthesis in 61 4 angiosperms , and this crosstalk of plant hormones shapes the physiological outcome of the 62 pathogenic attack (Robert-Seilaniantz et al. 2011). 63 One important group of defensive phenolic substances are condensed tannins (proanthocyanidins), 64 which are abundant in Norway spruce. These compounds are formed during the last step of 65 flavonoid biosynthesis and function both in constitutive and induced defence. Condensed tannins 66 form the major component of phenolics in needle mesophyll cells of Norway spruce (Soukupová et 67 al. 2000) and different types of tannins inhibit the growth of several fungi and bacteria in pure 68 culture (Kraus et al. 2003). They also appear to function as regulators of the fungal interactions of 69 foliage, as reported from Fremont cottonwood (Populus fremontii S. Wats.) (Bailey et al. 2005) and 70 Norway spruce (Rajala et al. 2014). The basal content of condensed tannins in trees is heritable; 71 therefore, the genotype explains much of the intraspecific variation in the concentration of 72 condensed tannins and other phenolic compounds, such as flavonoids (Mansfield et al. 1999; 73 Lamhamedi et al. 2000; Evensen et al. 2000; Schweitzer et al. 2008; Henery 2008). Furthermore, 74 the concentration of condensed tannins also varies depending on the organ, and the concentration is 75 more adaptive in shoots and leaves compared to roots, where it is relatively stable (Kosola et al. 76 2006). Upon infection with fungal pathogens the phenolic biosynthesis is induced (Likar and 77 Regvar 2008) and the accumulation of e.g. catechins, the building blocks of condensed tannins, has 78 been reported in Norway spruce (Evensen et al. 2000). These simple phenolics are gradually 79 converted to tannins and other insoluble polymers during an induced phenolic response (Brignolas 80 et al.1995; Evensen et al. 2000). 81 Plant-mediated interactions between the above- and below-ground communities of microbes have 82 been identified with angiosperms (de Román et al. 2011) and grasses (Mack and Rudgers 2008). 83 Recently, we found a slight negative correlation between the abundance of fungal needle 84 endophytes and the EMF richness of Norway spruce roots (Rajala et al. 2013). Also previously we 85 have observed opposite relationships between the growth rates of 14-yr-old Norway spruce clones 86 and the root and shoot associated fungal communities, such that the fast-growing spruces had higher 87 EMF diversity (Korkama et al. 2006) but possessed significantly less saprotrophic needle 88 endophytes (Korkama-Rajala et al. 2008) than the slow-growing spruces of equal age. However, 89 based on our most recent studies, the ability to form ectomycorrhizas does not differ between the 90 equally sized young spruce seedlings showing fast and slow growth performance later during their 91 life span (Velmala et al. 2014). The associated fungi may cause trade-offs between growth, 92 differentiation processes, and transferred resources as they bind host photosynthesised carbon and 93 nutrients in the environment of limited resources (Herms and Mattson 1992). We have observed 94 5 some differences in resource allocation between seedlings of fast- and slow-growing Norway spruce 95 origins (Velmala et al. 2014), and there might be differing needs of resource allocation between 96 growth and defence of these contrasting seed families. The positive relationship observed between 97 growth rate and the EMF richness of Norway spruce (Korkama et al. 2006) may be a result of 98 genetically different use of resources or varying responses to fungal infection. The cause of 99 differing EMF communities could also lie in the genetic susceptibility to pathogens, as it was 100 shown that the genetic resistance/susceptibility to herbivory affected the EMF community structures 101 of pinyon pines (Pinus edulis Engelm) (Sthultz et al. 2009). 102 To the best of our knowledge, no previous studies have addressed whether the induction of foliar 103 defences in conifers extends to the roots and affects root-associated EMF symbiosis. Host-fungal 104 relations may alter defence metabolism and may thereby affect tree resource allocation and further 105 interfere with other plant-microbe associations. Furthermore, the observed differences in the 106 associated fungal communities of seedlings with fast and slow growth during the later 107 developmental stage could reflect differences in the interactions between distant fungal 108 communities, including pathogens. 109 In our glasshouse experiment, we investigated whether the induction of foliar defences affect the 110 ectomycorrhizal colonization and richness of Norway spruce. Therefore, two types of seedlings 111 differing in long-term growth performance were infected with two necrotising fungal pathogens, B. 112 cinerea and Gibberella avenacea R.J. Cook (Synonym: Fusarium avenaceum (Fr.) Sacc.). The 113 experiment was designed to measure the responses of three well growing and three poorly growing 114 Norway spruce seed families before any differences in growth rates are visible. We hypothesise that 115 pathogen-induced stress will affect the ectomycorrhizal colonization and the EMF richness of 116 Norway spruce and that the EMF richness is lower in seedlings that are either more resistant to 117 fungal infection and thus foliar pathogens or show stronger induced responses to pathogenic 118 infection. Moreover, we postulate that genetically different Norway spruce seedlings vary in their 119 susceptibility to foliar pathogens and that the susceptibility to all fungal infections is higher in the 120 fast-growing seedling. We propose that there will be differences in the carbon allocation as a 121 response to pathogens and that the accumulation of condensed tannins varies in the needles after 122 foliar infection in genetically different seedlings. 123 6 Materials and methods124 Plant and fungal materials 125 The study was performed in the Suonenjoki nursery (62.625N, 27.122E) in eastern Finland with 126 seven Norway spruce seed families from which three were classified as fast-growing (good and 127 excellent growth) and three as slow-growing (stunted) families in long-term field trials (seed origins 128 as in Velmala et al. 2014). The seventh spruce family represented seed-orchard-seeds used in forest 129 regeneration in southern Finland (Online resource1 Table ESM1). In April 2011, the spruce seeds 130 were germinated in nursery containers on unfertilised light sphagnum peat PP03 (Kekkilä, Vantaa, 131 Finland) in a glasshouse. After six weeks, these seedlings were inoculated by transplantation into 132 Plantek-81F containers (cell vol 85 cm3) (BCC, Säkylä, Finland) filled with sieved forest humus 133 layer, which acted as a natural source of EMF inoculum. The humus was excavated from the 134 uppermost layer of fine sandy till of a Norway spruce stand out planted in 1993 at the nearby 135 Ruotsinkylä research area located in southern Finland. 136 Inoculation with the two fungal pathogens and with tap water only were performed in June when 137 the seedlings were indoors and were replicated in July and late August after the seedlings were 138 moved outside in mid-June. Eighteen seedlings from each of the seven seed families were randomly 139 placed in the three infection treatments. Before inoculation, the shoots were sprayed with tap water 140 to moisten the foliage. The seedlings were inoculated with a spore suspension of either Botrytis 141 cinerea isolate BcSjk1.1 (Petäistö et al. 2004) containing ca. 200 000 spores or the anamorphic 142 spores of Gibberella avenacea [Pielavesi nursery isolate (Fusarium avenaceum-G. avenacea)] 143 (Petäistö et al. 2012) containing ca. 90 000 spores. The inoculation occurred in June, July, and in 144 August. In August the spore suspension of G. avenacea contained a fourfold amount of spores. The 145 water only controls were treated with tap water by pipetting 200 µl of water terminally onto the 146 seedlings. After each inoculation, the seedlings were maintained indoors in the glasshouse, and 147 shoots were repeatedly sprayed with water to keep the foliage humid. Three days after the 148 inoculation, the seedlings were moved outdoors into a nursery field. An EMF re-inoculation of roots 149 was performed in August simultaneously with the foliar inoculations by adding 30 ml sieved forest 150 humus to the base of each seedling. 151 The B. cinerea spores were liberated from two-week-old cultures grown at 17 °C (in darkness for 3 152 days and then moved to ambient light) on potato dextrose agar medium with sterile water rubbed 153 with a glass rod. The G. avenacea spores were produced on autoclaved barley-corn with spruce and 154 pine needle homogenate (Petäistö and Kurkela 1993; Winder 1999) and grown for three weeks at 17 155 7 °C in light, after which an orange spore mass was collected in sterile water by rubbing the plate 156 with a glass rod. The number of spores in the filtered suspensions was estimated with a 157 haemocytometer (Fuchs-Rosenthal, Paul Marienfeld GmbH & Co. KG, Germany). The vitality of 158 each spore suspension was monitored with cultivation. 159 In general, the growth conditions of the seedlings during the summer adhered to the common 160 seedling production practises in Finland. The seedlings were fertilised with approximately 10 mg N 161 per seedling according to Kekkilä Forest-Superex (NPK 22-5-16) fertilisation program (Kekkilä, 162 Vantaa, Finland). Wintering of the seedlings was performed in an open nursery field under natural 163 snow cover. 164 Sampling 165 Seven replicate seedlings were sampled in late October after seven months of growth. The severity 166 of infection was determined by counting the number of damaged needles per seedling. Thereafter, 167 the shoots and roots were separated and the shoot heights measured. The level of EMF colonisation 168 (%) in the roots and the root tip densities (tips/mm) and the numbers were assessed under a 169 stereomicroscope. The following May, after wintering outdoors, the shoot length and number of 170 damaged needles of the remaining seedlings (seven families, three treatments, seven replicate 171 seedlings) were measured. Finally, after all sampling the roots and shoots were dried overnight at 172 60 ºC and weighed. 173 DGGE and sequence analysis 174 A randomised bulk sample (50 mg f.w.) of EMF fine root fragments from five replicate seedling 175 was freeze dried and homogenised in quartz sand with a FastPrep® (FP120; Qbiogene, Cedex, 176 France) and subjected to DNA extraction with NucleoSpin Plant II (Macherey-Nagel, Düren, 177 structions. The roots were lysed using the 178 CTAB lysis method based PL1 buffer and incubated for 30 min. Finally, the DNA was eluted with 179 50 µl and 100 µl PE buffer, precipitated with 0.6 vol of PEG-NaCl solution [20% PEG (w/v), 2.4 M 180 NaCl] on ice for 20 min, centrifuged (16 000 g for 20 min) and washed with 70 % ethanol. The dry 181 pellets were resuspended in diluted 30 µl TE buffer (1.5 mM Tris/HCl, 0.25 mM EDTA) and stored 182 at -20 °C. 183 To verify that the infection with the foliar pathogens was successful, we randomly selected eight 184 seedlings from both disease treatments and isolated the DNA from 80 mg (f.w.) needles. The freeze 185 dried needles were homogenised with two nuts and a screw using a FastPrep® (FP120; Qbiogene, 186 8 Cedex, France), and then subjected to the NucleoSpin Plant II (Macherey-Nagel, Düren, Germany) 187 DNA extraction protocol followed by PEG precipitation. 188 We used denaturing gradient gel electrophoresis (DGGE) (Muyzer et al. 1993) to analyse the fungal 189 communities, which is based on the amplification of the internal transcribed spacer 1 (ITS1) region 190 of rDNA (Andersson et al. 2003). The DGGE PCR products were generated with the primers ITS1F 191 (Gardes and Bruns 1993) with a 40 bp GC-clamp (Muyzer et al. 1993) and ITS2 (White et al. 192 1990). The cycling wa193 194 The thermal cycling conditions were as follows: an initial denaturation of 3 min at 95 °C followed 195 by 34 cycles of 30 s at 95 °C, 30 s at 57 °C, 60 s at 72 °C, and a final extension of 72 °C for 5 min. 196 The PCR products were electrophoresed with the DCode universal mutation detection system (Bio-197 Rad Laboratories, Hercules, CA, USA) in an acrylamide denaturing 18 58 % gradient gel (75 V, 60 198 °C, 16 h), stained with SYBR® Gold (Molecular Probes®; Life Technologies, Carlsbad, CA, USA) 199 200 USA) blue light. Image analysis and a binary matrix of band motility classes (occurrence of 201 operational taxonomic units, OTUs) were performed with the GelCompar II software (Applied 202 Maths NV, Sint-Martens-Latem, Belgium), version 5.1 with band matching optimisation of 0% and 203 position tolerance of 1%. 204 The identification of various OTUs representing the fungal community was based on Sanger 205 sequencing of at least two excised bands per OTU. Each DGGE band of interest was repeatedly 206 excised two to four times until the contaminant sequences could not be detected in the DGGE gel. 207 The pure bands were eluted with water and used as a template for PCR with the ITS1F and ITS2 208 primers, with 25 cycles as described above. The PCR fragments were purified and sequenced by the 209 Macrogen Sequencing Service (Macrogen Europe, The Netherlands) using the ITS1F-primer. The 210 sequences were revised in Geneious R6 (Biomatters, Auckland, New Zealand, available from 211 http://www.geneious.com/). The OTUs were identified by comparing the sequences (Online 212 resource1 Table ESM2; GenBank accession numbers KJ909938 KJ909957) against sequences in 213 UNITE (http://unite.ut.ee/) and the INSD database. 214 Measurement of condensed tannins 215 The dry needles were homogenised with a FastPrep® in 2-ml polypropylene tubes with 216 FastPrep®matrix A ceramic spheres and subjected to condensed tannin analysis using a 217 spectrophotometric, modified acid-butanol assay (proanthocyanidin assay, Waterman & Mole 1994) 218 9 as described by Kanerva et al. (2008). Briefly, the assay consists of a 70% acetone extraction 219 followed by HCl-catalysed depolymerisation of the condensed tannins in butanol to yield a pink-red 220 anthocyanidin product. However, in the present study, the extraction (3x5min, 40 ºC) of 0.1 g 221 homogenised needle material was performed with the Accelerated Solvent Extraction equipment 222 (ASE-350, Dionex, USA). As a calibrator, we used the condensed tannins extracted and purified 223 from Norway spruce needles according to the method of Kanerva et al. (2006). 224 Statistical analyses 225 To determine the differences in the growth parameters, the number of damaged needles, the 226 condensed tannin content in the needles, the levels of EMF colonisation and the EMF OTU richness 227 of the fast- and slow-growing seedlings in different treatments, we used linear and generalised 228 linear mixed models {lmer, glmer; lme4} (Bates et al. 2013). In the first model, the effect of the 229 growth group (fast or slow) on various parameters was studied separately in all treatments, and the 230 growth performance group was set as the fixed effect and seed origin was set as the random effect. 231 The second model solely evaluated the effect of treatment and did not distinguish between the fast 232 and slow growing seedlings; therefore, the treatment was set as the only fixed effect. The third 233 model included the treatment, growth performance group and their interaction as the fixed factors. 234 By including the interaction of the growth type and treatment, we determined whether the response 235 to treatment varied between the growth types. The interaction term was omitted from the model 236 when it was not at least marginally significant (P-value > 0.1). Seedling growth and needle damage 237 between autumn and spring were calculated with the growth performance group and year as the 238 fixed effects. The seed family was set as random in all of the models described above. The P-values 239 for the lmer-objects were calculated with the Satterthwaite approximation (Satterthwaite 1946) for 240 degrees of freedom {lmerTest} (Kuznetsova et al. 2012). For the counts, such as the OTU richness 241 and root tip number, we used a Poisson error distribution, and for the percentage, the binomial error 242 distribution. Wald chi-square likelihood-ratios were generated with {Anova; car} (Fox and 243 Weisberg 2011). 244 To assess the EMF community composition, a Bray-Curtis distance matrix (Bray and Curtis 1957) 245 was generated and exposed to 2-dimensional non-metric multidimensional scaling (NMDS) 246 {metaMDS: vegan} (Oksanen et al. 2013). The lowest stress was achieved through replicating the 247 loop 20 times with a maximum number of 40 starts. We also addressed the effect of seed origin and 248 other measured traits on the multivariate community data of the EMF with permutational 249 10 MANOVA with 4999 permutations and the seed origin as the stratum {adonis: vegan} (Oksanen et 250 al. 2013). 251 The linear relationships between the different traits were analysed using 252 statistics. All statistical analyses were performed in R 2.15.3 (R Development Core Team 2013), 253 and the graphs were created with packages ggplot2 (Wickham 2009) and vegan (Oksanen et al. 254 2013). 255 Results 256 Ectomycorrhizal fungal colonization and community composition 257 The inoculation with natural humus was successful, and the level of EMF colonisation (%) in the 258 roots of Norway spruce seedlings was greater than 80% (Table 1). In the slow-growing seedlings, 259 the condensed tannin content in the needles positively correlated with the root EMF colonisation % 260 in all treatments (G. avenacea: t19=3.40, r =0.61, P =0.003), (water only control t19=1.70, r =0.36, P 261 =0.106), (B. cinerea: t19=2.57, r =0.51, P =0.019) and the induced production of condensed tannins 262 for the G. avenacea (t19=3.87, r =0.66, P =0.001) and B. cinerea (t19=2.18, r =0.45, P =0.04) foliar 263 treatments. However, in the fast-growing seedlings, EMF colonisation (%) positively correlated 264 with the condensed tannin content (t19=2.85, r =0.55, P =0.010) and the induced level of condensed 265 tannins (t19=2.38, r =0.48, P =0.03) only when the seedlings were infected with B. cinerea. 266 The EMF OTU richness was not affected by the constitutive level of condensed tannins or the 267 fungal pathogen induced resistance in the foliage and was similar in all treatments and for both 268 growth performance groups (Table 1). Of the slow-growing families, the EMF OTU richness 269 positively correlated with the constitutive level of condensed tannins in the needles (water only 270 control: t13=2.76, r =0.60, P =0.02). The EMF OTU richness was lowest in the G. avenacea 271 treatment, but the reduction was not statistically significant (Table 1). Furthermore, there was a 272 marginally significant negative correlation between the G. avenacea induced production of 273 condensed tannins in the needles and the EMF OTU richness in the roots of the slow-growing 274 seedlings (G. avenacea: t13=-1.89, r =-0.46, P =0.08). Of the fast-growing seedlings, the EMF OTU 275 richness negatively correlated with the root biomass (G. avenacea: t13=-2.24, r =-0.53, P =0.04), but 276 the other root growth traits did not have any linear relationships with the EMF OTU richness. 277 The EMF community comprised Atheliaceaes, such as Amphinema byssoides, Tylospora 278 asterophora and Piloderma sphaerosporum, as well as Thelephora terrestris, Lactarius rufus, and 279 11 Cenococcum geophilum (Fig. 2). Two of the sequences corresponded to uncultured environmental 280 EMF root tip sequences in the UNITE database, and two other OTUs corresponded to 281 ectomycorrhiza associated fungi (Archaeorhizomyces (Rosling et al. 2011) and fungi inhabiting 282 Cenococcum sp. ectomycorrhizae). The other fungal OTUs corresponded to ascomycoteous moulds 283 and saprotropic soil fungi (Mortierella, Cryptococcus, Ilyonectria, Trichosporon, and Fomitopsis 284 species). All non-EMF OTUs were omitted from the EMF OTU count, but the total DGGE-based 285 fungal OTU richness is reported in Table 1. 286 The EMF community structures of the fast- and slow-growing Norway spruce seedlings greatly 287 overlapped and were independent of treatment (Fig. 2). There was a weak linear correlation 288 between the shoot:root ratio and the 2-dimensional ordination of the EMF community (r2=0.077, P 289 =0.03) (Fig. 2). Based on the permutational multivariate ANOVA, over 95% of the variation in the 290 EMF community remained unexplained with the measured traits. 291 Growth 292 As expected (Velmala et al. 2014), there were no significant differences in the growth between 293 seedlings of fast and slow origins during their first year of growth (Table 1, Online resource1 Table 294 ESM3). The shoot and root biomass had a strong and systematic positive relationship in the entire 295 dataset (t145=18.96, r=0.84, P <0.001). The foliar infection with Botrytis cinerea and Gibberella 296 avenacea increased the root (F2=6.186, P=0.002) and shoot (F2=4.378, P=0.015) biomass of the 297 seedlings compared to the uninfected water only control treatment in autumn (Table 1). Seedling 298 growth was significantly affected by the necrotising treatment with B. cinerea (Table 1), thereby 299 decreasing the shoot:root ratio (F2=7.867, P<0.001). The shoot height was differentially affected by 300 the necrotising pathogens for both the fast- and slow-growing seedlings. The fast-growing seedlings 301 were taller both in the water only control and B. cinerea treatments, but by contrast, for the G. 302 avenacea treatment, the slow-growing seedlings were taller (significant interaction of treatment and 303 growth type F2=3.251, P=0.042). The seedlings that were left overwintering did not show any 304 differences in shoot height the following spring either between the treatments (F2=0.487, P=0.616) 305 or growth performance groups (F1=1.947, P=0.235). Additionally, the shoot height did not differ 306 significantly between the autumn and spring samplings (F1=0.63, P =0.434, Table 1). 307 The root tip number of fast- and slow-growing seedlings was differentially affected by the foliar 308 treatment (significant interaction of treatment and growth type 2 2=419.0, P<0.001, Table 1). The 309 slow-growing seedlings had higher root tip number than the fast-growing seedlings for both the 310 water only control (F1=6.86, P=0.012, Table 1) and B. cinerea treatments (F1=3.17, P=0.082, Table 311 12 1), whereas for the G. avenacea treatment, no differences in the short root densities were observed. 312 There were no linear correlations between the root biomass and short root density for either growth 313 performance group in any of the treatments. Regardless of the treatment, the root tip density of the 314 seed-orchard seedlings (s1002) was similar to that of the fast-growing seedlings (Table 1). 315 Susceptibility to foliar pathogens: Constitutive and induced defence 316 Foliar pathogens, B. cinerea and G. avenacea, led to a successful infection and were identified in 317 the necrotic needles by visual necrosis and sequence analysis (Online resource1 Table ESM2). The 318 necrotising pathogens caused only moderate foliar damage, resulting in an increased number of 319 dead needles ( 2 2=81.299, P<0.001, Table 1). The necrotic infections proceeded slowly throughout 320 late summer and autumn, and during the winter the number of dead needles had increased 321 significantly ( 2 1=61.32, P<0.001, Table 1) for all treatments. During the autumn, the only 322 difference in the number of damaged needles was between the water only control treatment and 323 necrotising fungal treatments, and between the spruce families (Fig. 1). Overwintering revealed 324 visible differences in the number of dead needles between the growth performance groups. The fast-325 growing seedlings had significantly higher number of necrotic needles in the G. avenacea treatment 326 group than the slow-growing seedlings ( 2 1=10.173, P=0.001, Table 1). 327 The amount of condensed tannins in the needles did not vary statistically significantly between the 328 seedlings of different origin or between the treatments (Fig. 1, Table 1). The condensed tannin 329 content in the fast-growing seedlings correlated negatively with the needle damage during the 330 autumn for the G. avenacea infection (t19=-2.53, r=-0.5, P =0.02), and marginally significantly for 331 the infection with B. cinerea (t19=-1.77, r=-0.38, P =0.09). The foliar treatment did not significantly 332 induce the production of condensed tannins in needles in the entire dataset, partially due to high 333 within family variation for the level of condensed tannins. On average, the increase in the 334 condensed tannin content for the B. cinerea infection was 12% for the fast-growing seedlings and 335 21% for the slow-growing seedlings. These values were 4% and 22%, respectively, for the G. 336 avenacea infection (Fig. 1, Table 1). This induced level of condensed tannins was assessed by 337 dividing the concentration of condensed tannins under foliar treatment with the average 338 concentration of condensed tannins in the water only control treatment separately for each seed 339 origin. 340 For the G. avenacea infection, the root biomass of the slow-growing seedlings positively correlated 341 with the concentration of condensed tannins in the needles (t19= 2.31, r = 0.61, P <0.01) and the 342 induced level of tannins (t19= 3.01, r = 0.57, P <0.01). The condensed tannins did not have an 343 13 unambiguous linear correlation with the short root density; however, the fast-growing seedlings 344 infected with G. avenacea showed a positive relationship between the concentration of condensed 345 tannins and the total number of root tips (t19= 2.31, r = 0.46, P =0.03). 346 Discussion 347 Contrary to our hypothesis, the Norway spruce seedlings were similarly receptive to EMF for all 348 treatments. The EMF richness of the fast- and slow-growing seedlings did not differ, as in our 349 previous study with pure culture EMF inoculation of spruce seedlings with identical origins 350 (Velmala et al. 2014). The induced defences against the common grey mould Botrytis cinerea and 351 Gibberella avenacea did neither significantly alter the EMF associations in the roots, even though 352 induced defences triggered by foliar pathogenic fungi have repetitiously been reported in Norway 353 spruce (Krokene et al. 2001; Nagy et al. 2004; Swedjemark et al. 2007). It is good to keep in mind 354 that the DGGE based EMF OTU richness is an estimate of the true EMF richness, and it was not 355 possible to partition ectomycorrhizal abundances. Moreover we might have missed rare EMF 356 species, since pooling and PCR based techniques do not necessarily recover real species richness 357 and abundance of the fungal communities (Avis et al. 2010). 358 As hypothesised, the susceptibility to pathogenic fungal infection varied between the spruce 359 families and between the fast-and slow-growing seedlings when the follow-up period was extended 360 over winter. The fast-growing seedlings were slightly more susceptible to foliar damage than the 361 slow-growing seedlings, particularly for the G. avenacea infection. During defence activation, 362 resources that could be used for growth are redirected to defence, thereby creating a trade-off of 363 resources between growth and defence (Eyles et al. 2010; Schultz et al. 2013). Nevertheless, in the 364 present study, the foliar infection and consequent production of defence compounds did not have 365 any relevant cost for seedling growth during the first year. This may be explained by the rapid sink-366 source transitions between the carbon sinks created by various fungal points of infection (both 367 ectomycorrhizal and pathogenic fungi) along the plant (Schultz et al. 2013). In fact we observed a 368 positive relationship between growth and infection with foliar pathogenic fungi, and when exposed 369 to foliar stress by B. cinerea, the seedlings invested more resources belowground compared with the 370 uninfected water only control treatment. Some of these growth effects may result from changes in 371 hormone concentrations or ratios as phytohormones are suggested to be important players in 372 carbohydrate pathways and in dry-mass partitioning between shoots and roots (Rook and Bevan 373 2003). Changes in hormone balance, stimulated or produced by the fungal pathogen, may have 374 14 drastic effects; Exogenous gibberellin treatment of angiosperms have shown to increase growth and 375 also salicylic acid production, which in turn reduces jasmonic acid content and leads to increased 376 susceptibility to necrotizing fungi (Robert-Seilaniantz et al. 2011). Furthermore, reductions of 377 cytokinins, involved in the regulation of shoot and root growth, increase the ratio of auxin and 378 cytokinins which have been observed to stimulate root growth (Thomas et al. 1995). Plants also 379 have the tendency to respond to pathogenic attack by moving valuable resources away from the 380 diseased tissue (Schultz et al. 2013), as is a commonly outcome reported for foliar stresses (Barto 381 and Rilling 2010). In Velmala et al. (2014) we showed that after an intensive EMF inoculation, the 382 fast-growing seedlings invested slightly more carbon in the belowground biomass compared to the 383 shoot biomass than the slow-growing seedlings. In the present study the slow-growing seedlings 384 responded more robustly to foliar infection and were less damaged by the foliar pathogens after 385 winter. They also showed a marginal decrease in EMF richness after G. avenacea infection. It is 386 unlikely that G. avenacea reduced the ability of the seedling to support root symbiotic fungi via a 387 reduction in the photosynthetic capacity because the degree of foliar damage caused was very 388 moderate (approximately 5%) and because removal of the photosynthetic tissue will not reduce but 389 rather boost the carbon flow belowground (Barto and Rillig 2010). Therefore, the pathogen-390 triggered defence mechanisms in the shoots of slow-growing Norway spruce seedlings may have 391 been stronger than the defence mechanisms of the fast-growing seedlings. 392 The pathogen-triggered defence measured by the production of condensed tannins in the needles 393 was relatively weak. The synthesis of phenols, such as condensed tannins, is very energy intensive 394 and plants may prefer the production of other defence signalling molecules, such as salicylic acid 395 (more in Pfabel et al. 2012). Our study supports the view that conifers show very little response to 396 foliar induced defences compared to broadleaf trees, because they primarily rely on constitutive 397 defence and contain high levels of defensive secondary metabolites which decrease the need of 398 induced resistance (Wagner 1988; Mattson et al. 1988; Henery 2008). Notably, we measured the 399 constitutive and induced defence only in the needles because they are adaptive organs (Kosola et al. 400 2006) and we do not know whether the foliar treatments affected the phenol content of the roots. 401 The induced systemic resistance has shown to move from the roots to the shoots in Norway spruce 402 as e.g. root inoculation with the mycorrhiza helper bacterium Streptomyces sp. increased the needle 403 resistance of Norway spruce against B. cinerea (Lehr et al. 2007). However the systemic signal is 404 not necessarily bidirectional because in our study, the B. cinerea infection had no effect on the EMF 405 richness in the roots, and exposure to G. avenacea only marginally reduced the EMF richness in the 406 slow-growing seedlings. Similar results from a field experiment were obtained for Phytophthora-407 15 infected chestnut (Castanea sativa Mill.), in which no significant differences were observed for the 408 EMF communities between healthy and infected trees (Blom et al. 2009). Moreover, the negative 409 effects of systemic resistance of soy (Glycine max (L.) Merr.) on its mycorrhizal colonization has 410 shown to be temporary and last only a short period. Additionally, the established mycorrhizas show 411 no response to the induction of resistance (de Román et al. 2011). We performed the EMF 412 inoculation and foliar infections simultaneously, which may explain why we observed no long-term 413 effects of induced defence on the associated EMF community of Norway spruce seedling roots. 414 Norway spruce has a multifaceted defence system and single phenolic markers for resistance are 415 difficult to assess (Fossdal et al. 2012a). Similar to studies with strawberry (Fragaria x ananassa 416 Duch.) (Hébert et al. 2002) and grapevine (Vitis vinifera L.) (Iriti et al. 2005), the increase of 417 condensed tannins in the needles seemed to be a suitable marker for resistance activation against B. 418 cinerea and G. avenacea. However, the condensed tannins had contradictory relationships with the 419 different fungal species. Rajala et al. (2014) suggested that the condensed tannins reduced the 420 fungal richness in the needles most likely by excluding some species more than others. 421 Correspondingly, resistant tree clones do not always contain higher concentrations of phenols, such 422 as condensed tannins or flavonoids than the susceptible clones (Henery 2008; Evensen et al. 2000). 423 In poplar (Populus trichocarpa x deltoides) an increase in the proportion of condensed tannins in 424 leaves has been reported after EMF infection (Pfabel et al. 2012). In our study, the constitutive 425 condensed tannin content in the needles of the fast-growing seedlings did not prevent the G. 426 avenacea infection, and the condensed tannin concentration had even a positive relationship with 427 the EMF colonisation % in the unstressed seedlings. Therefore, rather than acting as broad-428 spectrum antifungal components, an adequate level of condensed tannins may indicate good health 429 and vitality of the seedling. Healthy plants can remain in a stressful environment longer but will 430 eventually succumb to the pathogen if they are not resistant. 431 Fisher et al. (2006) suggested that there might be a genetically based relationship between fine root 432 production and the content of foliar condensed tannins in poplar (Populus angustifolia James and P. 433 fremontii Watson) that may have adaptive significance. In this study, the fast-growing seedlings 434 showed this type of connection under G. avenacea stress, in which the seedlings with high levels of 435 condensed tannins in their shoots grew more fine roots. This trait may have adaptive significance in 436 nature because increased fine root production has been suggested to secure nutrient gain under 437 stressed conditions (e.g., Nadelhoffer et al. 1985, Hendricks et al. 2000). In general, the fine root 438 architecture of the seedlings in the uninfected water only control treatment was as expected based 439 on our previous study Velmala et al. (2014), and the slow-growing seedlings had denser root 440 16 systems and higher root tip density than the fast-growing seedlings. The necrotising pathogen 441 attacks levelled this difference such that the root tip density of the slow-growing seedlings 442 decreased to that of the fast-growing and seed-orchard (s1002) seedlings. 443 To conclude, we demonstrate that the induction of foliar defence does not affect the root-associated 444 EMF community of Norway spruce seedlings. There was also no direct and unambiguous 445 relationship between the susceptibility to foliar fungal infection and the belowground EMF species 446 richness of the seedlings. However, certain differences between the spruce groups were observed: 447 The fast-growing seedlings were more damaged by the necrotising foliar pathogens, and the high 448 concentration of condensed tannins in the needles of fast-growing seedlings did not provide 449 resistance against B. cinerea and G. avenacea. The slow-growing seedlings were less damaged by 450 the pathogens, and they showed a somewhat stronger response to pathogenic fungi in the needles. 451 Moreover, the G. avenacea triggered defence appeared to be harmful to the root associated EMF 452 symbionts of slow-growing seedlings because the defence response marginally significantly 453 reduced the EMF richness. These trends indicate differing strategies of resource allocation between 454 fast- and slow-growing spruce families which may indicate underlying host effects for the selection 455 of symbionts for the spruce. 456 Acknowledgments 457 This work was funded by the Academy of Finland (project 128229) and Otto A. 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Springer, New York 635 Winder RS (1999) The influence of substrate and temperature on the sporulation of Fusarium avenaceum 636 and its virulence on marsh reed grass. Mycol Res 103:1145 1151 637 638 21 Table 1 Traits of Norway spruce seedlings inoculated with forest humus and exposed to two 639 necrotising foliar diseases Mean ± standard deviation for separate treatments and fast- and slow-640 growing seedlings. The letters in the right corner of the mean values indicate different mean values 641 between the treatments (P<0.05). The underlined numbers emphasised with italics indicate 642 statistically significant differences (P<0.05), and only underlined numbers (P<0.1) indicate 643 marginally significant differences between fast- and slow-growing spruce seedlings within each 644 treatment. The seed orchard seeds, s1002, are presented only as a reference; therefore, these 645 seedlings are omitted from the mean values calculated for the fast- and slow-growing seedlings. 646 Figure captions 647 Figure 1 The number of damaged needles (a), the condensed tannin concentration in needles 648 (b), the normalized condensed tannin content (c) and the EMF OTU richness (d) in three fast- 649 and three slow-growing Norway spruce seed families exposed to two necrotising foliar 650 diseases Light grey bars indicate the mean values of fast-growing seedlings and dark grey bars the 651 mean values of slow-growing seedlings. Vertical error bars show the standard error based on 652 nonparametric bootstrapping (n=7 in each bar). Statistically significant differences can be found 653 between families with non-overlapping error bars. 654 655 Figure 2 Two-dimensional NMDS of the ectomycorrhizal fungal communities of fast- and 656 slow-growing Norway spruce seedlings exposed to two necrotising foliar diseases Ellipses are 657 drawn based on standard errors of the mean scores of fast- (light grey ellipses) and slow-growing 658 (dark grey ellipses) seedlings. Treatments are separated by line type: dotted line illustrates the water 659 only control treatment without necrotising fungal pathogens. The ectomycorrhizal fungal OTU 660 centroids are marked with a triangle. The left-pointing arrow show the direction of increasing 661 shoot:root ratio (r2=0.08, P=0.03) on the NMDS plot. 662 Electronic supplementary material 663 Table ESM1 Growth and origin information of Norway spruce (Picea abies (L.) Karst.) seed 664 families used in the study Families 612, 298, 1162 represent fast-growing seedlings and families 665 1183, 394 and 427 slow-growing seedlings. Seed orchard s1002, was used as an outer reference 666 only, and was omitted from the calculations made for the fast- and slow-growing origins. The 667 information of these spruce plus trees is archived in the forest genetic register maintained at the 668 Finnish Forest Research Institute. Growth performance is assessed from 14 yr old trees from 7 to 10 669 experimental fields. The seedling information overlaps with Velmala et al. 2014, New Phytol 201: 670 610 622. 671 Table ESM2 Description and ISDN sequence accession numbers of fungal species inhabiting 672 roots and needles of Norway spruce (Picea abies (L.) Karst.) seedlings Identification of OTUs is 673 based on BLAST search from the UNITE and INSD databases. 674 Table ESM3 The effects of foliar treatment and growth performance group on growth, root 675 characteristics and ectomycorrhizal fungal communities of Norway spruce seedlings. Results 676 are based on general and generalised linear mixed models with foliar treatment and growth group 677 and their interaction as explanatory variables and seed origin as a random. Statistically significant 678 P-values are bolded. 679 Table 1 Traits of Norway spruce seedlings inoculated with forest humus and exposed to two necrotising foliar diseases Foliar treatment Mean ± sd n=49 Slow, n=21 Fast, n=21 s1002, n=7 n=49 Slow, n=21 Shoot biomass (g) 0.62±0.2 0.59±0.18 0.67±0.2 0.56±0.27 0.70±0.28 0.7±0.29 Root biomass (g) 0.20±0.09a 0.19±0.07 0.22±0.1 0.18±0.11 0.27±0.14b 0.27±0.14 Shoot height autumn (mm) 135.4±31.1 132.7±27.7 142.6±29.9 121.9±42.0 142.0±42.8 137.9±41.1 Short root density (root tips/mm) 0.83±0.21 0.92±0.17 0.76±0.22 0.74±0.22 0.81±0.23 0.87±0.24 Shoot height following spring (mm)* 142.5±38.1 146.4±25.0 156.3±34.5 123.5±46.4 132.0±45.7 134.6±34.3 Shoot:root ratio 3.27±0.73b 3.22±0.77 3.22±0.67 3.55±0.83 2.79±0.68a 2.81±0.7 Number of root tips (pcs) 1675±902a 1808±825 1632±832 1405±1334 2253±1490c 2391±1409 Damaged needles summer (pcs) 0.35±0.72a 0.52±0.81 0.19±0.68 0.29±0.49 3.16±2.82b 3.52±3.03 Damaged needles autumn (pcs) 0.82±1.24a 1±1.38 0.67±1.24 0.71±0.76 3.92±2.83b 4.05±3.01 Damaged needles following spring (pcs)* 5.39±2.87 a 5.04±2.26 5.93±2.54 5.2±3.7 10.31±6.39 b 10.52±6.54 Condensed tannins (mg/g d. w.) 88.7±33.7 81.5±36.7 95.8±29.7 - 100.8±33.5 95.2±37.0 EMF OTU richness (pcs)** 4.63±1.46 4.4±1.59 5.2±1.08 3.6±1.52 4.69±1.45 5.13±1.19 ECM colonisation (%) 88.8±11.5 88.1±13.8 91.9±7.2 81.4±12.4 85.6±14.1 82.7±14.5 Total fungal OTUs 9.69±3.04 9.40±3.40 9.87±2.50 10±3.94 10.23±3.12 9.73±3.03 * Due to the destructive sampling in autumn, these traits are measured from a set of overwintering seedlings the following spring ** EMF richness was assessed from 5 replicate seedlings Water only control Botrytis cinerea Fast, n=21 s1002, n=7 n=49 Slow, n=21 Fast, n=21 s1002, n=7 0.81±0.18 0.33±0.21 0.71±0.22 0.71±0.2 0.75±0.2 0.6±0.29 0.33±0.11 0.1±0.06 0.23±0.08ab 0.23±0.08 0.24±0.08 0.18±0.08 166.3±18.5 81.1±37.7 148.27±30.4 151.5±28.7 148.2±31.1 138.7±35.8 0.75±0.22 0.79±0.21 0.83±0.22 0.83±0.2 0.82±0.26 0.87±0.23 157.6±36.7 102.7±48.7 136.6±44.5 139.5±41.3 162.9±29.8 106.1±42.9 2.58±0.61 3.35±0.56 3.21±0.52b 3.19±0.59 3.23±0.41 3.19±0.64 2625±1526 724±300 1829±751b 1827±693 1903±764 1616±943 3.48±2.77 1.14±1.46 2.59±2.35b 2.38±2.6 2.57±2.23 3.29±2.14 4.24±2.88 2.57±1.9 3.04±2.33c 2.86±2.59 3.14±2.2 3.29±2.14 11.63±7.09 8.73±5.28 9.33±5.5 b 8.7±4.79 11.89±4.37 7.31±6.32 106.4±29.4 - 97.5±37.0 96.1±35.3 98.9±39.5 - 4.47±1.60 4.0±1.58 4.23±1.88 4.27±1.79 4.07±2.07 4.6±2.07 88.2±13.4 86.0±15.5 89.3±8.7 91.9±7.3 85.6±9.3 93.1±6.5 11.00±2.83 9.4±4.28 9.03±3.11 8.07±2.79 10.00±3.37 9.2±2.95 Gibberella avenacea Velmala et al 2014.pdf Velmala_Infection_PS_2014.pdf