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Article

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

by
Andrzej Szczepkowski
1,*,
Waldemar Kowalczuk
2,
Katarzyna Sikora
3,
Marta Damszel
4 and
Zbigniew Sierota
5
1
Department of Forest Protection, Institute of Forest Sciences, Warsaw University of Life Sciences—SGGW, ul. Nowoursynowska 159, 02-776 Warszawa, Poland
2
EKOSYSTEM S.C. ul., Wspaniała 1, 05-400 Otwock, Poland
3
Department of Forest Protection, Forest Research Institute, Sękocin Stary, ul. Braci Leśnej 3, 05-090 Raszyn, Poland
4
Department of Entomology, Phytopathology and Molecular Diagnostics, University of Warmia and Mazury, ul. Prawocheńskiego 17, 10-720 Olsztyn, Poland
5
Department of Forestry and Forest Ecology, University of Warmia and Mazury in Olsztyn, Plac Łódzki 2, 10-727 Olsztyn, Poland
*
Author to whom correspondence should be addressed.
Forests 2022, 13(2), 229; https://doi.org/10.3390/f13020229
Submission received: 13 December 2021 / Revised: 17 January 2022 / Accepted: 26 January 2022 / Published: 3 February 2022
(This article belongs to the Special Issue Forest Health Management: Interaction between Forest Trees, Pathogens and Environment)
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Abstract

:
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.

1. Introduction

In spruce (Picea abies (L.) H. Karst.) stands, the pathogen Heterobasidion parviporum (Niemela & Korhonen) is an important wood destroyer [1,2,3,4]. Together with H. annosum (Fr.) Bref. and Armillaria solidipes Peck = A. ostoyae Romagn. (Herink), it is one of the main causal agents of diseases in harvested forests in the Northern Hemisphere [1,5,6,7]. Wood-inhabiting fungi, both pathogens and saprotrophs, decompose cellulose and lignin in the cell walls through oxidoreduction enzymes secreted extracellularly during substrate colonization [8]. The decomposition of wood leads to the release of D-glucose and its oxygenation, binding water and carbon dioxide [9,10,11,12]. These processes produce large amounts of energy and various metabolites, including nitrogen compounds, that can be used by other organisms [13,14,15,16,17,18]. As a result of the infection of live spruce trees by H. parviporum and the associated persistent decomposition of the heartwood, also after the felling of the trees [19,20], attempts to detect the presence of wood rot on standing trees at an early stage have been made, albeit with varying degrees of success [21,22,23]. For example, computed tomography allows the assessment of both changes in tree structure and the extent of wood loss caused by H. parviporum [24,25,26].
Decomposing tissue appears to provide favorable conditions for the colonization by other fungi along with natural succession [27,28]. Fungi that decompose spruce wood have been largely described in terms of their distribution at different sites [29], the infectivity of root residues [30], the genetic aspects of infection [31,32], or root endophytes as potential biocontrol agents [33], among other issues. However, the biota accompanying H. parviporum in decaying wood [34,35] have rarely been recorded, with only 95 search results in Google Scholar.
Since H. parviporum is a major cause of infection and death of mature trees in spruce stands in many countries [36,37,38], the aim of the present study was to determine the proportion of this pathogen in commercial spruce stands over 50 years old in the Borecka Forest, northeastern Poland, that have previously been infested by H. parviporum. We determined the fungal biota inhabiting the wood of trees and stumps in selected stands growing on old forest sites and on post-agricultural sites. We hypothesized that: (i) infection with H. parviporum can be detected early by tomographic examination of the logs, and (ii) stumps of felled and dead trees with internal rot are, on post-agricultural soil, a more adequate food source for various fungi of different trophic groups than stumps on old forest soil.

2. Materials and Methods

2.1. 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 km2) 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 (1–30). 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).

2.2. 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].

2.3. 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].

2.4. 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 species-specific 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.

2.5. 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 Table 2 and Table 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.

3. 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 (Table 2 and Table 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.
The taxa found belonged to Ascomycota (8 species) and Mucoromycota (1), but mainly to Basidiomycota (70 taxa), with the representative orders Agaricales (30) and Polyporales (13) predominating. The sporocarps belonged to a total of 18 orders and 45 families at least. Among the identified taxa (59 saprotrophs, 14 parasites), four species (Helvella macropus, Paxillus involutus, Tomentella bryophila, T. radiosa) are classified as ectomycorrhizal fungi, and two species (Hypocrea pulvinata, Tremella encephala) are mycoparasites (Table 2 and Table 3).
In the old forest sites, 45 species were identified, of which 21 were only found in these sites (e.g., Amphinema byssoides, Armillaria borealis, Gloeophyllum odoratum, Phlebiopsis gigantea, Pholiota squarrosa, Pluteus pouzarianus, Pseudohydnum gelatinosum, or Sistotrema brinkmannii), whereas 57 species were recorded in the post-agricultural sites, of which 34 taxa only occurred in these sites (e.g., Bjerkandera adusta, Kuehneromyces mutabilis, Mycena galericulata, Pholiota flammans, Pleurotus dryinus, P. ostreatus, P. pulmonarius, Trichomolopsis rutilans, Xylodon flaviporus). Overall, 24 species occurred on both site types (e.g., Amaropostia stiptica, Armillaria cepistipes, Cyanosporus caesius, Heterobasidion parviporum, Hypholoma capnoides, H. fasciculare, Onnia tomentosa, Postia ptychogaster, Stereum sanguinolentum). Only three species were found on the wood of spruce trees growing on both soil types: Amaropostia stiptica, Heterobasidion parviporum, and Onnia tomentosa. For the post-agricultural soils, 15 species were recorded, whereas in the forest soils, we found 6 species (Table 2 and Table 3).
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) (Table 2 and Table 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 (Table 2 and Table 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 (Table 2 and Table 3), both in forest (NCBI sequences OL652577, −80) and agricultural soils (NCBI sequences OL652578, −79, −81, −82, −83).
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.
The six species collected were red-listed fungi. Two of them (Onnia tomentosa, Pleurotus pulmonarius) are listed in the threatened category and described as vulnerable (V), whereas four species (Entoloma byssisedum, Galerina triscopa, Physisporinus vitreus, and Postia ptychogaster) are rare (R). Of the identified species, 20 (Amphinema byssoides, Armillaria borealis, A. cepistipes, Clitopilus hobsonii, Entoloma byssisedum, Galerina triscopa, Hyphoderma roseocremeum, Hyphodontia arguta, H. pallidula, Onnia tomentosa, Physisporinus vitreus, Pleurotus pulmonarius, Pluteus pouzarianus, Postia ptychogaster, P. tephroleuca, Ramaria apiculata, Sistotrema brinkmannii, Trechispora hymenocystis, T. nivea, and T. mollusca) are included in the “Register of Protected and Endangered Fungal Species in Poland (GREJ)”.

4. 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 post-agricultural 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].

5. 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 “dead-wood”, 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.

Author Contributions

A.S., M.D., K.S. and Z.S. substantially contributed to the conceptualization, resources, writing of the original draft, review and editing of the text; W.K., A.S. and M.D. performed the field experiments; K.S. and A.S. analyzed the data. All authors have read and agreed to the published version of the manuscript.

Funding

This research was partially supported by the Warmia and Mazury University in Olsztyn, Poland (Project No. 30.610.019-110).

Data Availability Statement

Sequences OL634955-56; OL652577-83; OL691107-OL691112 are available at http://www.ncbi.nlm.nih.gov(accessed on 12 December 2021).

Acknowledgments

The authors thank Agata Młodzińska, Bioidea Poland, for her constructive bio-informatic additions, the Czerwony Bór and Borki Forest Districts administration for help in the field works and the anonymous reviewers for their valuable comments and suggestions.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Localization of the eight evaluated plots in Czerwony Dwór and Borki Forest Districts in Poland (Map source: [41]).
Figure 1. Localization of the eight evaluated plots in Czerwony Dwór and Borki Forest Districts in Poland (Map source: [41]).
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Figure 2. Diagram showing the selection and location of 30 trees and stumps within the conventional survey plot.
Figure 2. Diagram showing the selection and location of 30 trees and stumps within the conventional survey plot.
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Figure 3. One of the trees cut down for DNA analysis of fungi inside the spruce trunk. The crown of the tree showed no symptoms of defoliation or discoloration, whereas evaluation via PICUS Sonic Tomography showed the presence of stem internal rot. Photo by M. Damszel.
Figure 3. One of the trees cut down for DNA analysis of fungi inside the spruce trunk. The crown of the tree showed no symptoms of defoliation or discoloration, whereas evaluation via PICUS Sonic Tomography showed the presence of stem internal rot. Photo by M. Damszel.
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Forests 13 00229 g004 550
Figure 4. Measurement with PICUS Sonic (a), and tomogram of the trunk presented in Figure 3 at a height of 0.6 m (b). Photo by W. Kowalczuk.
Figure 4. Measurement with PICUS Sonic (a), and tomogram of the trunk presented in Figure 3 at a height of 0.6 m (b). Photo by W. Kowalczuk.
Forests 13 00229 g004
Forests 13 00229 g005 550
Figure 5. Alpha diversity in arable soil (red) and forest soil (blue) taxa groups based on the number of observed species, Chao1, ACE, Shannon, Simpson, and Fisher index; a, b indicate significant differences between groups.
Figure 5. Alpha diversity in arable soil (red) and forest soil (blue) taxa groups based on the number of observed species, Chao1, ACE, Shannon, Simpson, and Fisher index; a, b indicate significant differences between groups.
Forests 13 00229 g005
Forests 13 00229 g006 550
Figure 6. Symptoms of heartwood rot caused by H. parviporum on successive 1-m sections of a spruce trunk. Photo by M. Damszel.
Figure 6. Symptoms of heartwood rot caused by H. parviporum on successive 1-m sections of a spruce trunk. Photo by M. Damszel.
Forests 13 00229 g006
Table
Table 1. Description of the assessed compartments in the two forest types.
Table 1. Description of the assessed compartments in the two forest types.
Forest
District		Plot No.
Subdistrict Compartment		Soil TypeStand
Age		No. of Trees on Survey PlotTrees with Decay by PICUS (%)No. of Stumps/with Decay Symptoms (Including Stumps with Hollows and Basidiomata
of H. parviporum
and Armillaria spp.)
Czerwony
Dwór		1—Dunajki 102lForest5314413.339/33 (13)
2—Kaliniszki 85hForest6311833.343/36 (10)
3—Dunajki 59kForest748256.735/29 (12)
5—Dunajki 58cArable5310936.764/44 (11)
6—Dunajki 44aArable6311733.351/43 (20)
7—Dunajki 43gArable7412913.355/53 (19)
Borki4—Knieja Ł. 49bForest1115170.034/32 (10)
8—Lipowa G. 6aArable1097173.318/18 (15)
Average
number of stumps		Forest
Arable		 43.3
39.2		37.8/32.5 (11.3)
47.0/39.5 (16.3)
Table
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.
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.
Stand LocationForest Soil/Old ForestArable Soil/Post-Agricultural SoilNumber of Stands with Taxa Present
Site No.1234Mean
1–4		5678Mean
5–8
Number of Stumps39433534386451551847
ASCOMYCOTA
Helotiales
Gelatinodiscaceae
Ascocoryne cylichnium (Tul.) Korf S7.77.0003.73.113.73.65.66.56
Hypocreales
Hypocreaceae
Hypocrea citrina (Pers.) Fr. S00000001.800.51
Hypocrea pulvinata Fuckel M02.3000.6000001
Pezizales
Helvellaceae
Helvella macropus (Pers.) P. Karst.
E		0000002.0000.51
Pyronemataceae
Scutellinia scutellata (L.) Lambotte S000000005.61.41
BASIDIOMYCOTA
Agaricales
Crepidotaceae
Crepidotus malachius Sacc. var. trichifer Hesler & A.H. Sm. S0000002.0000.51
Entolomataceae
Clitopilus hobsonii (Berk.) P.D. Orton S04.7001.2000001
Entoloma byssisedum (Pers.) Donk S0000002.0000.51
Hymenogastraceae
Galerina marginata
(Batsch) Kühner S		5.105.702.71.63.91.801.35
Galerina triscopa (Fr.) Kühner S02.308.62.7001.800.53
Galerina sp. S2.62.3001.2001.800.53
Gymnopilus penetrans
(Fr.) Murrill S		5.17.02.98.86.01.60016.74.66
Gymnopilus picreus (Pers.) P. Karst. S0005.91.502.005.61.93
Incertae sedis
Cystoderma carcharias (Pers.) Fayod S0000002.0000.51
Tricholomopsis rutilans
(Schaeff.) Singer S		0000002.01.801.02
Lycoperdaceae
Apioperdon pyriforme (Schaeff.) Vizzini S02.33.75.83.06.32.01.822.28.07
Macrocystidiaceae
Macrocystidia cucumis
(Pers.) Joss. S		0000002.0000.51
Marasmiaceae
Gymnopus androsaceus
(L.) Della Magg. &
Trassin. S		000001.60000.41
Mycenaceae
Mycena galericulata
(Scop.) Gray S		000001.62.01.801.43
Mycena galopus (Pers.) P. Kumm. S2.6002.91.43.12.01.801.75
Mycena sp. S2.64.75.72.94.06.303.602.56
Pleurotaceae
Pleurotus pulmonarius (Fr.) Quél. S0000002.0000.51
Pluteaceae
Pluteus atromarginatus (Konrad) Kühner S002.92.91.5000002
Pluteus pouzarianus Singer S2.608.602.8000002
Physalacriaceae
Armillaria borealis *
Marxmüller & Korhonen P		048.80012.2000001
Armillaria cepistipes *
Velen. P		56.4034.35.924.239.162.710.95.629.67
Schizophyllaceae
Schizophyllum commune Fr. S0002.90.7000001
Strophariaceae
Hypholoma capnoides (Fr.) P. Kumm.
S		7.79.38.606.412.52.03.604.56
Hypholoma fasciculare
(Huds.) P. Kumm. S		10.3004.73.86.307.311.16.25
Hypholoma lateritium (Schaeff.) P. Kumm
S		02.3000.6000001
Kuehneromyces mutabilis (Schaeff.) Singer & A.H. Sm.
S		000003.10000.81
Pholiota flammans
(Batsch) P. Kumm.
S		000001.63.9001.42
Atheliales
Atheliaceae
Amphinema byssoides (Pers.) J. Erikss. S0002.90.7000001
Auriculariales
Auriculariaceae
Exidia nigricans (With.) P. Roberts S000000005.61.41
Incertae sedis
Pseudohydnum gelatinosum (Scop.)
P. Karst. S		02.32.901.3000002
Boletales
Coniophoraceae
Coniophora arida (Fr.) P. Karst. S04.7001.2000001
Hygrophoropsidaceae
Hygrophoropsis aurantiaca (Wulfen) Maire S0005.91.5001.85.61.93
Paxillaceae
Paxillus involutus (Batsch) Fr. E02.3000.6000001
Dacrymycetales
Dacrymycetaceae
Calocera viscosa (Pers.) Fr. S02.305.92.051.60000.43
Dacrymyces stillatus Nees S5.17.02.98.86.07.80002.05
Gloeophyllales
Gloeophyllaceae
Gloeophyllum odoratum (Wulfen) Imazeki S0002.90.7000001
Gomphales
Gomphaceae
Ramaria apiculata (Fr.) Donk S00011.82.9000001
Hymenochaetales
Hymenochaetaceae
Onnia tomentosa (Fr.) P. Karst. P0002.90.7000001
Hyphodontiaceae
Hyphodontia arguta (Fr.) J. Erikss. S0000000011.12.81
Hyphodontia pallidula
(Bres.) J. Erikss. S		000000005.61.41
Incertae sedis
Trichaptum abietinum
(Pers. ex J.F. Gmel.) Ryvarden S		0002.90.7000001
Schizoporaceae
Xylodon flaviporus (Berk. & M.A. Curtis ex Cooke) Riebesehl & Langer S000001.601.800.92
Xylodon nesporii (Bres.) Hjortstam & Ryvarden S000000005.61.41
Polyporales
Dacryobolaceae
Postia ptychogaster
(F. Ludw.) Vesterh S		7.7005.93.4001.85.61.94
Postia tephroleuca (Fr.) Jülich S000001.60000.41
Fomitopsidaceae
Fomitopsis pinicola (Sw.) P. Karst. P04.7001.21.601.800.63
Hyphodermataceae
Hyphoderma roseocremeum (Bres.) Donk
S		2.60000.7000001
Incertae sedis
Amaropostia stiptica (Pers.) B.K. Cui, L.L. Shen &Y.C Dai P02.32.901.30005.61.43
Incrustoporiaceae
Skeletocutis amorpha (Fr.) Kotl. & Pouzar S0002.90.7000001
Meruliaceae
Phlebia tremellosa
(Schrad.) Nakasone & Burds. S		00000001.800.51
Physisporinus vitreus
(Pers.) P. Karst. S		002.900.7000001
Rigidoporus sanguinolentus (Alb. & Schwein.) Donk
S		0002.90.7000001
Phanerochaetaceae
Bjerkandera adusta
(Willd.) P. Karst. S		0000002.0000.51
Phlebiopsis gigantea (Fr.) Jülich S2.6002.91.4000002
Polyporaceae
Cyanosporus caesius (Schrad.) McGinty P5.12.3001.96.301.802.04
Xenasmataceae
Xenasmatella vaga (Fr.) Stalpers S002.95.92.2001.816.74.64
Russulales
Stereaceae
Heterobasidion parviporum * Niemelä & Korhonen P5.14.75.711.86.81.69.87.311.17.58
Bondarzewiaceae
Stereum sanguinolentum (Alb. & Schwein.) Fr. P2.62.302.92.04.73.93.65.64.57
Thelephorales
Thelephoraceae
Tomentella bryophila
(Pers.) M.J. Larsen E		000000005.61.41
Tomentella radiosa
(P. Karst.) Rick E		0000000011.12.81
Trechisporales
Hydnodontaceae
Trechispora hymenocystis (Berk. & Broome) K.H. Larss. S0000002.05.501.92
Trechispora nivea (Pers.) K.H. Larss. S2.60000.70005.61.42
Trechispora mollusca
(Pers.) Liberta S		0000002.0000.51
Tremellales
Tremellaceae
Tremella encephala Pers. M000000005.61.41
Total: 69 taxa (Ascomycota—5, Basidiomycota—64)
Total: 69 taxa (S—55, P—8, E—4, M—2)
Total: 395 recordsforest soil—185arable soil—210
S—218 recordsforest soil—105 arable soil—113
P—170 records forest soil—78 arable soil—92
E—5 recordsforest soil—1arable soil—4
M—2 recordsforest soil—1arable soil—1
* from basidiomata, mycelium, or rhizomorphs (PCR-confirmed).
Table
Table 3. Frequency (%) of fungal taxa on Norway spruce trees within plots on old forest (1–4) and post-agricultural soils (5–8) (trophic categories: P—parasites and S—saprotrophs); * W—in wood inside trunk (PCR-confirmed).
Table 3. Frequency (%) of fungal taxa on Norway spruce trees within plots on old forest (1–4) and post-agricultural soils (5–8) (trophic categories: P—parasites and S—saprotrophs); * W—in wood inside trunk (PCR-confirmed).
Stand LocationForest Soil/Old ForestArable Soil/Post-Agricultural SoilNumber of Stands with Taxa Present
Site No.12345678
Number of Trees144118825110911712971
ASCOMYCOTA
Helotiales
Hamatocanthoscyphaceae
Xenopolyscytalum pinea
Crous S		00000001.4 *
W		1
Incertae sedis
Scytalidium lignicola
Pesante
P		00000.9 *0001
Hypocreales
Nectriaceae
Mariannaea pinicola L. Lombard & Crous S00000001.4 *
W		1
BASIDIOMYCOTA
Agaricales
Physalacriaceae
Armillaria cepistipes
Velen.
P		00000000.9 *
W		1
Pleurotaceae
Pleurotus dryinus (Pers.) P. Kumm. P00000.9001.42
Pleurotus ostreatus (Jacq.) P. Kumm. P000000.9001
Strophariaceae
Hypholoma capnoides (Fr.) P. Kumm.
S		000000.9 *001
Hypholoma fasciculare
(Huds.) P. Kumm. S		000000.9001
Pholiota squarrosa (Vahl) P. Kumm. P00.80000001
Cantharellales
Hydnaceae
Sistotrema brinkmannii
(Bres.) J. Erikss.
S		0.7*002.0 *W00002
Dacrymycetales
Dacrymycetaceae
Dacrymyces stillatus Nees
S		00000.90001
Hymenochaetales
Hymenochaetaceae
Onnia tomentosa (Fr.) P. Karst. P0005.9000.802
Incertae sedis
Trichaptum abietinum
(Pers. ex J.F. Gmel.) Ryvarden S		00.80000001
Oxyporaceae
Oxyporus ravidus (Fr.) Bondartsev & Singer P000000.9001
Incertae sedis
Incertae sedis
Resinicium bicolor (Alb. & Schw. ex Fr.) Parm. P000000.9 *001
Polyporales
Incertae sedis
Amaropostia stiptica (Pers.) B.K. Cui, L.L. Shen &Y.C Dai P001.200.90002
Russulales
Bondarzewiaceae
Heterobasidion parviporum * Niemelä &
Korhonen P		0002.0 *
W		00.9 *0.8 *03
MUCOROMYCOTA
Mucorales
Mucoraceae
Mucor hiemalis Wehmer
S		00000001.4 *
W		1
Total: 18 taxa (Ascomycota—3, Basidiomycota—14, Mucoromycota—1)
Total: 18 taxa (S—8, P—10)
Total: 22 records forest soil—8 arable soil—14
S—7 records forest soil—3 arable soil—4
P—15 recordsforest soil—5 arable soil—10
* from basidiomata, mycelium, or rhizomorphs (PCR-confirmed).
Table
Table 4. Values of some ecological indices describing the fungal communities in the forest (s1–s4) and arable (s5–s8) soils.
Table 4. Values of some ecological indices describing the fungal communities in the forest (s1–s4) and arable (s5–s8) soils.
Soil TypeObservedChao1se.chao1ACEse.ACEShannonSimpsonFisher
s11822.66674.483223.9132.40332.26520.80249.5999
s22130.16677.370731.44362.73652.43830.829812.4085
s314216.629623.01752.16132.19790.82229.4903
s42532.33335.671834.09092.91083.0940.949123.1609
s52132.259.532832.86893.05012.4440.84829.8691
s62147.2518.736475.09724.81222.07080.733710.7594
s7234414.729951.29853.80282.92180.932522.5337
s82139.213.138142.41812.9392.91310.937526.5619
Table
Table 5. Correlation coefficient and Spearman p-values for the compared variables (counts).
Table 5. Correlation coefficient and Spearman p-values for the compared variables (counts).
Counts All StumpsPICUS+
Trees		H. parviporum + Armillaria spp. Stumps
Fungi on stumps-R = 0.58, p = 0.13R = −0.09, p = 0.83R = −0.096, p = 0.82
CountsFungi on stumpsAll stumpsPICUS+
trees		H. parviporum + Armillaria spp. stumps
Forest soil vs. Arable soilR = −0.77, p = 0.23R = 0.2, p = 0.92R = 0.2, p = 0.92R = −0.63, p = 0.37
CountsArmillaria spp.Hypholoma spp. H. parviporum
Forest soil vs. Arable soilR = 0.8, p = 0.33R = 0.4, p = 0.75R = −0.26, p = 0.74
CountsNo. of trees on survey plotTrees with decay by PICUS (n)
Forest soil vs. Arable soilR = 0.2, p = 0.92R = 0.2, p = 0.92
CountsNo. of trees on survey plotStand age
Trees with decay by PICUS (n)R = −0.96, p = 0.00011R = 0.63, p = 0.097
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Szczepkowski, A.; Kowalczuk, W.; Sikora, K.; Damszel, M.; Sierota, Z. Fungi Occurring in Norway Spruce Wood Decayed by Heterobasidion parviporum in Puszcza Borecka Stands (Northeastern Poland). Forests 2022, 13, 229. https://doi.org/10.3390/f13020229

AMA Style

Szczepkowski A, Kowalczuk W, Sikora K, Damszel M, Sierota Z. Fungi Occurring in Norway Spruce Wood Decayed by Heterobasidion parviporum in Puszcza Borecka Stands (Northeastern Poland). Forests. 2022; 13(2):229. https://doi.org/10.3390/f13020229

Chicago/Turabian Style

Szczepkowski, Andrzej, Waldemar Kowalczuk, Katarzyna Sikora, Marta Damszel, and Zbigniew Sierota. 2022. "Fungi Occurring in Norway Spruce Wood Decayed by Heterobasidion parviporum in Puszcza Borecka Stands (Northeastern Poland)" Forests 13, no. 2: 229. https://doi.org/10.3390/f13020229

APA Style

Szczepkowski, A., Kowalczuk, W., Sikora, K., Damszel, M., & Sierota, Z. (2022). Fungi Occurring in Norway Spruce Wood Decayed by Heterobasidion parviporum in Puszcza Borecka Stands (Northeastern Poland). Forests, 13(2), 229. https://doi.org/10.3390/f13020229

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