Ectomycorrhizal Fungal Assemblages of Nursery-Grown Scots Pine are Inﬂuenced by Age of the Seedlings

: Scots pine ( Pinus sylvestris L.) is the most widely distributed pine species in Europe and is relevant in terms of planted areas and harvest yields. Therefore, each year the demand for planting stock of Scots pine is exceedingly high, and large quantities of seedlings are produced annually throughout Europe to carry out reforestation and afforestation programs. Abundant and diverse ectomycorrhizal (ECM) symbiosis is critical for the success of seedlings once planted in the ﬁeld. To improve our knowledge of ECM fungi that inhabit bare-root nursery stock of Scots pine and understand factors that inﬂuence their diversity, we studied the assemblages of ECM fungi present across 23 bare-root forest nurseries in Poland. Nursery stock samples were characterized by a high level of ECM colonization (nearly 100%), and a total of 29 ECM fungal taxa were found on 1- and 2-year-old seedlings. The diversity of the ECM community depended substantially on the nursery and age of the seedlings, and species richness varied from 3–10 taxa on 1-year-old seedlings and 6–13 taxa on 2-year-old seedlings. The ECM fungal communities that developed on the studied nursery stock were characterized by the prevalence of Ascomycota over Basidiomycota members on 1-year-old seedlings. All ecological indices (diversity, dominance, and evenness) were signiﬁcantly affected by age of the seedlings, most likely because dominant ECM morphotypes on 1-year-old seedlings ( Wilcoxina mikolae ) were replaced by other dominant ones (e.g., Suillus luteus , Rhizopogon roseolus , Thelephora terrestris, Hebeloma crustuliniforme ), mostly from Basidiomycota, on 2-year-old seedlings. Across all nurseries, negative correlations were identiﬁed for diversity metrics and soil N or C, indicating that mineral and organic fertilization contributes to the differences in the ECM fungal communities in nurseries. We discuss the ecological and practical implications of the composition and diversity of ECM fungi occurring on bare-root planting stock of Scots pine.


Introduction
Scots pine (Pinus sylvestris L.) is a keystone species in the forest ecosystems of Eurasia. This species is among the most important conifers in forestry and is globally widespread, ranging from western Europe to the east of Russia, and from northern Scandinavia to the mountains of southern Europe. In northern Eurasia, Scots pine plays an essential role in forest ecosystems through the maintenance of biodiversity, conservation of genetic resources, and provision of valuable habitat for wildlife while also providing social benefits such as recreation. Economically, this species is a crucial timber resource and provides raw material for the paper and pulp industry [1]. Scots pine frequently forms large monoculture forests, especially in the boreal regions, but it can also be found as an admixture with many other tree species across its wide range. The extent of Scots pine forests in Europe exceeds 28 million ha [1], and approximately 5.5 million ha of this area is found in Poland [2].
Because of its wide distribution and its economic importance, Scots pine is considered a highly desirable species for many kinds of European forest reforestation and afforestation programs. Throughout Europe, nurseries annually produce large quantities of Scots pine planting stock, and a single modern nursery can produce several million plants each year [3]. Though natural forest regeneration or production of container-grown stock material are widely developing, most of the seedling stock in several European countries, Poland included, is still produced in bare-root forest nurseries, which is estimated to be significantly less expensive and capable of higher yields [4,5]. Bare-root forest-tree seedlings are grown in fields much like any agricultural crop and are termed bare-root because roots are exposed at field planting [6,7]. In Poland, bare-root production constitutes over 1.3 billion tree seedlings per year. Scots pine seedlings constitute a significant part of this production and serve as the main source of seedlings for reforestation of near 60,000 ha per year [2].
Like most forest trees, Scots pine has been recognized as an obligate mycotroph, known as a host of 200-300 ectomycorrhizal (ECM) fungal species [8][9][10][11][12][13][14][15]. Ectomycorrhizal fungi play a crucial role in tree growth and form a vital link between tree roots and soil substrate. They are almost entirely dependent on the photosynthates of their host plants [16]. In return, ECM fungi assist plants in the acquisition of soil nutrients and water by increasing the volume of soil explored for resources [17][18][19][20]. An important function of ECM symbiosis, especially in the early stages of tree development, is protection from soil-borne pathogens and pinewood nematode infections [21][22][23]. Furthermore, when colonized with ECM fungi, seedlings are known to more efficiently tolerate different environmental stresses, such as drought or high temperatures [24,25]. Abundant seedling colonization by nursery-adapted ECM fungi also minimizes transplanting shock and positively influences the establishment and survival of young trees for at least several years after outplanting [26,27]. Forest tree seedlings with multiple ECM fungal symbionts can withstand a wider range of planting sites than seedlings with only one species of mycorrhizal fungus [28][29][30]. Nursery managers have long recognized the importance of well-developed ECM symbiosis for healthy seedling growth. Therefore, bare-root nursery procedures such as lifting, sorting, packing, storing, and transporting seedlings should be performed with care to minimize damage to the fine-root system and their mycorrhiza. Mycorrhizae destroyed by rough handling, desiccation, or heating will have to be replaced at the planting site at a cost of seedling energy and nutrients and will thus decrease outplanting success [31].
Before the molecular era, our knowledge about ECM fungal communities in forest nurseries was mainly based on morphotyping (morphological and anatomical identification) of mycorrhizal root tips [32][33][34], fungal isolation [35,36], or a combination of both of these methods [37,38]. Significant advances have been made since it was demonstrated that direct sequencing of fungal DNA from root tips could be a powerful and sensitive tool for the identification of potentially all root-inhabiting fungi [39,40]. This tool also appears to be an efficient approach to determining ECM fungi in forest nurseries, where the appearance of fruiting bodies is very rare due to the age of the seedlings and intensive nursery management (mostly weed control). Thus, the use of DNA sequencing of barcode genes, such as the ITS rDNA, for the identification and discovery of fungal specimens directly from ectomycorrhizas has become common and appears to be a relatively fast and inexpensive approach that requires far less specialized knowledge than the microscopic study of morpho-anatomical features [41,42], which often generates numerous ambiguities. Owing to this, there has been an unprecedented increase in the number of publications on the ECM fungal community structure of different forest ecosystems over the past two decades. Several studies have explored the mycorrhizal status of nursery planting stock of tree species that predominate in temperate forests, such as Pinus sylvestris [33,[43][44][45][46], Picea abies (L.) H.Karst. [45,47,48], Larix decidua Mill. [49], Quercus spp. [50], Fagus sylvatica L. [51], Betula pendula Roth, Tilia cordata Mill., and Carpinus betulus L. [52]. Among the tree species produced in forest nurseries in Poland, Scots pine was the first species analyzed molecularly in terms of its ECM assemblages [44]. After fifteen years, we found these assemblages worthy of reexamination using a much higher sampling effort that covers a variety of forest nurseries and in-depth molecular analyses that comprise recent advances in the use of better taxonomic reliability and annotations in public DNA repositories. To assist with meeting this need, we performed extensive research of fungal communities associated with 1-and 2-year-old P. sylvestris seedlings produced in 23 bare-root forest nurseries in Poland. We hypothesized that the composition of ECM communities of both seedling age groups would be similar, with a significant shift in abundance of some fungal species in older seedlings. We used this knowledge to shed light on the ecology of identified ECM fungi in the context of pioneer ECM communities under the influence of characteristic soil properties of forest bare-root nurseries.

Nurseries and Sampling of Seedlings
One-and two-year-old Scots pine seedlings were sampled in October 2015 from forest bare-root nurseries belonging to 23  . The size of sampled nurseries, ranging from 4.5-9.8 ha, and their areas were divided into several compartments with four to six standard nursery seedbeds each. A typical Polish bare-root nurseries crop cycle was applied comprising both a green manure crop and a year of leaving the soil fallow. Before sowing, Scots pine seeds were treated with fungicide T 75 DS/WS (with thiram as an active substance) at the rate of 5 g kg −1 . Sowing was conducted with the single-seed seeder in rows 0.005 m apart, with spacing in a row 0.037 m in April 2013 and 2014 (2-and 1-year-old seedlings respectively). If necessary, to control the damping-off of seedlings, TOPSIN M 500 SC or MILDEX 71.1 WG fungicides were used. No herbicides or insecticides were used in any nursery. Weed control was performed mechanically. Nitrogen fertilizers were applied at annual levels of 35-65 kg N ha −1 , depending on the nursery, following a schedule designed to satisfy the nutrient requirements of the seedlings based on a soil analysis of each nursery. Before the study, 1-and 2-year-old seedlings were grown for 17 and 29 months, respectively. One-year-old seedlings were sampled from all 23 nurseries, while 2-year-old seedlings were sampled from 15 nurseries. In each nursery, five sub-samples, each comprised of five seedlings lifted from the Scots pine seedbeds of a given age, were randomly sampled. The average of these five replicates served as the unit of analysis (nursery stock sample). A total of 38 nursery stock samples and 950 seedlings were analyzed. Seedlings were removed from the nursery together with adjacent bulk soil and transported immediately to the laboratory in plastic bags.

Soil Analysis
In the lab, soil and roots were separated. The root-free soil from each sample was thoroughly mixed and sieved (2-mm mesh) for the determination of pH (in H 2 O and KCl) and soil chemical analyses. The pH of the soil samples was determined by mixing 20 mL of soil with 40 mL of deionized water or 1 M KCl. After 1 h, the pH was measured with a calibrated pH meter equipped with a glass electrode. Soil for analysis of total element concentrations was ground using a ball mill (Analysette 3 Spartan Pulverisette 0, Fritsch GmbH, Idar-Oberstein, Germany). The total N and C contents were measured using the elemental combustion system CHNS-O (Constech Analytical Technologies Inc., Valencia, CA, USA). Total K, Mg, and P were extracted from soil by digestion in hot concentrated HClO 4 (Digestor 40 Auto, Foss Tecator, Sweden). The Mg, K, and P concentrations were measured by an ionic chromatograph DX-100 (Dionex Corporation, Sunnyvale, CA, USA) and a Varian BQ 20 atomic absorption spectrophotometer with a graphite cuvette.

Ectomycorrhizal Assessment and Identification
The root system was gently washed in tap water to remove most of the soil and organic debris. The clean roots were cut into approximately 2.5-3.0 cm long sections and placed into a Petri dish filled with water. Sections were randomly selected, and the numbers of all active root tips colonized by each morphotype were counted. Successive root sections were examined until 300-350 root tips had been counted in each of the replicates. Morphological typing of the ECM root tips was examined under a stereomicroscope at 10 to 60 times magnification (Zeiss Stemi 2000-C, Carl Zeiss, Germany). Ectomycorrhizas were separated into morphotypes based on macroscopic features (color of mantle, type of ramification, and the presence of extramatrical hyphae, rhizomorphs, and cystidia) and cross-referenced with a database used by the staff of our Laboratory of Symbiotic Associations at the Institute of Dendrology. To distinguish colonized and uncolonized root tips, especially in the case of 1-year-old seedlings, we regularly performed anatomical surveys of cross-sections (using a compound microscope under 400 or 1000× magnification) by freehand sectioning of short roots from both the terminal and proximal portions of intact laterals of tested seedlings. Each distinct mycorrhizal morphotype was described and photographed for further reference. Morphotypes from each nursery were considered as an independent sample and, as such, were placed separately in Eppendorf tubes in water and stored at −20 • C until processing for DNA analysis. The identification of previously selected and preserved morphotypes was based on the molecular analysis of two to three mycorrhizal tips of each unique morphotype. Total DNA was extracted using the GeneMATRIX Plant and Fungi DNA Purification Kit (EURx, Gdańsk, Poland), following the manufacturer's protocols. The fungal internal transcribed spacer (ITS) region was amplified with ITS1F/ITS4 primers, using a Type-it Microsatellite PCR Kit (Qiagen GmbH, Hilden, Germany). The PCR products were sequenced at the Laboratory of Molecular Biology in the Adam Mickiewicz University in Poznan using a CEQ 20000XL automatic sequencer (Beckman Coulter Inc., Brea, CA, USA) with an ITS4 primer. The obtained sequences were edited using BioEdit 7.2 and compared with reference fungal ITS sequences from the UNITE [53] and GenBank databases (www.ncbi.nlm.nih.gov) using BLAST [54]. Species-level identification of the ectomycorrhizae was defined as sharing >97% of the ITS region [55]. ITS regions that had a similarity between sequences of ≥95% were classified at the genus level, ITS regions that had a similarity between sequences of ≥90% were classified at the family level, and ITS regions that had a similarity between sequences <90% were classified at higher levels. Because in the case of Laccaria the ITS region is more conserved than in other genera, a 98% species-level threshold was applied for both Laccaria identified in our study [56].

Statistical Analysis
The diversity of the ectomycorrhizas on the Scots pine seedlings was expressed as the number of identified ECM taxa (taxa richness), relative abundance, and frequency. The relative abundance of identified ECM fungal taxa was calculated as the number of tips of a given fungal taxa per total number of ECM tips extracted per nursery stock sample. The relative abundance of a given taxon was calculated as the number of ECM root tips of a taxon, divided by the total number of ECM root tips. The relative frequency of a given ECM fungal taxon was calculated as the number of nurseries in which it was found, divided by the total number of nurseries. To assess the sufficiency of the sampling effort, the first-order jackknife estimator was calculated with the EstimateS 9.1 software [57] using 100 randomized runs without sample replacement. The diversity of ECM fungal communities was evaluated using Shannon diversity (H), Simpson's diversity (1-D), Simpson's dominance (D), and evenness indices using PAST 1.89 software [58]. Comparison of ECM fungal composition was calculated using the Jaccard and Bray-Curtis similarity coefficients. Both coefficients were used in the analysis of similarity (ANOSIM) to determine whether the ECM fungal communities differed between seedlings with different ages. Data were standardized and square root transformed prior to analysis. The advantages of the ANOSIM test are that it does not assume any underlying distribution to the data and avoids using similarity indices to directly compare sets of communities. Nonmetric multidimensional scaling (NMDS) was used to provide a visual summary of the pattern of Jaccard and Bray-Curtis values. Similarity of percentages (SIMPER) was carried out to investigate which taxa were responsible for observed differences. All absolute data were square root transformed prior to the analysis performed with PAST 1.8 software [58].
The differences in the chemical composition of soils, the mean fungal taxa richness, and the ecological indices between the nursery stock samples of different ages were analyzed using analysis of variance (ANOVA) (the assumption of normality of the data and homogeneity of variances were tested by the Shapiro-Wilk test and Levene's test, respectively). Post hoc comparisons of means between the sites were performed using Tukey's test at a significance level of p < 0.05. For relative abundances, no homogeneity of variance was found, and differences in the relative abundance of ECM fungal taxa between 1-and 2-year-old seedlings were therefore tested using the Kruskal-Wallis test. Relationships between relative abundance of individual ECM fungal taxa and soil variables were examined using the Spearman rank correlation, and relationships between taxa richness, diversity, dominance, evenness, and soil parameters were tested using the Pearson correlation. Computations were performed using the statistical software package Statistica, version 5.5 (StatSoft Inc., Tulsa, OK, USA).

Results
Significant differences between 1-and 2-year-old nursery beds were found in the concentrations of C and C/N ratio. Mean values of N, P, K, Ca, and Mg did not differ significantly. The pH was acidic and ranged between 4.18 and 6.32 (in KCl); however, there were no significant differences between nurseries (Table 1). Different letters indicate significant differences between nurseries at p < 0.05 (Tukey's test).
The degree of mycorrhizal colonization ranged from 84-100% for 1-year-old Scots pine seedlings with a mean of 97.1%, and 98.5-100% for 2-year-old seedlings with a mean of 99.8% (Table 2). Based on the morphotyping of 10,548 mycorrhizas, 37 provisional morphotypes were distinguished. DNA isolation was done from 754 mycorrhizal tips (3-114 tips from each provisional morphotype, depending on its frequency), and PCR products were obtained for 665 samples (88.2%). The overall successful sequencing rate was 95.3% (634 of 665 sequenced amplicons). Based on sequence analysis, 29 ECM fungal taxa were identified, with 27 taxa shared between 1-and 2-year-old seedlings (Table 2, Figure 1). One taxon, Tuber sp. 2, was noted exclusively on 1-year-old seedlings, while another taxon was found exclusively on 2-year-old seedlings (Laccaria tortilis (Bolton) Cooke). The jackknife 1 estimator of ECM richness was 35.6 for 1-year-old seedlings and 30.8 for 2year-old seedlings ( Table 2). Fourteen identified ECM fungal taxa belonged to Ascomycota and 15 taxa belonged to Basidiomycota. From 29 detected fungal taxa, 19 were identified to species level, six to genus, and four to order or family level ( Table 2).
The taxa richness of identified ECM fungal taxa in individual nurseries was variable and ranged from 3 to 10 for 1-year-old seedlings and from 6 to 13 taxa for 2-year-old seedlings, depending on the nursery (Figure 2A,B). The average taxa richness per nursery was significantly higher for 2-year-old seedlings ( Table 2). On 1-year-old seedlings, the mean relative abundance of the members of Ascomycota was significantly higher than the abundance of fungi from Basidiomycota, while 2-yearold seedlings were dominated by ECM fungal symbionts belonging to Basidiomycota ( Figure 3A,B).    Table 2). Wilcoxina mikolae was a dominant mycorrhizal species on 1-year-old seedlings (mean relative abundance of 74.8%, with a range of 38.5-98.5%), but less abundant on 2-year-old seedlings (mean relative abundance of 15.4%, with a range of 2.5-33.1%). The frequency of this fungus was very high irrespective of the age of the seedlings, as it was recorded in each of the analyzed nurseries (Table 2; Figure 2A,B). The next most frequent and abundant fungal partner of Scots pine seedlings was S. luteus. The mean relative abundance of this species increased with seedling age from 2.5% on 1-year-old seedlings (ranging between 0-7.1%) to 31.4% on 2-year-old seedlings (ranging between 10.1-55.4%; Table 2; Figure 2A,B). Despite the low abundance of this species on 1-year-old seedlings, S. luteus was detected on more than 65% of nursery stock samples. On 2-year-old seedlings, S. luteus was observed in all investigated nurseries. Another fungal species with relatively high frequency on 1-year-old seedlings and high abundance and frequency on 2-year-old seedlings was R. roseolus. Most of the fungal taxa detected on 1-year-old seedlings were characterized by low relative abundance (below 1%) and low frequency. From 28 taxa present on 1-year-old seedlings, 16 were observed in only one or two nurseries. In contrast, the frequency of fungi found on 2-year-old seedlings was higher than on 1-year-old seedlings. Only 5 of 28 taxa were characterized by low frequency (i.e., were identified in one or two nurseries).
The ANOSIM and NMDS ordinations were used to evaluate the influence of the age of the Scots pine seedlings on ECM fungal communities. The NMDS ordination based on the Jaccard index indicated a lack of separation of the ECM fungal communities on 1-and 2-year-old seedlings (R = 0.10, p = 0.09, stress = 0.15; Figure 4A). In contrast, the NMDS analysis carried out with the Bray-Curtis index showed complete separation of ECM fungal communities associated with 1-and 2-year-old seedlings. The ANOSIM confirmed significant differences among the seedlings of different ages (R = 0.96, p = 0.0001, stress = 0.09; Figure 4B). SIMPER analysis showed that W. mikolae, S. luteus, and R. roseolus were the principal species associated with the dissimilarity of ECM fungal communities among the 1and 2-year-old seedlings (with a 38.7%, 17.7%, and 11.1% contribution to dissimilarity, respectively; Table 3).
For 1-year-old seedlings, the strongest positive significant correlations were found between the relative abundance of W. mikolae and N and C content in the nursery bed soil and between the abundance of Cenococcum geophilum Fr. and C content. Positive relationships were also observed between the relative abundance of S. granulatus and R. luteolus and soil pH. Suillus granulatus was negatively correlated with soil C level. Relative abundance of W. mikolae colonizing 2-year-old seedlings was positively correlated with N, P, and K level and negatively correlated with Mg. For 2-year-old seedlings, positive correlations were also noted between the abundance of C. geophilum and C level and C/N ratio and between Pustularia sp. and P content. Soil pH (in H 2 O) and N and K negatively correlated with Hebeloma crustuliniforme abundance. The N content in the soil negatively influenced the fungal taxa richness of the 2-year-old seedlings. Dominance coefficient for 1-year-old seedlings was positively correlated with N, P, and K, and diversity was negatively correlated with N, P, and K. In the case of 2-year-old seedlings, dominance and diversity coefficients were either positively or negatively correlated with N content and soil pH. Evenness of tested fungal communities of 1-year-old seedlings was negatively influenced by K and Mg content (Table 4). Table 3. Similarity of percentages (SIMPER) output indicating the percentage of contribution of ectomycorrhizal to 1-and 2-year-old Scots pine seedlings in forest bare-root nurseries (an accumulative contribution to 90% is presented).  Table 4. Spearman's rank correlation between relative abundance of ectomycorrhizal fungal species, and soil parameters and Pearson's correlation between taxa richness and diversity indices and soil parameters in forest nurseries (data presented only for species for which significant correlations were noted).

Discussion
High diversity of ECM fungi spontaneously colonizing root seedlings is a required parameter of the nursery stock. In the observed ECM communities of 1-year-old and 2year-old Scots pine seedlings originating from bare-root forest nurseries, we distinguished altogether 29 ECM fungal taxa (28 in each age class). This is relatively high compared to previous studies targeting ECM fungal communities in bare-root forest nurseries of coniferous species (Scots pine, Norway spruce Picea abies (L.) Karst., and European larch Larix decidua Mill.), where 7 to 21 taxa were identified [44,45,[47][48][49]. This result is likely due to a better sampling effort (a higher number of surveyed nurseries) than what has been carried out in previous research. Fungal species richness estimator (first-order jackknife) showed that our estimated sampling completeness (percent of observed species) constitutes approximately 78% and 91% of the estimated species richness for 1-year-old and 2-year-old seedlings, respectively. Thus, we believe that we encountered most (but not all) of the fungal species and that the potential of Scots pine seedlings to harbor ECM symbionts in the condition of bare-root forest nursery is still slightly higher than we found. Additional sampling would likely detect some of the unobserved fungal species that were possibly overlooked due to their irregular spatial or temporal distribution. We did not find some species previously identified by other authors on Scots pine seedlings (e.g., Hebeloma cavipes Huijsman, or H. helodes J. Favre) [37,44]; however, other species of Hebeloma were found (e.g., H. sacchariolens Quél., H. velutipes Bruchet, and H. crustuliniforme).
One of the most characteristic features of the ECM fungal community that developed on the studied nursery stock was the prevalence of Ascomycota over Basidiomycota members on 1-year-old seedlings ( Figure 3A). Previous studies have also shown a high affinity of nursery stock to harbor ascomycete ECM fungi on Scots pine and European larch in Polish and Lithuanian bare-root forest nurseries [44,45,49], often to the extent of the ascomycete dominating in the ECM fungal community [50][51][52].
Among ascomycete fungi, W. mikolae was the most represented species in the mycorrhizal fungal community of 1-year-old seedlings, both in terms of frequency and abundance ( Table 2). Wilcoxina spp. were also noted as a predominant symbiont for young seedlings in different natural settings [59,60]. It is often suggested that this type of symbiosis appears in disturbed habitats when the host plant is subjected to certain physiological stress [16,61,62]. It is highly probable that during the first months of growth in nursery conditions Scots pine seedlings are subjected to particular stress, mostly connected with high doses of nitrogen fertilization applied as part of the nursery practice [33]. The results of the Spearman's correlation confirmed this by showing a direct positive relationship between the abundance of W. mikolae and nitrogen content in the nursery bed soil. Previous studies also suggested that high fertility, high pH levels, and high water availability were qualitatively correlated with the occurrence of Wilcoxina-type symbiosis in some nursery studies [33,63]. Our results also indicated that W. mikolae abundance was positively related to carbon content in the nursery bed soil. This is likely a result of the high amount of composted material used by nursery managers to prevent organic matter reduction during nursery production. Other results have also indicated that the addition of compost fertilizer increases the contribution of members of Ascomycota to the fine root colonization of Norway spruce nursery seedlings [48]. The predominance of W. mikolae and other ascomycetes (e.g., Meliniomyces bicolor, Cadophora finlandica (C.J.K. Wang & H.E. Wilcox) T.C. Harr. & McNew, Peziza sp., Pyrenomycetaceae) on 1-year-old seedlings may also suggest that these taxa have a distinct life strategy that promotes the colonization of young nursery seedlings that other ECM fungal species may not have. Wilcoxina spp. are known for persistent propagules (thick-walled chlamydospores) that remain viable in the soil for extended periods [62]. In pine seedling bioassays, W. mikolae had the most viable spore banks, with 70% survival after 6 years of field incubation [64]. This characteristic may give W. mikolae an advantage over other ECM symbionts with less viable spores in nursery conditions. Studies comparing the benefits of mycorrhizal association between different ECM fungi (basidiomycetes and ascomycetes and non-mycorrhizal) have found Wilcoxina taxa to rank high in terms of seedling biomass [65] and nitrogen uptake [66]. LoBuglio and Wilcox [67] found that Pinus resinosa seedlings inoculated with ectendomycorrhizal E-strain isolate, later identified as Wilcoxina, and planted on iron tailings tended to show better survival rates than controls.
Structurally, W. mikolae forms so-called ectendomycorrhiza. This type of symbiosis can be distinguished from typical ectomycorrhizas by characteristic features such as a very thin, often fragmentary, mantle, poorly developed Hartig net, and intracellular penetration of hyphae in the epidermal and cortical cells of the mycorrhizal root tips [68]. Such structural features of ectendomycorrhizas may be of importance during the first months of nursery growth and explain why ECM basidiomycetes with more abundant external structures contribute poorly to the species assemblage present in the 1-year-old planting stock. It is generally assumed that ECM morphotypes with high fungal biomass in the mantle sheathing of the short roots (e.g., suilloid mycorrhizas) require more carbon from the host tree than do thin-mantled and smooth types with less external mycelium (e.g., W. mikolae) [69,70]. This indicates that the ability to form ECM structures is controlled, at least partly, by the developmental stage of the host plant [12]. The lower input of carbon from 1-year-old seedlings with a very restricted photosynthetic surface area may be a factor limiting the development of a wider spectrum of fungi with more abundant mycelia. Other authors have also suggested a relationship between the diversity of ECM fungi and additional energy input (e.g., carbohydrates) due to the enhanced net photosynthetic rate supplied by the host plant [71][72][73].
Edaphic conditions, mostly increased fertility, which can inhibit the development of more diverse ECM communities on pine nursery stock, are rather short-lived. Seedling nutrient reserves decline quickly due to dilution in tissue nutrient concentrations and lower fertilizer supply when the seedlings grow older [33,74]. Consequently, the relationship between Asco-and Basidiomycota fungal symbionts fundamentally changes in the second season of seedling growth in nursery conditions ( Figure 3B), mostly in favor of suilloid taxa. This is in line with a well-known principle of colonization that both the composition of a fungal community and the relative abundance of the members within it gradually change when the medium is changed [75,76]. We predicted that the age of the seedlings would influence the ectomycorrhizal communities of bare-root Scots pine seedlings, and our results supported our predictions. All ecological indices (diversity, dominance, and evenness) were significantly affected by the age of the seedlings, most likely because dominant ECM morphotypes in 1-year-old seedlings (W. mikolae) were replaced by other dominant ones (e.g., S. luteus, R. roseolus, T. terrestris, H. crustuliniforme), chiefly from Basidiomycota.
Individual nursery stock samples were characterized by a high level of ECM colonization (nearly 100%). However, the taxa richness (3-10 taxa on 1-year-old seedlings and 6-13 taxa on 2-year-old seedlings) and composition of taxa appeared to be a characteristic feature of each nursery. Differences in ECM communities among individual nurseries may be explained by (i) differences in management practices, such as soil management and seedbed preparation, irrigation and fertilizer formulas, pest and weed control, crop rotation cycles, and transplanting, and (ii) environmental factors, such as site-specific soil characteristics, age and locality of the nursery, composition of forest-tree species surrounding the nursery, and local microclimate [77]. Other factors, including competitive interactions among different ECM species and the availability of different sources of inoculum [78], may also contribute to the observed differences in the ECM fungi communities among the different nurseries.
Our results provide some insights for forest management practices. We found that species diversity, which is an important seedling attribute after outplanting [27,29], was negatively correlated with nitrogen levels in the nursery soil. Therefore, we recommend limiting nitrogen fertilization in nursery practices. Moreover, despite the prevalent use of 1-year-old seedlings for reforestation and afforestation, we argue that a wider use of 2-year-old seedlings should be considered. Older seedlings, with high species diversity of ECM fungi and a high abundance of suilloid mycorrhizas, should be preferred, especially for renewing difficult and dry areas. Ectomycorrhizas formed by fungi belonging to the Suillus and Rhizopogon genera are characterized by abundant extramatrical mycelium and the presence of thick and well-developed rhizomorphs that play an essential role in the transport of water to the host plant over long distances and, in some cases, may be treated as a water reservoir [79]. Results of inoculation experiments with fungi that form rhizomorphs have demonstrated that such seedlings are more tolerant to drought [80,81].

Conclusions
This study aimed to answer important research questions regarding whether ECM fungal diversity is related to seedling age, soil nutrient levels, and the pH of the nursery beds. We showed that Scots pine nursery stock is well colonized by early-stage ECM fungal species (e.g., Wilcoxina, Rhizopogon, Suillus, Hebeloma), well adapted to the age of the seedlings, and characterized by rapid growth and perhaps enhanced resource exploitation [82]. Achieving robust, highly colonized early-stage EMF communities on planting stock should be a goal of each nursery manager. Therefore, we argue that commonly measured seedling attributes (e.g., shoot height, stem diameter, root mass, shoot-to-root ratio, drought resistance, mineral nutrient status) considered crucial in determining the health and successful establishment of seedlings after their transplanting into forest stands [63] could include information about the ECM status of planting stock.

Data Availability Statement:
The data used in this study can be accessed by contacting the corresponding authors.