Fungi Occurring in Norway Spruce Wood Decayed by Heterobasidion parviporum in Puszcza Borecka Stands (Northeastern Poland)

: In many spruce stands, trees are frequently attacked by the pathogen Heterobasidion parviporum , albeit without visible symptoms in the crown. In the present work, the results of the presence of stem rot, assessed by PICUS Sonic Tomography, and the fungal biota on trees and stumps in eight plots in the Puszcza Borecka Forest are described. The plots were located in stands on original forest soil (4) and on post-agricultural soil (4), where around a stump with H. parviporum symptoms (signs of internal rot and basidiocarps), 30 trees were selected and examined for internal rot. Wood samples were collected from two selected trees for fungal molecular analysis. A total of 79 fungal taxa were found, including 57 taxa in plots on post-agricultural soil and 45 on forest soil. There were 395 fungal records on stumps and 22 records on trees, therein, from the inner parts of felled trunks. Signiﬁcant differences in the Chao-1 diversity index indicate that the origin of the soil—post-agricultural or forest soil—inﬂuenced the alpha diversity of the fungal communities in the forests studied. The values of the Shannon and Simpson indices show that the two communities were similar in terms of species numbers. The presence of basidiomata of H. parviporum and two species of Armillaria (mainly A. cepistipes ) in samples on all plots is striking, although Armillaria spp. was detected more frequently. Most of the species identiﬁed were typical saprotrophs, although rare species were also found, such as Entoloma byssisedum , Onnia tomentosa , Physisporinus vitreus , Postia ptychogaster , and Ramaria apiculata . The presence of H. parviporum in the inner woody parts was conﬁrmed by PCR analysis, and decay was detected even up to a stem height of 6 m. Armillaria was the dominant genus in the studied stands and plays a signiﬁcant and underestimated role in heartwood decay of old spruce trees in Puszcza Borecka Forest.


Research Plots
The study was performed in summer 2017 (PICUS examination) and autumn 2021 (stumps examination) in spruce stands (over 50 years old) in northeastern Poland (Figure 1) in the Borki and Czerwony Dwór Forest Districts (FDs), which belong to the Białystok Regional Directorate. Both FDs are mainly located in the Borecka Forest, a large forest complex (230 km 2 ) in the lake area of the 842. 86 Ełk Lake District (Masurian Lakeland) [39]; part of Borecka Forest is a dedicated nature reserve. According to the annual reports of the State Forests [40], both forest areas show significant damage by fungal pathogens, mainly Heterobasidion spp. and Armillaria ostoyae. Based on personal communication with foresters and an inventory, eight threatened stands were selected, four on former agricultural land and four old forest stands ( Figure 1, Table 1). The old forest stands were selected randomly, whereas the stands on former agricultural sites were located on the edge of Borecka Forest. The numbers of the compartments were obtained from the Forest Data Bank [41].
In each of these stands, spruce stumps with symptoms of internal decay typical of H. parviporum or with basidiomata were searched. One randomly selected stump served as the reference for further evaluation, and the tree closest to it was selected as the plot center (No. 1 in Figure 2). Subsequently, 30 consecutive trees were selected in a spiral and numbered . The trees showed no signs of crown thinning or dieback. The undergrowth and the small trees in the lower floors of the plot were omitted. The diameter of this area was measured and represented the survey plot ( Figure 2). Within each plot, all spruce stumps and all trees were counted and examined for the presence of sporocarps (Table 1).

Computed Tomographic Analysis
All 240 trees (30 trees in each of the 8 plots) were examined with PICUS Sonic ® to determine the presence or absence of internal rot ( Figure 3).
Tomographic assessment of wood decay inside the tree trunk [42,43] was performed at a height of 0.1 and 0.6 m above the ground (Figure 4), using the Sonic Tomograph-PICUS 3 Q74 EXP and the PICUS GMS Calliper 3 measuring device [26].

Assessment of Sporocarps
The collected specimens, growing in the root necks of all standing trees and on tree stumps within each plot, were identified using standard mycotaxonomic methods [44]. Species were identified using keys [45][46][47][48][49], and dried specimens were stored in the fungarium of the Forest Protection Department of the Warsaw University of Life Sciences (SGGW). The nomenclature of fungi followed the Index Fungorum database [50]. Threat categories were assigned according to the "Red List of Macrofungi in Poland" [51]; endangered and rare fungal species were listed according to Kujawa et al. (2021) [52].

Preparation of Pure Cultures and Molecular Analyses
Two trees showing signs of decay previously indicated by PICUS were randomly selected from the measurement plots (one from forest plot 49b and the other from arable site plot 6a). The trees were felled in October 2020. After debarking the trunk, five replicates of wood samples were cut at a height of 0.1 and 0.6 m, placed in sterile glass tubes, and stored in the refrigerator prior to further analyses. In the laboratory, the samples were surface-sterilized, cut into small inocula, and placed on 2% malt extract agar (MEA) supplemented with 50 mg/L streptomycin. Cultures were incubated at 22 • C for 7 days and sub-cultured as necessary. The resulting colonies were transferred to plates containing 2% MEA to obtain pure cultures for DNA analyses.
Fungal genomic DNA from mycelium, rhizomorphs, and sporocarps was extracted according to Kubiak et al. (2016) [53]. For the identification of fungal isolates obtained from decaying wood, the ITS rDNA region was amplified and sequenced using the primers ITS1F and ITS4 [54,55]. Identification of Heterobasidion specimens was performed by speciesspecific PCR with the primer pairs MJ-F/MJ-R (H. annosum) and KJ-F/KJ-R (H. parviporum) [56]. Identification of Armillaria species was performed based on sequencing of a portion of the translation elongation factor 1 alpha (tef 1-alpha, amplified according to Szewczyk et al. (2015) [57]. The amplified regions had a size of 100 bp (H. annosum), 350 bp (H. parviporum), and 600-650 bp (tef 1-alpha). Both strands of PCR products of the ITS region and the tef 1-alpha gene were sequenced using a 3730XL DNA analyzer (Applied Biosystems, Waltham, MA, USA) at Genomed (Warsaw, Poland). Nucleotide sequences were read and edited using FinchTV v. 1.4.0 (Geospiza Inc., Seattle, WA, USA) and aligned with sequences publicly available in GenBank (http://www.ncbi.nlm.nih.gov; 12 January 2022), using the BLASTn algorithm to confirm the taxonomy of the fungi studied.

Statistical Analyses
To determine the species diversity in the studied groups, several indices were calculated: Chao1, Ace, Shannon, Simpson, and Fisher. Alpha biodiversity was analyzed using the phyloseq package R [58]. Statistical analysis of the selected indices was performed using the R function pairwise.wilcox.test as Wilcoxon sum in pairs. Chi-square statistical analysis was performed for the frequencies presented in Tables 2 and 3. Statistical analysis was performed using the Pearson chi-square test, and differences were considered statistically significant at p < 0.1. Correlation analysis for some of the tested results was performed using the ggpubr library of the R package. The method chosen was the non-parametric Spearman correlation. The alpha biodiversity of community samples describing individual taxa within a defined area or a collective list of species present in a given area was assessed. Both ACE and Chao1 are richness estimators, whereas Shannon and Simpson are diversity indices. The Fisher index was theoretically adjusted for sampling bias. Table 2. Frequency (%) of fungal taxa on Norway spruce stumps within plots on old forest sites (1-Statistical analysis was performed using the Pearson chi-square test, and differences were considered statistically significant at p < 0.1. Correlation analysis for some of the tested results was performed using the ggpubr library of the R package. The method chosen was the non-parametric Spearman correlation. The alpha biodiversity of community samples describing individual taxa within a defined area or a collective list of species present in a given area was assessed. Both ACE and Chao1 are richness estimators, whereas Shannon and Simpson are diversity indices. The Fisher index was theoretically adjusted for sampling bias4) and post-agricultural sites (5-8) (trophic categories: E-ectomycorrhizal fungi, M-mycoparasites, P-parasites, S-saprotrophs.   Karst. E

Results
Computed tomographic analysis of the stems showed the presence of internal rot in 13.3-73.3% of the trees studied (Table 1). On average, the percentage of trees with internal damage was 43.3% on forest sites and 39.2% on former agricultural sites. For both soil types, the differences in the proportions of trees in which H. parviporum was detected by PICUS were not significant (pchi-sq. > 0.05).
The average percentage of stumps with decay symptoms (hollow) and/or basidiomata of both pathogens was 11.3 and 16.3%, respectively (Table 1). In both categories, the proportion of stumps with rot increased with stand age. It is noteworthy that the crowns of all trees with internal wood decay found by tomography showed no signs of thinning or discoloration of the needles.
In the eight studied plots representing the economic spruce stands of Borecka Forest, a total of 79 fungal taxa were found. In total, 417 records (395 on stumps and 22 on trees), including from the inner parts of the felled trunks, were obtained (Tables 2 and 3). The occurrence of fungi inhabiting the wood of spruce growing on former agricultural sites (57 taxa) was higher than that of spruce growing on forest sites (45 taxa), with 224 versus 193 records.
Significant differences in the Chao1 diversity index indicate ( Table 4) that the origin of the soil-farm or forest soil-influences the alpha diversity of the fungal communities in the studied forests. The values of the Shannon and Simpson indices showed that the two communities were similar in terms of species number, with p = 0.88. The probability for the Chao1 index, which counts singletones and doubletones, was at the 0.05 limit, indicating a difference between the groups in the low-identified species (where we had one or two of the stumps). Regarding the ACE indicator, which is a non-parametric method for estimating the total number of species based on the coverage (size) of the sample, a probability at the 0.05 limit means that between groups of samples, the distribution of coverage (size) varies ( Figure 5). Fungi belonging to four trophic groups were found on the studied tree stumps: saprotrophs (55 taxa), parasites (8), ectomycorrhizal (4), and mycoparasites (2) based on 395 records (185 in forest soil, 210 in arable soil). On the examined trees, 8 saprotrophs and 10 parasites were found based on 22 records (8 in forest soil, 14 in arable soil) (Tables 2 and 3).
In the saprotroph group, three fungal species of the genus Hypholoma were found, among others: H. capnoides and H. fasciculare occurred in six plots and H. lateritia in one plot (Tables 2 and 3). The shares of stumps with basidiomata of H. capnoides were similar for post-agricultural soil (11 stumps) and forest soil (10 stumps). Hypholoma fasciculare basidiomata were found more frequently on stumps in post-agricultural soil (10 stumps) compared to stumps in forest soil (6 stumps) ( Table 2).  The presence of root pathogens (H. parviporum and two species of Armillaria) in stumps and trees on all plots is striking, although Armillaria spp. were detected more frequently (122 stumps and trees versus 25 stumps and trees). Although the presence of A. borealis was confirmed via mycelium under bark and basidiomata with PCR (NCBI sequences OL634955 and −56), only in spruce stumps in one plot (No. 2) on forest soil, A. cepistipes was detected in most plots (Tables 2 and 3 There was no significant correlation between the presence of a particular taxa in arable soil or forest soil, while the highest correlation was found for Armillaria spp. (R = 0.8, p = 0.33), all fungi R = −0.77, p = 0.23) and H. parviporum + Armillaria spp. stumps (R = −0.63, p = 0.37). Weak correlations were detected for Fungi on stumps vs. All stumps (R = 0.58, p = 0.13), and PICUS+ vs. All stumps (R = −0.49, p = 0.21). The analyzes performed also showed a strong negative correlation (p < 0.1) between trees with rot confirmed tomographically (PICUS+) (n) and the number of trees in the study plot (R = −0.96, p = 0.00011), and a positive correlation between trees with rot (PICUS+) (n) and stand age (Table 5). Basidiomata were found more frequently on stumps than in the root necks of standing trees. In wood samples taken from different sections of two felled trees that showed symptoms of wood decay on tomographic examination, genetic analysis confirmed the presence of the DNA of the pathogens with 100% identity: H. parviporum (NCBI sequence OL691107-OL691110), A. cepistipes (see above), both at 0.1 and 0.6 m, as well as Sistotrema brinkmannii (NCBI sequence OL691111) and Resinicium bicolor (NCBI sequence OL691112). On the other hand, the presence of H. parviporum was not confirmed at a height of 6.0 m, despite visible symptoms of wood decay ( Figure 6). The presence of both pathogens, whose sequences were submitted to GenBank, was confirmed from both perimeters of the surface.

Discussion
In this study, the presence of fungi was identified by classical methods. Root pathogens were detected using genetic methods, both on 821 trees, including 240 PICUS-trees, and on 339 stumps in both site types (four stands on old forest soil and four stands on postagricultural soil). Based on our results, the wood of both standing spruce with living crowns and stumps is commonly colonized by fungi belonging to different trophic groups, with different interactions among them [59][60][61]. The difference in the occurrence of different taxa (values of Chao1 and ACE) may be caused by differences in the structure of spruce wood due to different soil fertility levels (arable was made fertile in the past) [62,63].
A similar number of wood-inhabiting species (58), compared to our study (57), in spruce stands on former agricultural land was found in a 3-year study in Slovakia in spruce stands aged 21-51 years [64]. It is worth noting that in the cited studies, typical spruce pathogens, such as Armillaria spp. and Heterobasidion spp., only sporadically occurred, in contrast to our results. We detected numerous species of pathogens (Heterobasidion parviporum and Armillaria spp.) as well as saprotrophs and symbiotrophs. Kubart et al. (2016) [65] point out that the presence of stump fungi is common in spruce stands in different regions of Sweden, whereas saprotrophic fungi, especially Resinicium bicolor, Fomitopsis pinicola, and Hyphodontia spp., which were also found in our studies, are present in large numbers.
It is interesting to note that the occurrence of fungi on spruce stumps and trees growing on an old arable soil was more numerous and diverse than on forest soils. This is probably due to the greater fertility of arable soils, which favors the formation of a wider annual increment [66,67], as well as to different fungal communities present in the environment of both types of stands in the past and today [68,69].
The analysis showed the strong negative correlation between the number of trees with tomographically confirmed decay and all stumps on the plots and positive correlation with stand age, which can be attributed to the systematic removal of dead trees by the forest administration and thus the increasing number of stumps. The weak correlation between all stumps and the number of inhabiting taxa is understandable. It is surprising that communities frequency within a dense forest complex, i.e., a Borecka forest, differ considerably depending on the type of soil (forest or arable) on which the stands grow. For similar trends in colonization of stumps and logs in a habitat, see Kubartova et al. (2012) [70].
Among the identified fungi, several taxa are effective competitors of food root pathogens (including Hypholoma, Pleurotus, and Phlebiopsis) and have therefore been studied and used in the biological protection against these pathogens [71][72][73][74][75]. The relatively high proportion of Hypholoma species suggests that fungi of this genus are widespread and important organisms that can limit the population of root pathogens in spruce stands [76,77].
Interestingly, several fungi described by Wojewoda and Ławrynowicz (2006) [51] as threatened/vulnerable (Onnia tomentosa, Pleurotus pulmonarius) or rare (Entoloma byssisedum, Galerina triscopa, Physisporinus vitreus, Postia ptychogaster) were identified in this study. In Poland, O. tomentosa is known from about a dozen sites, mainly from large, well-preserved forest complexes in the mountains (in the south of the country) and in the north [78,79]; this species has not been reported from the Borecka Forest. According to Ryvarden et al. (2017) [49], terrestrial basidioma often develop in large numbers from roots in old spruce stands. In our study, basidiocarps were found in both old (111 years old, forest plot 4) and a much younger, 74-year-old tree stand (arable plot 7). P. pulmonarius is known from numerous sites in Poland [79] and does not seem to be a threat to any species. It grows on various deciduous trees and in North America also on conifers, e.g., on Abies [80,81]. In Poland, P. pulmonarius has rarely been reported on conifers, e.g., Picea abies [82,83], and recently, it has been found on the trunk of a fallen Pinus sylvestris in a wind-damage area in Kampinos National Park [84]. The occurrence of numerous rare fungal species in spruce stands, both in managed and old forests, has been reported previously [85][86][87][88][89]. Gori et al. (2013) [90] found that in the Alps, spruce infection and wood rot can last for up to 80 years, with variable growth decline depending on climatic altitudes and the effects of drought [91]. The trees tested showed extensive heartwood decay, suggesting that infection began in the younger age classes. In the Borecka Forest, the proportion of H. parviporum-positive trees was 13.3-70.3%, although the trees were older than 50 years. The large number of undergrowth trees in the studied plots indicates a high density of root systems and an easy penetration of the mycelium of both root pathogens (H. parviporum and Armillaria spp.) into the trees due to numerous secondary infections through the roots [37,[92][93][94]. In Norway, significant threats are caused by both Heterobasidion fungi, with a high proportion of H. parviporum, 98.5% [95]. The authors found that 68.2% of the trees were infected in 44-year-old regenerated spruce stands.
The Armillaria species found here (A. cepistipes and A. borealis) are mainly described as weak pathogens of deciduous trees and as opportunistic pathogens or saprotrophs of conifers [96][97][98][99]. In the current study, A. cepistipes was identified by genetic analyses of DNA from underneath bark, rhizomorphs, and basidiocarps collected in all stands studied, but surprisingly, more frequently in stands established on post-agricultural soils than on forest soils, which is described by other authors [100][101][102].
In a previous study, both H. parviporum and A. cepistipes have also been re-isolated from within the wood after felling standing trees whose crowns showed no symptoms of disease [103]. This confirms the thesis that without computed tomographic assessment of the interior of the wood, trees infected with the pathogen can grow unproductively for decades as commodities in managed spruce stands [19,37,104,105]. Another question is their ecological role in the ecosystem, where the pathogen and the resulting decomposition of wood during forest succession can provide suitable opportunities for the development of other organisms such as other fungi, birds, bats, insects, and squirrels [106][107][108][109][110].
Rigerte et al. (2019) [34] found 11 taxa described only as fungal root endophytes in spruce, none of which were identified in the present study. Numerous saprotrophs and symbiotrophs found on stumps and trees indicate that trees formerly infested by root pathogens are still attractive as food sources and are not discriminated against by the enzymes and metabolites of the pathogens [111].

General Conclusions
Our results show that trees older than 50 years, regardless of the type of soil on which the spruce stands grow (forest, post-agricultural), are subjected to advanced internal wood rot. This means that the infection of the roots took place many years earlier and that the development of the pathogen, detected by tomography inside the trunk, takes at least a dozen years. Consequently, in some valuable spruce stands, the degree of infestation of spruce with root pathogens should be determined by computed tomography at the age of 30-40 years.
Trees or stem sections showing advanced heartwood decay could be termed "deadwood", and some of them should be left in the stand from an ecological point of view to contribute to an increased biodiversity.
Because of the significant proportion of fungi of the genus Armillaria in the studied stand, which is generally underestimated in risk assessments, monitoring methods for assessing the presence of these pathogens in spruce stands need to be revised, including the severity of rhizomorph occurrence in the soil.