Article Ectomycorrhizal Fungal Assemblages of Nursery-Grown Scots Pine are Influenced by Age of the Seedlings Maria Rudawska * and Tomasz Leski Institute of Dendrology, Polish Academy of Sciences, Parkowa 5, 62-035 Kórnik, Poland; [email protected] * Correspondence: [email protected] Abstract: 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. Citation: Rudawska, M.; Leski, T. Across all nurseries, negative correlations were identified for diversity metrics and soil N or C, Ectomycorrhizal Fungal Assemblages indicating that mineral and organic fertilization contributes to the differences in the ECM fungal of Nursery-Grown Scots Pine are communities in nurseries. We discuss the ecological and practical implications of the composition Influenced by Age of the Seedlings. and diversity of ECM fungi occurring on bare-root planting stock of Scots pine. Forests 2021, 12, 134. https://doi. org/10.3390/f12020134 Keywords: symbiosis; ectomycorrhizal diversity; Pinus sylvestris; fungal diversity Received: 21 December 2020 Accepted: 21 January 2021 Published: 25 January 2021 1. Introduction Scots pine (Pinus sylvestris L.) is a keystone species in the forest ecosystems of Eurasia. Publisher’s Note: MDPI stays neutral This species is among the most important conifers in forestry and is globally widespread, with regard to jurisdictional claims in ranging from western Europe to the east of Russia, and from northern Scandinavia to published maps and institutional affil- the mountains of southern Europe. In northern Eurasia, Scots pine plays an essential iations. 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 Copyright: © 2021 by the authors. forests, especially in the boreal regions, but it can also be found as an admixture with many Licensee MDPI, Basel, Switzerland. other tree species across its wide range. The extent of Scots pine forests in Europe exceeds This article is an open access article 28 million ha [1], and approximately 5.5 million ha of this area is found in Poland [2]. distributed under the terms and Because of its wide distribution and its economic importance, Scots pine is considered conditions of the Creative Commons a highly desirable species for many kinds of European forest reforestation and afforestation Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ programs. Throughout Europe, nurseries annually produce large quantities of Scots pine 4.0/). planting stock, and a single modern nursery can produce several million plants each year [3]. Forests 2021, 12, 134. https://doi.org/10.3390/f12020134 https://www.mdpi.com/journal/forests Forests 2021, 12, 134 2 of 16 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 [8–15]. 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 [17–20]. An important function of ECM symbiosis, especially in the early stages of tree development, is protection from soil-borne pathogens and pinewood nematode infections [21–23]. 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 [28–30]. 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 identifica- tion) of mycorrhizal root tips [32–34], 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 [33,43–46], 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 va- riety 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 Forests 2021, 12, 134 3 of 16 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