Structure and Abundance of Fusarium Communities Inhabiting the Litter of Beech Forests in Central Europe

: Members of the genus Fusarium and related genera are important components of many ecosystems worldwide and are responsible for many plant diseases. However, the structure of beech litter-inhabiting Fusarium communities and their potential role in reducing the natural regeneration of European beech are not well understood. To address this issue, we examined Fusarium communities in the litter of uneven-aged, old-growth beech-dominated forests in the Carpathians (Poland) and in the Alps (Austria), and in a managed beech stand (Poland). The fungi inhabiting beech litter were investigated using beechnuts and pine seedlings as bait. The pathogenicity of the most common species was investigated by inoculating beech germinants. Fusarium spp. were identiﬁed based on morphology and DNA sequence comparisons of RPB2 and TEF1- α genes, combined with phylogenetic analyses. Twelve fungal species were identiﬁed from 402 isolates, including nine known and three currently undescribed species. The isolates resided in three species complexes within the genus Fusarium . These were the F. oxysporum (one taxon), F. sambucinum (three taxa), and F. tricinctum (six taxa) species complexes. In addition, one isolate was assigned to the genus Neocosmospora , and one isolate could be placed within the genus Fusicolla . The most frequently isolated fungi from beechnuts and beech germinants were F. avenaceum (Fr.) Sacc., F. sporotrichioides Sherb. and Fusarium sp. B. The structure and abundance of species within Fusarium communities varied by beech forest type. The species richness of Fusarium spp. was greatest in old-growth beech-dominated stands, while abundances of Fusarium spp. were higher in managed beech-dominated stands. Pathogenicity tests showed that all four Fusarium species isolated from beechnuts and beech germinants could cause germinants to rot beech, suggesting that these fungi may play a negative role in the natural beech regeneration. in beech that they may play a negative role in the natural regeneration of The suggest that the structure and abundance of Fusarium communities inhabiting the litter may vary depending on the beech forest composition. further studies on the presence of Fusarium spp. on different forest trees are Although, an increasing number of studies on soil fungal communities in forest ecosystems have recently in many countries, the results of our study highlight the fact that only a small proportion of litter are currently known. This survey of Fusarium spp. in beech revealed three new Fusarium species belonging to the Fusarium tricinctum species complex (FTSC) which the taxonomic status needs to be clariﬁed in further studies. supports the view that the diversity of Fusarium species and related genera in forest litter in Central

The number of germinated beechnuts may be reduced by fungal pathogens inhabiting the litter layer. A recent study showed that habitants of beech litter, namely Neonectria and Ilyonectria species are pathogenic and may adversely affect the natural regeneration of F. sylvatica [13]. The relatively small number of Fusarium spp. reported from forestry systems might leave the impression that forests do not harbor a large diversity of these fungi, although it may also indicate that forest plants have been poorly explored in this regard. We are inclined towards the second option and hypothesize that there are many Fusarium spp. including undiscovered species associated with juvenile stages of forest trees. As part of a fungal diversity survey of beech litter in Central Europe conducted in 2011 [13], several members of the genus Fusarium occurring in larger numbers were recorded. Therefore, the objectives of this study were: (i) to characterize the diversity of Fusarium spp. and related genera in beech litter, (ii) to characterize pathogenicity of several isolates of the most frequently recovered Fusarium spp. associated with beech litter. Accordingly, this study will provide knowledge for understanding the role of Fusarium species on natural beechnuts germination and hence the survival of beech natural regeneration.

Study Area
The research conducted from 2010-2012 was carried out in three study sites: natural uneven-aged old-growth beech forests in the Carpathians (Babia Góra National Park, Poland, 49 • 33 N, 18 • 34 E) and the Alps (Rothwald, Dürrenstein, Austria, 47 • 47 N, 15 • 04 E) and one even-aged (60-80 years old) managed beech forest in the Krakowsko-Częstochowska Highland (Zabierzowski Forest, Zabierzów, Poland, 50 • 09 N, 19 • 78 E). Both old-growth stands were composed of European beech, silver fir, and Norway spruce, with beech the most abundant tree species. In the Western Carpathians (Babia Góra), the elevation of the study area was between 940 and 1010 m, and the bedrock was Carpathian flysch, a mixture of sandstone and shales. The mean annual temperature was approximately 4 • C, and the annual precipitation ranged between 1300 and 1400 mm. In the Alps (Rothwald), the elevation of the study area was between 900 and 1400 m, and the bedrock was primarily dolomite and banked limestone. The mean annual temperature was between 4 and 5 • C, and the mean annual precipitation was 2200 mm. In the Zabierzowski Forest, the elevation was between 290 and 310 m; the bedrock was loess covering deeper layers of limestone. The climate was mild with a mean annual temperature of approximately 8 • C and a mean annual precipitation of 700 mm. The stand was composed of 70% European beech; the other tree species were European ash, sycamore (Acer pseudoplatanus L.), and pedunculate oak. Detailed information regarding these locations can be found in Jankowiak et al. [13].

Fungal Isolation
Fusarium spp. were collected via trapping using beechnuts (experiment 1, in situ) and Scots pine (Pinus sylvestris L.) seedlings (experiment 2, in the laboratory). In situ experiments were conducted using a network of permanent sample plots established in Babia Góra National Park in 1995 and in Rothwald in 2001 for monitoring seed production and dispersal by forest trees [46]. In the Zabierzowski Forest, the experiment was conducted along a line transect as described by Jankowiak et al. [13]. In the laboratory experiment, the litter used was taken from some permanent sample plots established in Babia Góra. Pine seedlings were used to isolate a broad-spectrum of pathogenic fungi, including those related to conifers. The methodological details for both experiments have been described previously by Jankowiak et al. [13]. Experiment 1: In the stands, fifty or 25 beechnuts were placed inside perforated plastic boxes (0.08 m 3 or 0.02 m 3 ), mixed with leaf litter collected near to the site of the box in the sample plot, and finally the box was filled with litter. Then, the boxes were covered with a thin layer of litter and left in the forest. The boxes were placed in the autumn (October-November) in 38 sample plots in Babia Góra, 30 sample plots in Rothwald, and 10 sample plots in the Zabierzowski Forest. After wintering, the boxes were recovered in the spring (late April to late May) and taken to the laboratory. All beechnuts and beech germinants were collected and analysed. The numbers of healthy and diseased specimens were counted. The beechnuts and beech germinants affected by necrosis, and other discoloration were used for fungal isolation ( Figure 1A-D). including those related to conifers. The methodological details for both experiments have been described previously by Jankowiak et al. [13]. Experiment 1: In the stands, fifty or 25 beechnuts were placed inside perforated plastic boxes (0.08 m 3 or 0.02 m 3 ), mixed with leaf litter collected near to the site of the box in the sample plot, and finally the box was filled with litter. Then, the boxes were covered with a thin layer of litter and left in the forest. The boxes were placed in the autumn (October-November) in 38 sample plots in Babia Góra, 30 sample plots in Rothwald, and 10 sample plots in the Zabierzowski Forest. After wintering, the boxes were recovered in the spring (late April to late May) and taken to the laboratory. All beechnuts and beech germinants were collected and analysed. The numbers of healthy and diseased specimens were counted. The beechnuts and beech germinants affected by necrosis, and other discoloration were used for fungal isolation (Figure 1 A-D). Experiment 2: Litter collected from 16 sample plots in Babia Góra was mixed with a sterile peat-vermiculite substrate (a 2:1 ratio of litter to substrate) and then placed in plastic pots (volume 330 ml). Thirty surface-sterilised P. sylvestris seeds had been sown in each of the five replicate pots (30 seeds per pot). The experiment was conducted in a phytotron chamber for 10 weeks. During that period, any P. sylvestris seedlings with damping-off symptoms were collected for fungal isolation.
Samples taken from both experiments, i.e., symptomatic tissues of beechnuts, beech germinants, and P. sylvestris roots were surface sterilized for 10 s in 96% ethyl alcohol followed by 3 min in 4% v/v sodium hypochlorite (NaOCl) (Chempur, Piekary Śląskie, Poland). Next, the tissues were rinsed three times for 3 min in sterile distilled water, dried on sterile blotting paper, and finally cut into smaller fragments (5 mm × 5 mm) and placed onto 2% malt extract agar (MEA) medium (Biocorp, Warszawa, Poland) containing 2 μg mL −1 tetracycline hydrochloride (Sigma-Aldrich, St. Louis, MO, USA). In summary, fungi Experiment 2: Litter collected from 16 sample plots in Babia Góra was mixed with a sterile peat-vermiculite substrate (a 2:1 ratio of litter to substrate) and then placed in plastic pots (volume 330 mL). Thirty surface-sterilised P. sylvestris seeds had been sown in each of the five replicate pots (30 seeds per pot). The experiment was conducted in a phytotron chamber for 10 weeks. During that period, any P. sylvestris seedlings with damping-off symptoms were collected for fungal isolation.
The isolation plates were incubated in the dark at 22 • C for one week. Isolates were then transferred to fresh 2% MEA by hyphal tip transfer for purification. Isolates with fusarioid spores were retained for further study and stored at 4 • C.

Morphological Identification
Isolates identified as Fusarium spp. were purified by single-spore culturing. Singlespore derived isolates were cultivated on potato dextrose agar (39 g PDA l −1 , Biomaxima, Poland) and synthetic low nutrient agar (SNA) [47] and were grouped into morphotypes based on their macro-and microscopic features [48]. Depending on the number of isolates belonging to the same morphotype, 1-15 isolates per morphotype were chosen for molecular identification. The isolates are maintained in the culture collection of the Department of Forest Ecosystems Protection, the University of Agriculture in Krakow, Poland (Table 1).

DNA Extraction, Amplification and Phylogenetic Analysis
DNA was extracted using the Genomic Mini AX Plant Kit (A&A Biotechnology, Gdynia, Poland) according to the manufacturer's protocol. DNA was amplified in a 25 µL reaction mixture containing 0.25 µL of Phusion High-Fidelity DNA polymerase (Finnzymes, Espoo, Finland), 5 µL of Phusion HF buffer (5X, 0.5 µL of dNTPs (10 mM), 0.75 µL of DMSO (100%) and 0.5 µL of each primer (25 µM). Amplification of the gene regions was performed under the following conditions: a denaturation step at 98 • C for 30 s followed by 35 cycles of 5 s at 98 • C, 10 s at 52-64 • C (depending on the optimal melting temperature of the primers and fungal species) and 30 s at 72 • C and a final chain elongation step at 72 • C for 8 min. The amplification reactions were performed using a LabCycler thermocycler (SensoQuest Biomedical Electronics GmbH, Göttingen, Germany). For sequencing and phylogenetic analyses, two loci were amplified: RNA polymerase second largest subunit (RPB2), and the translation elongation factor 1-alpha (TEF1-α). The primers used for PCR and sequencing of the various gene regions were as follows: RPB2-5F2 Forests 2021, 12, 811 6 of 18 and fRPB2-7cR or fRPB2-7cF and fRPB2-11aR [49,50] for RPB2; EF1/EF2 [51] for TEF1-α. The isolates were identified to the species level by conducting Basic Local Alignment Search Tool (BLAST) searches with Fusarium-ID [52] and GenBank sequence data. BLAST searches [53] using the BLASTn algorithm were performed to retrieve similar sequences from GenBank (http://www.ncbi.nlm.nih.gov, accessed on 1 April 2021). The reference sequences came from several taxonomic reports [54][55][56][57][58], including ex-type cultures of Fusarium spp. Datasets were curated with the Molecular Evolutionary Genetic Analysis (MEGA) v6.06 program [59]. The first analysis based on TEF1-α sequences was conducted to assess the preliminary identification of the isolates and their phylogenetic affinity among the different species complexes (SC) of Fusarium and related genera. In this study, TEF1-α combined with RPB2 were used for phylogenetic analysis of four Fusarium SC and for the genus Necosmospora.
Phylogenetic trees were inferred for each of the datasets using three different methods: Maximum likelihood (ML), Maximum Parsimony (MP), and Bayesian inference (BI). For ML and BI analyses, the best-fit substitution models for each aligned dataset were established using the corrected Akaike Information Criterion (AICc) in jModelTest 2.1.10 [62,63]. ML analyses were carried out with PhyML 3.0 [64], utilizing the Montpelier online server (http://www.atgc-montpellier.fr/phyml/, accessed on 15 April 2021). The ML analysis included bootstrap analysis (1000 bootstrap pseudoreplicates) to assess node support values and the overall reliability of the tree topology.
MP analyses were performed using PAUP* 4.0b10 (Swofford D.L., Sunderland, MA, USA) [65]. Gaps were treated as the fifth state. Bootstrap analysis (1000 bootstrap replicates) was conducted to determine the levels of confidence for the nodes within the inferred tree topologies. Tree bisection and reconnection (TBR) was selected as the branch swapping option. The tree length (TL), Consistency Index (CI), Retention Index (RI), Homoplasy Index (HI), and Rescaled Consistency Index (RC) were recorded for each analysed dataset after the trees were generated.
BI analyses using Markov Chain Monte Carlo (MCMC) methods were carried out with MrBayes v3.1.2 [66]. The four MCMC chains were run for 10 million generations applying the best-fit model for each data set. Trees were sampled every 100 generations, resulting in 100,000 trees. The Tracer v1.4.1 program [67] was utilized to determine the burn-in value for each dataset. The remaining trees were utilized to generate a 50% majority-rule consensus tree, which allowed for calculating posterior probability values for the nodes.
All sequences generated in this study were deposited in NCBI GenBank (Table 1) and are presented in the phylogenetic trees. All analyses were first run independently for each gene partition. The resulting trees were visually compared for topological incongruences. Gene partitions showing no topological incongruence were combined for the final analyses presented here. The different partitions and conditions used for each analysis are shown in Table 2.

Pathogenicity Tests
Fusarium avenaceum, F. sambucinum Fuckel, F. sporotrichioides, and Fusarium sp. B associated with symptomatic beechnuts and/or beech germinants were used for inoculation (two isolates per species) ( Table 1). Five healthy germinants (about 10-15 mm long) were placed on a 9-cm plastic Petri plate covered with sterile moistened blotting paper. The germinants were inoculated with 5-mm plugs taken from the margin of a 10-14-dayold fungal culture growing on a PDA medium. Five germinants were inoculated with sterile PDA as negative controls. The plates were wrapped with Parafilm ® (Amcor, Zürich, Switzerland) and stored at 15 • C in the dark. After 14 days, the number of germinants with visible necroses were recorded. Fusarium was re-isolated from symptomatic germinants on a PDA medium. The experiment was repeated twice.

Fungal Isolation
In total, 402 fungal isolates resembling Fusarium spp. have been collected. Of these, 375 isolates were isolated from beechnuts and beech germinants (Experiment 1), whereas 27 isolates were obtained from pine roots (Experiment 2).

Fungal Identification and Phylogenetic Analysis
Comparison of the TEF1-α sequences with sequences in GenBank and FUSARIUM-ID database confirmed their phylogenetic affinities among the different species complexes (SC) of Fusarium and related genera. The isolates were distributed into three Fusarium SC, namely the F. oxysporum SC (FOSC, one isolate), F. sambucinum SC (FSAMSC, six isolates), and the F. tricinctum SC (FTSC, 29 isolates). Three isolates (25HS, 26HS, and 50HS) belonged to the genus Neocosmospora, and one isolate (16HS) represented the genus Fusicolla.
Within the F. oxysporum SC, one isolate was identified as F. oxysporum sensu stricto ( Figure 2). The F. sambucinum SC was represented by three species. In this species complex, RPB2 and TEF1-α sequences of one isolate were identical to that of F. graminearum Schwabe, three isolates represented F. sporotrichioides, while two isolates were F. sambucinum (Figure 3). The RPB2 and TEF1-α sequences for 29 isolates in the F. tricinctum SC showed that these isolates represented three known and three unknown taxa. The known species were represented by F. acuminatum (one isolate), F. avenaceum (15 isolates), and F. tricinctum (one isolate) (Figure 4). Two isolates named as Fusarium sp. A, and nine isolates named as Fusarium sp. B formed two distinct lineages most closely related to F. avenaceum (Figure 4). Within the FTSC, Fusarium sp. A formed a new lineage, while isolates of Fusarium sp. B belonged to the FTSC 5 ( Figure 4). One of the isolates referred to herein as Fusarium sp. C, formed a separate clade close to F. acuminatum and F. tricinctum (Figure 4). The genus Neocosmospora was represented by one species: Neocosmospora solani ( Figure 5).  (Figure 4). The genus Neocosmospora was represented by one species: Neocosmospora solani ( Figure 5).

Frequency of Isolation of Fusarium spp. and Related Genera from Beech Litter
Experiment 1: The members of the F. tricinctum SC were the most commonly isolated, and F. avenaceum was the dominant member among the Fusarium spp. isolated, found in 9.3% to 65.2% of beechnuts and beech germinants. This fungus had the highest isolation frequency (65.2%) from the Zabierzowski Forest. The lowest isolation frequency of F. avenaceum (9.3%) was obtained in Babia Góra (Table 3). Fusarium sp. B, the next most frequently isolated Fusarium species from beechnuts and beech germinants were isolated from 2.1 to 5.8% of specimens (Table 3). Additionally, F. sporotrichioides was isolated at varying frequencies having the highest isolation frequency (10.1%) in the Zabierzowski Forest, and the lowest isolation frequency (from 0% to 0.8%) in Babia Góra and Rothwald (Table 3). Other Fusarium species, Fusicolla sp., and Neocosmospora solani were sporadically isolated (from 0.1% to 2.9%) ( Table 3). The genera Fusarium, Fusicolla, and Neocosmospora were most commonly isolated in the managed forest, Zabierzowski Forest (79.7%). In contrast, the frequencies of these fungi in the natural forests (Babia Góra and Rothwald) were considerably lower (19.3% and 16.8%, respectively) ( Table 3). Table 3. Isolation frequency (%) of Fusarium and related genera from the beech litter collected at the three study sites. Experiment 2: Only three Fusarium species were found in the litter from Babia Góra. Like experiment 1, the most abundant species was F. avenaceum, found in 16.7% of dying P. sylvestris seedlings (Table 3). Fusarium graminearum and F. sporotrichioides were sporadically isolated (1.4%). Isolates of F. graminearum have been detected only in this experiment (Table 3).

Pathogenicity
Inoculation of beech germinants with isolates of F. avenaceum, F. sambucinum, F. sporotrichioides, and Fusarium sp. B resulted in extensive necrosis on all germinants; no necrosis occurred in control germinants ( Figure 6A-F). All the fungal species were successfully re-isolated from the inoculated germinants.

Discussion
This study resulted in the recovery of 402 isolates of Fusarium and related genera from natural and semi-natural beech-dominated woodlands in Central Europe, where these fungi are largely unstudied. The fungi included species of Fusarium, Neocosmospora solani, and Fusicolla sp. The phylogenetic analysis showed that these isolates could be assigned to 12 distinct taxa, of which four represented undescribed species; and F. avenaceum, F. sporotrichioides, and Fusarium sp. B were the most commonly detected. Isolates of F. avenaceum, F. sambucinum, F. sporotrichioides, and Fusarium sp. B were also determined to be virulent on beech germinants.
This study not only illustrated the widespread nature of Fusarium distribution in the litter of beech forests in Central Europe, but also established, for the first time that Fusarium spp. can be major germinants pathogens in the natural regeneration of beech in this area. The pathogenicity tests in this study showed that Fusarium spp. caused severe symptoms on the beech germinants. These findings highlight the importance of beech

Discussion
This study resulted in the recovery of 402 isolates of Fusarium and related genera from natural and semi-natural beech-dominated woodlands in Central Europe, where these fungi are largely unstudied. The fungi included species of Fusarium, Neocosmospora solani, and Fusicolla sp. The phylogenetic analysis showed that these isolates could be assigned to 12 distinct taxa, of which four represented undescribed species; and F. avenaceum, F. sporotrichioides, and Fusarium sp. B were the most commonly detected. Isolates of F. avenaceum, F. sambucinum, F. sporotrichioides, and Fusarium sp. B were also determined to be virulent on beech germinants.
This study not only illustrated the widespread nature of Fusarium distribution in the litter of beech forests in Central Europe, but also established, for the first time that Fusarium spp. can be major germinants pathogens in the natural regeneration of beech in this area. The pathogenicity tests in this study showed that Fusarium spp. caused severe symptoms on the beech germinants. These findings highlight the importance of beech litter as a disease reservoir for germinating beechnuts. This agrees with findings from a study in Poland [13], wherein the same samples from beech forests, isolation of pathogenic Ilyonectria spp. and Neonectria spp. ranged from 32 to 48%. Our work confirmed that beechnuts lying on the litter surface or buried within the litter are exposed to a wide range of potentially damaging agents. Among them, fungal pathogens belonging to Fusarium spp., Ilyonectria spp., and Neonectria spp. seems to be very important for beechnuts viability and therefore have a negative role in the natural regeneration of beech. We suggest that this is an important mechanism contributing to the lack of seedlings after mast years when the beechnuts that survived until spring on the forest floor were numerous but almost no seedlings are present.
In the present study, among the 10 recorded Fusarium species, there were five species namely F. avenaceum, F. oxysporum, F. sambucinum, F. sporotrichioides [69], and N. solani [8] that had been previously detected on beechnuts after harvest and after drying in Poland. These pathogens have also been reported as causal agents of damping-off in tree seedlings in forest nurseries [16,18,25]. It cannot be excluded that Fusarium spp. inhabiting beech litter may be disseminated via seed and spread within forest nurseries. A similar phenomenon in seed-borne Fusarium pathogens of Pinus ponderosa Dougl. ex C. Lawson were observed by Salerno et al. [70], who showed that many different Fusarium species are carried on seeds of P. ponderosa in Argentina that may serve as inoculum sources for damping-off and root rot diseases in forest nurseries.
Fusarium avenaceum was the most frequently encountered species in our study, found in association with beechnuts and beech germinants at all study sites. It indicates that F. avenaceum is a major agent responsible for the decline of beechnuts in Central Europe. This fungus can cause damage to many agricultural crops worldwide, including forest nurseries [19,71,72]. Fusarium avenaceum has also been recorded in the natural regenerating of Eucalyptus seedlings with causing damping-off symptoms in Australia [37]. This species has also been isolated from the roots of forest trees in Iran [36], forest litter in Sri Lanka [41], oak and sycamore litter in the UK [32], and from symptomatic shoots of Q. robur in Poland [73,74].
Fusarium sporotrichioides and Fusarium sp. B were also isolated relatively frequently from infected beechnuts and beech germinants in this survey. The pathogenicity tests in this study showed that isolates of both Fusarium species were able to induce necrosis on beech germinants, which may suggest that these species have the potential to reduce the natural regeneration of beech. Fusarium sporotrichioides is widespread across tropical and temperate regions [75] and is commonly associated with seedling diseases in forest nurseries [16], wilt symptoms and needle dieback on mature trees [76] or cankers [77]. This fungus was also found in beechnuts [8] and insect-damaged acorns of Q. robur [78]. In turn, an undescribed species named as Fusarium sp. B is the member of Fusarium tricinctum species complex (FTSC 5), and the phylogenetic analysis showed that this taxon is closely related to F. avenaceum. The RPB2 and TEF1-α sequences of Fusarium sp. B obtained in this study were identical to isolates of Fusarium sp. reported from Turkey (NRRL 52730, NRRL 52227) [55,58]. Interestingly, isolates matching Fusarium sp. B have been recovered from the symptomatic stems of young seedlings in naturally regenerated oaks in Poland [79], indicated that this Fusarium species is commonly distributed in hardwood forests in Poland. The Fusarium tricinctum species complex (FTSC) was also represented by two other unknown taxa: Fusarium sp. A and Fusarium sp. C. All unknown Fusarium species detected in this study (Fusarium sp. A-C) probably represent new taxa and will be described elsewhere.
Fusarium oxysporum, the most common Fusarium damping-off pathogen in bareroot forest nurseries [3,16], was detected only sporadically in diseased tissues of beechnuts and beech germinants, although it is known from other types of forests. It has been recovered in forest litter and soil in many regions of the world (e.g., [39,40,43,44,[80][81][82]) as well as in the roots of forest trees in Iran [36]. This study provides the first information about the presence of F. oxysporum in the litter in beech forests.
There were large quantitative and qualitative differences in the Fusarium spp. composition between the two examined types of forests, i.e., natural old-growth beech forests with a small admixture of A. alba and P. abies (Babia Góra, Rothwald), and managed beech forest with a small admixture of different hardwood species (Zabierzowski Forest). The highest species richness value was found in a natural old-growth beech forest in Babia Góra (10 species) while the lowest species richness value occurred in a managed beech forest in Zabierzowski Forest (4 species). It is possible that old beech forest enriched with conifers could promote some Fusarium species. On the contrary, the least complex Fusarium community was found in managed and relatively tree species poor Zabierzowski Forest. Recent studies have shown that, the most important factor that shape soil microbiome community assemblages are the tree hosts promoted by complex interactions, including alteration of the microclimate (temperature and moisture), production of litter, production of root exudates, or direct interactions with root-symbiotic and root-associated microorganisms [83,84].
The abundance of various Fusarium spp. also differed among the forest types. The abundance of Fusarium spp., especially F. avenaceum, F. sambucinum, and F. sporotrichioides in managed beech-dominated stand was considerably higher than in an old growth beechdominated forest. The high Fusarium spp. abundance in the managed beech-dominated stand may be due to their host preference for hardwood trees. A comparative analysis of the occurrence of Fusarium spp. and Cylindrocarpon-like fungi (Ilyonectria spp. and Neonectria spp.) that have been collected from the same sites by Jankowiak et al. [13] showed that Cylindrocarpon-like fungi had no host preferences although the occurrence of Ilyonectria species appeared to be more closely related to the presence of conifers in temperate forests.

Conclusions
We showed that Fusarium spp. are diverse and important components of the litter mycobiota in beech forests and that they may play a negative role in the natural regeneration of beech. The results suggest that the structure and abundance of Fusarium communities inhabiting the litter may vary depending on the beech forest composition. However, further studies on the presence of Fusarium spp. on different forest trees are needed. Although, an increasing number of studies on soil fungal communities in forest ecosystems have recently been conducted in many countries, the results of our study highlight the fact that only a small proportion of litter fungi are currently known. This survey of Fusarium spp. in beech litter revealed three new Fusarium species belonging to the Fusarium tricinctum species complex (FTSC) for which the taxonomic status needs to be clarified in further studies. This supports the view that the diversity of Fusarium species and related genera in forest litter in Central Europe is high.

Data Availability Statement:
The data presented in this study are available on request from the first author.