Acta	Silvae	et	Ligni	135	(2024),	53–63
53
Original scientific article  / Izvirni znanstveni članek 
THE EFFECT OF FOREST GAPS ON THE DIVERSITY AND COMPOSITION OF 
BACTERIAL COMMUNITIES IN MIXED-TYPE FOREST SOILS ACROSS THE 
CARPATHIAN MOUNTAINS
VPLIV GOZDNIH VRZELI NA PESTROST IN SESTAVO BAKTERIJSKIH ZDRUŽB 
V TLEH MEŠANIH GOZDOV KARPATOV
Nejc SUBAN¹, Olivera MAKSIMOVIĆ2,, Nataša ŠIBANC3, Tijana MARTINOVIĆ4,5, Eva DARENOVA6, Matjaž 
ČATER7,8, Tine GREBENC9
(1) Slovenian Forestry Institute, Department of forest physiology and genetics, nejc.suban@gozdis.si
(2) Slovenian Forestry Institute, Department of forest physiology and genetics, olivera.maksimovic@gozdis.si
(3) Slovenian Forestry Institute, Department of forest physiology and genetics, natasa.sibanc@gozdis.si
(4) Slovenian Forestry Institute, Department of forest physiology and genetics, tijana.martinovic@gozdis.si
(5) Laboratory of Environmental Microbiology, Institute of Microbiology of the Czech Academy of Sciences, Czech Republic, tijana.
martinovic@biomed.cas.cz
(6) Global Change Research Institute CAS, Czech Republic, darenova.e@czechglobe.cz
(7) Department of Silviculture, Faculty of Forestry and Wood Technology, Mendel University, Czech Republic, matjaz.cater@mendelu.cz
(8) Slovenian Forestry Institute, Department of forest physiology and genetics, matjaz.cater@gozdis.si
(9) Slovenian Forestry Institute, Department of forest physiology and genetics, tine.grebenc@gozdis.si
ABSTRACT
Mixed forests of European beech (Fagus sylvatica L.) and silver fir (Abies alba Mill.) play a vital ecological role in Central and 
South-Eastern Europe. This study investigates the diversity and composition of soil bacterial communities in these forests, 
focusing on rhizosphere and bulk soils under varying canopy structures. Soil samples were collected from eight sites along the 
Carpathian Mountains, including managed forests and the remnants of old growth. Metabarcoding of bacterial communities 
revealed that alpha diversity (species richness, Shannon index, and evenness) was significantly affected by sampling location 
but not by forest canopy structure or soil type (rhizosphere and bulk soil). The lowest bacterial diversity was found in the 
old-growth forest of the Beskidy region, while the highest was recorded in managed forest in Vrancea. Beta diversity analy-
ses showed minimal variation between rhizosphere and bulk soil bacterial communities, with geographic distance being the 
strongest predictor of community composition. Actinobacteriota and Proteobacteria were the dominant phyla across all sites, 
with higher relative abundance of Actinobacteriota in all rhizosphere samples compared to bulk soil. Complex combinations 
of various environmental conditions at each sampling location, including soil parameters (mainly pH and C:N ratio), the age 
of forest gaps, the type and intensity of disturbances, and species composition of above-ground vegetation, can strongly affect 
soil bacterial communities. A closer examination of additional environmental variables would be necessary to better explain 
the observed differences in the diversity and composition of bacterial communities.
Key words: forest gaps, forest management, soil microbiome, soil, rhizosphere, Carpathians, temperate forest
IZVLEČEK
Mešani gozdovi navadne bukve (Fagus sylvatica L.) in bele jelke (Abies alba Mill.) so eden najpomembnejših ekosistemov na ob-
močju srednje in jugovzhodne Evrope. S pristopom molekularne identifikacije smo analizirali diverziteto in sestavo talnih bakte-
rijskih združb na osmih lokacijah vzdolž Karpatov. Metagenomska analiza bakterijskih združb je pokazala, da je na alfa diverziteto 
(bogastvo vrst, Shannonov indeks in enakomernost) pomembno vplivala le lokacija vzorčenja, ne pa tudi struktura gozdne krošnje 
ali vrsta tal (rizosfera in zemlja, oddaljena od vpliva korenin). Najmanjšo bakterijsko vrstno pestrost smo potrdili v pragozdnem 
rezervatu Beskidy, največjo pa v gospodarskem gozdu v Vranceji. Analiza beta diverzitete je pokazala minimalne razlike med bak-
terijskimi združbami rizosfere in zemlje zunaj rizosfere, pri čemer je bila geografska oddaljenost glavni dejavnik vpliva na sestavo 
bakterijske združbe. Actinobacteriota in Proteobacteria sta bila prevladujoča rodova bakterij na vseh analiziranih lokacijah, pri 
čemer je bila relativna pojavnost rodu Actinobacteriota v vseh vzorcih rizosfere višja kot v zemlji zunaj območja rizosfere. Kom-
binacije različnih okoljskih razmer, predvsem pH tal in razmerje med C in N, ter drugih dejavnikov, kot so starost gozdne vrzeli, 
intenziteta in vrsta motnje, ki je povzročila njen nastanek, ter vrstna sestava vegetacije na vzorčenih lokacijah, lahko pomembno 
vplivajo na mikroorganizme v tleh. Da bi bolje pojasnili razlike v pestrosti in sestavi bakterijskih združb, bi bilo treba v analize 
vključiti dodatne spremenljivke.
Ključne besede: gozdne vrzeli, gospodarjenje z gozdom, mikrobna združba tal, tla, rizosfera, Karpati, gozdovi 
zmernega klimatskega pasu
UDC 181.351(045)=111 Received / Prispelo: 21. 10. 2024
DOI 10.20315/ASetL.135.5 Accepted / Sprejeto: 29. 11. 2024
54
Suban	N.,	Maksimović	O.,	Šibanc	N.,	Martinović	T.,	Darenova	E.,	Čater	M.,	Grebenc	T.:	The	effect	of	forest	gaps	on	the	...
1 INTRODUCTION
1 UVOD
European beech (Fagus sylvatica L.) and silver fir 
(Abies alba Mill.) are crucial components of Central and 
South-Eastern European forest ecosystems and are ex-
pected to remain key species in mid- and high-altitude 
European forests (Leuscher, 2009; Dobrowolska et al., 
2017). European beech exhibits high adaptability and 
ecological plasticity, thriving primarily in temperate 
regions due to its sensitivity to drought and high tem-
peratures (Jump et al., 2006; Colin et al., 2017; Levanič 
et al., 2023). It plays a vital role in providing ecosystem 
services such as timber production, carbon sequestra-
tion, biodiversity preservation, and maintaining soil 
fertility, soil stability, and water resources (Duncker et 
al., 2012). In contrast, silver fir is predominantly found 
in colder temperate regions, particularly in the Alps 
and Carpathians, where lower elevations have recently 
seen declines in growth due to climatic extremes, air 
pollution, and subsequent pest and pathogen attacks 
(Bošela et al., 2018; Čater and Levanič, 2019; Čater et 
al., 2024).
While trees are key drivers of atmospheric carbon 
uptake, soil microbial communities are fundamental 
to ecosystem function, stability, and productivity (van 
der Heijden et al., 2008; Baldrian et al., 2017a, 2017b; 
Mercado-Blanco et al., 2018). Microbes, as pathogens, 
symbionts, mutualists, and decomposers, regulate car-
bon cycling, as well as nutrient cycling and availability 
through processes such as litter decomposition, min-
eral weathering, nitrogen fixation, and nutrient uptake 
(Uroz et al., 2013; Lladó et al., 2017). Fungi, particu-
larly in temperate and boreal forests, are the primary 
decomposers of recalcitrant organic matter (Voríšková 
et al., 2013; Kohout et al., 2021). The role of bacte-
ria, however, is less clear, varying between taxa that 
rely on low molecular weight carbon compounds and 
those capable of decomposing complex biopolymers 
(Lladó et al., 2017; Algora et al., 2022) since complexi-
ty of lignocellulose demands that no single enzyme or 
bacterial strain is able to fully degrade plant biomass 
(López-Mondéjar et al., 2019). Forest tree species in-
fluence soil microbial communities through root exu-
dates, litter deposition, and fine root turnover, affect-
ing community composition and related ecosystem 
services (Martinović et al., 2022).
The Carpathian Mountains, one of the largest for-
ested mountain chains in Central Europe, feature di-
verse forest types dominated by beech and mixed coni-
fer stands at elevations between 500 and 1450 meters 
(Dinca et al., 2022; Knorn et al., 2013). This region, un-
derexplored in terms of soil biodiversity and ecosys-
tem functions (Guerra et al., 2020), has been largely 
excluded from large-scale soil sampling efforts, such 
as the LUCAS survey (Labouyrie et al., 2023). Comple-
mentary to Dařenova et al. (2024), who studied the 
drivers of soil CO2 efflux in beech-silver fir forests, this 
study analyzes bacterial community diversity in the 
rhizosphere and bulk soil under varying forest canopy 
structures in mixed forests of European beech and sil-
ver fir along the Carpathian Mountains. Using the same 
sampling design, we employed metabarcoding of soil 
environmental DNA to assess the diversity of selected 
bacterial taxa (Vasar et al., 2022).
Trees under the open and closed canopy show dif-
ferential growth rates (Orman et al., 2021; Čater et al., 
Fig. 1: Sampling locations along the Carpathian arc (adapted 
from Čater et al., 2024)
Slika 1: Lokacije vzorčenj vzdolž karpatske gorske verige 
(prilagojeno po Čater et al., 2024)
	 Acta	Silvae	et	Ligni	135	(2024),	53–63
55
2024), and the understorey vegetation community is 
different (Dařenova et al., 2024). A higher amount of 
solar radiation and precipitation reaching the ground 
under open forest canopy, as opposed to a closed one, 
presents more favourable conditions for soil microbi-
al activity (Jianxin et al., 2016; Chen and Yang, 2015). 
However, the area under open canopy receives a smal-
ler input of organic matter due to lower root density 
and litterfall from trees (Griffiths et al., 2010; Kohout 
et al., 2018) while receiving more organic matter from 
herbaceous plants that usually develop abundantly un-
der canopy openings. We hypothesized that bacterial 
beta diversity would differ significantly between soils 
under open and closed canopies, as well as between 
bulk and rhizosphere samples. In a study across Euro-
pean countries (Labouyrie et al., 2023), microbial ri-
chness and diversity were found to increase from less 
disturbed (i.e. woodlands) to more managed areas (i.e. 
grasslands and croplands). We hypothesized that bac-
terial alpha diversity would be significantly lower in 
the protected old-growth forests (Buzau and Beskidy) 
compared to managed sites.
2 METHODS
2 METODE
2.1 Study sites and research design
2.1 Območja raziskave in zasnova raziskave
A total of 48 soil samples was collected from eight 
sites along the Carpathian arc in Romania, Slovakia, 
and the Czech Republic (Adamič et al., 2023; Čater et 
al., 2024) (Fig. 1). All sites were located above 800 m 
on Cambisols and were selected for their favorable 
climate and soil conditions for beech and fir regene-
ration (Dařenova et al., 2024). Following the approach 
of Han et al. (2020) and Lyu et al. (2022), three plots 
of different light intensities were selected per site, ca-
tegorized as closed canopy, forest edge, and open gap 
(Dařenova et al., 2024; Čater et al., 2024). Two sites 
(Buzau and Beskidy) were unmanaged old-growth 
forest remnants, while the others had been managed 
with low-intensity silvicultural systems for over a cen-
tury (Adamič et al., 2023). In each plot, three bulk soil 
and three rhizosphere samples (top 10 cm) were col-
lected using standardized probes (Grebenc and Krai-
gher, 2007). The soil samples were kept at 4°C during 
transport to the institute’s laboratory. Bulk soil and 
root samples were then separated under a binocular 
microscope, and roots were washed under tap water to 
remove soil particles (Mrak et al., 2019). Samples were 
freeze-dried and stored at -20°C until further analyses.
Mean monthly temperature and precipitation data 
from 1901 to 2020 were obtained via kriging from 
the Royal Netherlands Meteorological Institute’s Cli-
mate Explorer web page (http://climexp.knmi.nl) to 
calculate mean annual temperature and precipitation 
(Dařenova et al., 2024).
2.2 Molecular analysis
2.2 Molekularna analiza
DNA was extracted from 250 mg of soil adjacent to 
roots (rhizosphere soil) and bulk soil using the DNeasy 
Plant Mini Kit (Qiagen, Hilden, Germany). Bacterial 
metabarcoding was conducted using the 341f/805r 
primer pair, with PCR conditions as follows: 94°C for 5 
min, followed by 30 cycles of 94°C for 30 s, 56°C for 30 
s, and 72°C for 30 s, with a final extension of 7 min at 
72°C. DNA was purified, quantified, and sequenced on 
an Illumina MiSeq platform (350 bp paired-end) (Unuk 
Nahberger et al., 2019).
2.3 Bioinformatics and statistical analysis
2.3 Bioinformatika in statistična analiza
Raw sequences were processed with SEED v2.1.2 
(Větrovský et al., 2013), with minimum quality thresh-
olds set at 30 for sequences and 12 for base pairs. 
Chimeric sequences were removed using USEARCH 
Table 1: Regions of each sampling location, with corre-
sponding geographical coordinates, altitude, average annual 
air temperature, and total annual precipitation
Preglednica 1: Območja vzorčnih lokacij s pripadajočimi zem-
ljepisnimi koordinatami, nadmorsko višino, povprečno letno 
temperature in povprečno skupno letno količino padavin
Plot number Region Altitude (m) Longitude Latititude Average annual air temperature (°C)
Total annual 
precipitation (mm)
1 Gorj 985 22.916944° 45.169444° 4.7 1073
2 Arges 995 24.651111° 45.460278° 7.4 812
3 Buzau 1038 26.228889° 45.614167° 6.8 744
4 Vrancea 830 26.603889° 46.001389° 8.3 603
5 Neamt 950 26.168333° 46.854167° 5.8 704
6 Suceava 850 25.683333° 47.468333° 5.4 738
7 Bardejov 880 21.016562° 49.254738° 7.2 758
8 Beskidy 820 18.416805° 49.402483° 7.1 744
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Suban	N.,	Maksimović	O.,	Šibanc	N.,	Martinović	T.,	Darenova	E.,	Čater	M.,	Grebenc	T.:	The	effect	of	forest	gaps	on	the	...
(Edgar, 2010), and sequences were clustered at a 97% 
similarity threshold using VSEARCH (Rognes et al., 
2016). BLAST analysis was performed against UNITE 
(Kõljalg et al., 2013) and NCBI databases, with an e-
value cutoff of 1e-50 and 92% similarity. The sequenc-
es are deposited in the data repository of the Slovenian 
forestry institute.
Statistical analyses were conducted in R (v. 4.3.2) 
using the phyloseq, vegan, ggplot2, multcomp, indic-
species, and pairwiseAdonis packages. Alpha diversity 
indices (species richness, Shannon index, and even-
ness) were compared between regions, forest stand 
types, and soil sample types using one-way ANOVA 
and Tukey HSD tests. Homogeneity of variance was 
assessed with Levene’s test. Beta diversity was as-
sessed using pairwise PERMANOVA on Bray-Curtis dis-
tance matrices (number of permutation = 999), with 
Hellinger transformation for standardization. Commu-
nity composition was visualized via NMDS ordinations. 
We used a statistical approach developed by De Cáceres 
et al. (2012) to identify indicator species at the given 
locations. The relative abundances of the OTUs at each 
sampling site were calculated from the phloseq object 
by dividing the number of each respective OTU by the 
sum of all OTU counts at the location as described in 
McMurdie and Holmes (2013). The soil parameter 
values are extracted from the study by Dařenova et al. 
(2024) since the analyses were preformed on the same 
soil samples used in our metagenomic study.
3 RESULTS
3 REZULTATI
ANOVA and subsequent Tukey HSD tests indicated 
that only the sampling location (region) had a stati-
stically significant effect (p<0.05) on bacterial rich-
ness, evenness, and Shannon diversity index (results 
are presented in Fig. 2; the p-values from the ANOVA 
test are presented in Table 1 in Supplementary Data). 
Levene’s test confirmed the homogeneity assumpti-
on of variance across the groups of the region varia-
ble (p=0.031). A total of 73,038 OTUs were identified 
with sequence similarity greater than 82%, and 52,987 
OTUs with similarity greater than 92%. The lowest di-
versity was observed in the Beskidy region, where the 
mean species richness (a) and its standard deviation 
was 3,117 ± 888.6, the mean Shannon index (b) was 
6.5 ± 1, and the mean evenness index (c) was 0.81 ± 
0.1. In contrast, the highest diversity was recorded in 
Vrancea, with a mean species richness (a) of 5,656 ± 
Fig. 2: Species richness (a), Shannon index (b), and evenness 
(c) values, along with grouping of these values by Tukey’s 
HSD test of statistical significance for bacterial communities 
in soil samples collected from eight sites along the Carpathi-
an arc
Slika 2: Ocene vrstnega bogastva (a), Shannonovega indeksa 
(b) ter indeksa enakomernosti (c) za bakterijske združbe v 
tleh. Vrednosti so združene v skupine glede na statistično 
značilnost razlik med posameznimi lokacijami, ocenjeno s 
HSD Tukeyevim testom variance.
	 Acta	Silvae	et	Ligni	135	(2024),	53–63
57
234.3, a mean Shannon index (b) of 8.1 ± 0.1, and a 
mean evenness index (c) of 0.83 ± 0.01. Tukey’s HSD 
test revealed statistically significant differences (adju-
sted p-value<0.05) in alpha diversity indices between 
Beskidy and the other regions (Bardejov, Buzau, Suce-
ava, Neamt, and Vrancea), while Gorj and Arges were 
only found to be statistically different from Suceava, 
Vrancea, and Neamt in terms of species richness (Fig. 
2; p-values from Tukey’s HSD test are presented in Ta-
ble 2 in the Supplementary Data).
The NMDS stress values (0.104) suggested a relia-
ble representation of the NMDS dissimilarity matrix. 
The first NMDS plot (Fig. 3a), depicting communities 
under different forest canopy types, showed no discer-
nible clustering along the axes. However, the second 
plot (Fig. 3b) exhibited a slight clustering of rhizo-
sphere samples along the NMDS2 axis within the more 
widely scattered bulk soil data points. PERMANOVA 
results revealed that sample type had a statistically 
significant effect (p<0.001; see Table 3 in Supplemen-
tary Data) although it explained only 4.4% of the vari-
ance, whereas geographic distance, expressed through 
a space variable consisting of three significant PCNM 
vectors, explained the largest proportion of variation 
(13.2%) (Table 3 in Supplementary Data).
Bar plots of OTU relative abundance at each sam-
pling location (Fig. 4) showed that Actinobacteriota 
was the dominant phylum across all sampling loca-
tions, followed by Proteobacteria. Other important 
phyla included Planctomycetota, Verrucomicrobiota, 
Firmicutes, Chloroflexi, Acidobacteriota, Myxococcota, 
Gemmatimonadota, and Bacteroidota. Notably, Actino-
Fig. 3: NMDS graphs of bacterial community composition at 
each sampling location. a) bacterial community composition 
under different forest canopy types; b) bacterial community 
composition from different soil sample types (rhizosphere 
soil and bulk soil).
Slika 3: Grafi NMDS ordinacije vrstne sestave bakterijskih 
združb na posameznih vzorčnih lokacijah. a) sestava bak-
terijskih združb v tleh pod različno pokrovnostjo drevesnih 
kroženj; b) sestava bakterijskih združb glede na tip tal (rizos-
ferna tla in zemlja, oddaljena od korenin).
Fig. 4: Relative abundance of bacterial phyla at each sam-
pling location (a) and sample type (b)
Slika 4: Relativna pojavnost bakterijskih filogenetskih debel 
na posameznih lokaciji (a) ter v obeh tipih tal (b)
58
Suban	N.,	Maksimović	O.,	Šibanc	N.,	Martinović	T.,	Darenova	E.,	Čater	M.,	Grebenc	T.:	The	effect	of	forest	gaps	on	the	...
bacteriota exhibited higher relative abundance in the 
rhizosphere compared to bulk soil. However, the relati-
ve abundance of bacterial indicator genera in both the 
rhizosphere and bulk soil was extremely low (0.08% 
and 0.015%, respectively).
4 DISCUSSION
4 RAZPRAVA
Our findings suggest that soil bacterial communi-
ty composition does not differ significantly between 
closed and open forest canopies. This may be due to 
the limited variation in environmental conditions ob-
served at the sampling sites, as reported by Dařenova 
et al. (2024). For example, soil temperature increases 
from closed canopy plots to gap edges and open cano-
py areas, but statistically significant differences were 
only observed at site 1 (Gorj) in 2022 and site 5 (Ne-
amt) in 2023, with the highest temperatures recorded 
at forest edges. Soil water content was generally higher 
under open canopies, though the pattern varied among 
plots. Furthermore, the content of carbon (C) and ni-
trogen (N), as well as the C:N ratio, showed no signi-
ficant trends across plots or sites, and pH values also 
lacked consistent patterns (Dařenova, 2024). Given 
the importance of soil properties, particularly pH and 
the C:N ratio, in predicting bacterial richness, diversity, 
and composition (Lauber et al., 2008, 2009; Wang et 
al., 2022; Labouyrie et al., 2023), these results likely 
explain the lack of pronounced differences in bacteri-
al community structure between forest canopy types. 
Recent studies have shown that higher soil pH increa-
ses both bacterial richness and Shannon index values, 
while higher calcium carbonate content and C:N ratios 
have the opposite effect (Labouyrie et al., 2023). Bac-
terial beta diversity is similarly influenced by pH, follo-
wed by the C:N ratio (Labouyrie et al., 2023).
Our hypothesis that alpha diversity indices would 
be lowest in unmanaged old-growth forests (Buzau 
and Beskidy) was not fully supported by the data. Whi-
le alpha diversity was significantly lower in the Beski-
dy region, the Buzau site grouped with regions exhi-
biting the highest diversity. This incongruence may 
be attributed to factors such as above-ground species 
composition, forest gap age and size, and the intensity 
of disturbance events, which can influence microbial 
diversity (Urbanová et al., 2015; Yang et al., 2017; Chen 
et al., 2022; Labouyrie et al., 2023). Variation in these 
factors across sites may also explain the lack of signi-
ficant differences in bacterial community composition 
between closed and open forest canopies. Post-distur-
bance forest development, characterized by increasing 
tree biomass and shifts in ground vegetation compo-
sition, can alter microbial activity, biomass, and com-
munity composition (Wang, 2022; Chauvat et al., 2003; 
Lucas-Borja and Delgado-Baquerizo, 2019).
Our results indicate no significant differences in 
bacterial alpha diversity between bulk soil and rhizo-
sphere samples. While some studies on herbaceous 
plants have reported higher bacterial diversity in rhi-
zosphere soils compared to bulk soils (Laurent and 
Aragno, 1999; Wei et al., 2023), our findings are more 
consistent with a study conducted in mountain forest 
ecosystems, which found no significant differences in 
alpha diversity between rhizosphere and bulk soils 
(Cui et al., 2019). However, PERMANOVA did reveal a si-
gnificant effect of soil sample type on bacterial commu-
nity composition, aligning with results from temperate 
mixed forests in Europe. For instance, Uroz et al. (2016) 
found that bacterial communities differed between 
rhizosphere and bulk soils regardless of tree species, 
while other studies have noted shifts in Acidobacterial 
taxa linked to preferences for leaf or needle litter from 
beech, spruce, or fir trees (Urbanová et al., 2015; Nacke 
et al., 2016). Consistent with European soil studies (La-
bouyrie et al., 2023), including soils in mixed-forests 
dominated by European beech Proteobacteria, Actino-
bacteria, and Acidobacteria were among the most abun-
dant phyla in our samples. However, the unusually high 
abundance of Planctomycetota in our samples, compa-
red to previous studies where this phylum was found 
in much lower abundances (Siles and Margesin, 2016; 
Bárta et al., 2017; Baćmaga et al., 2022), may indicate a 
methodological or technical bias in our data.
5 SUMMARY
5 POVZETEK
Mešani gozdovi zmerno toplega podnebnega pasu, 
v katerih prevladujeta navadna bukev (Fagus sylvatica 
L) in bela jelka (Abies alba Mill.), so na območju srednje 
in jugovzhodne Evrope ekonomsko najpomembnejši 
in geografsko najbolj razširjeni gozdni ekosistemi. Vse 
bolj pogosta sušna obdobja in visoke temperature ne-
gativno vplivajo na naravno obnovo in uspešnost preži-
vetja omenjenih vrst, predvsem jelke in smreke, na niž-
jih nadmorskih višinah. Poleg dreves, ki imajo ključno 
vlogo pri kroženju ogljika v naravi, vplivajo mikrobne 
združbe v tleh na vse bistvene biogeokemijske proce-
se, omogočajo kroženja hranil in energije ter delovanje 
ekosistemov. Drevesa, z izločanjem produktov foto-
sinteze prek koreninskega sistema in opada odmrlega 
rastlinskega materiala vplivajo na aktivnost mikrobnih 
združb in njihovo raznovrstnost. Biodiverziteta tal in 
delovanje mikrobnih združb sta na območju Karpatov 
pomanjkljivo raziskana. V raziskavi smo s pomočjo 
	 Acta	Silvae	et	Ligni	135	(2024),	53–63
59
metagenomskih pristopov opisali bakterijske združbe 
v tleh ter s Shannonovim indeksom, indeksom vrstne 
pestrosti in enakomernosti ocenili njihovo diverziteto. 
Primerjali smo bakterijske združbe v rizosferi in ze-
mlje zunaj območja korenin (bulk soil) ter bakterijske 
združbe odvzetih na območjih različne intenzitete za-
stiranja matičnega sestoja.
Vzorčenje smo opravili na osmih lokacijah vzdolž 
Karpatskega gorovja v Romuniji, na Slovaškem in Če-
škem. Izbrane vzorčne lokacije so na nadmorskih viši-
nah od 800 do 1100 metrov v mešanih gozdovih bukve 
in jelke. Iz pridobljenih podatkov sekvenciranja vzor-
cev zemlje smo identificirali 73.038 delovnih operacij-
skih enot (OTU) s podobnostjo sekvence (similarity), 
večje od 82 % glede na referenčno sekvenco, in 52.987 
delovnih operacijskih enot s podobnostjo, večjo od 92 
% baznih parov. S testom ANOVA smo pokazali, da le 
prostorska razporeditev vzorcev statistično značilno 
vpliva na vrednosti indeksov alfa diverzitete (vrstno 
bogastvo, Shannonov indeks in enakomernost združ-
be). Največ bakterijskih vrst smo zaznali v gospodar-
skem gozdu na vzorčni lokaciji Vrancea, najmanj vrst 
pa na najbolj severni lokaciji Beskidy. Z analizo beta 
diverzitete združb smo ocenili, da tip talnega vzorca 
statistično značilno pojasni le manjši del variance v 
vrstni sestavi bakterijske združbe (4,4 %), medtem ko 
je vpliv zemljepisne širine med lokacijami veliko večji 
(13,2 %). V vseh vzorcih so bili najpogosteje zastopa-
ne vrste iz rodu Aktinobacteria, njihova pogostost pa 
je večja v rizosfernih tleh kot v prostih tleh. Drugi pre-
vladujoči taksoni so vključevali Proteobacterie, Planc-
tomycetota, Verrucomicrobiota in Acidobacteriota.
V raziskavi mikrobnih združb na območju Karpa-
tov nismo odkrili značilnih razlik med bakterijskimi 
združbami v tleh pod zastorom krošenj ali na odprtem, 
čemur je morebiti botroval zelo majhen razpon vre-
dnosti okoljskih spremenljivk med različnimi lokacija-
mi. Razlike v povprečnih letnih temperaturah in sku-
pnih količinah padavin niso statistično značilno vpli-
vale na indekse diverzitete ter tudi talni parametri (pH 
in razmerje C:N), izmerjeni in testirani v sklopu študije 
Dařenove et al. (2024), niso sledili trendom geografske 
širine med vzorčnimi lokacijami. Predvidevali smo, da 
bo vrstno bogastvo najmanjše na lokacijah pragozdnih 
rezervatov, kar smo potrdili na eni izmed lokaciji (Be-
skidy), medtem ko je bila bakterijska združba druge 
lokacije (Buzau) pestra in vrstno bogata. Vrstna sesta-
va rastlin, način gospodarjenja z gozdom ter razlike 
v intenziteti in vrsti motenj, ki so botrovale nastanku 
vrzeli, so nekateri od dejavnikov, ki lahko vplivajo na 
nekatere neskladnosti izsledkov naših analiz. Tudi pri-
merjave alfa diverzitete med rizosfero in prostimi tle-
mi niso pokazale pomembnih razlik med združbami, 
čeprav lahko rastline s svojimi koreninskimi eksudati 
ustvarjajo razmere za specifično bakterijsko združbo, 
ki se razlikuje od tiste v prostih tleh. Z analizo tekso-
nomske pestrosti in pojavnosti smo ocenili, da največji 
delež zastopanosti v celotni združbi ponazarjata sku-
pini Actinobacteriota in Proteobacteriota. Zaznali smo 
tudi velik deležih skupine Planctomycetota, ki v drugih 
primerljivih študijah nikoli ni bila prepoznana s toli-
kšno pojavnostjo, kar pripisujemo bodisi specifičnim 
rastiščnim razmeram bodisi morebitni pristranskosti 
uporabljene metode.
ACKNOWLEDGEMENTS
ZAHVALE
This work was supported by Slovenian Research 
and Innovation Agency research projects J4-4547, J4-
3086, Research Core funding P4-0107 at the Slovenian 
Forestry Institute and the Czech Science Foundation 
(GAČR 21-47163L). NS was supported by a scholarship 
from the The Public Scholarship, Development, Disabi-
lity, and Maintenance Fund of the Republic of Slovenia 
through call no. 327. No animals, plants, or fungi were 
damaged during this study.
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Suban	N.,	Maksimović	O.,	Šibanc	N.,	Martinović	T.,	Darenova	E.,	Čater	M.,	Grebenc	T.:	The	effect	of	forest	gaps	on	the	...
SUPPLEMENTARY DATA
DODATNI PODATKI
Table 1: Adjusted p-values from Tukey’s HSD test for pair-
wise comparisons of the alpha diversity index values at the 
sampling locations
Preglednica 1: Prilagojene p-vrednosti HSD Tukeyevega tes-
ta za primerjavo vrednosti indeksov alfa diverzitete za pare 
vzorčnih lokacij
Richness Shannon Evenness
2_Arges-1_Gorj 0.997 1 1
3_Buzau-1_Gorj 0.137 0.497 0.822
4_Vrancea-1_Gorj *0.006 0.097 0.358
5_Neamt-1_Gorj *0.005 0.134 0.465
6_Suceava-1_Gorj *0.008 0.103 0.353
7_Bardejov-1_Gorj 0.310 0.540 0.787
8_Beskidy-1_Gorj 0.807 0.380 0.261
3_Buzau-2_Arges 0.459 0.590 0.719
4_Vrancea-2_Arges *0.037 0.133 0.264
5_Neamt-2_Arges *0.036 0.180 0.356
6_Suceava-2_Arges *0.049 0.140 0.259
7_Bardejov-2_Arges 0.734 0.633 0.678
8_Beskidy-2_Arges 0.381 0.302 0.355
4_Vrancea-3_Buzau 0.906 0.983 0.994
5_Neamt-3_Buzau 0.903 0.994 0.999
6_Suceava-3_Buzau 0.942 0.985 0.994
7_Bardejov-3_Buzau 1 1 1
8_Beskidy-3_Buzau *0.003 *0.003 *0.008
5_Neamt-4_Vrancea 1 1 1
6_Suceava-4_Vrancea 1 1 1
7_Bardejov-4_Vrancea 0.687 0.975 0.997
8_Beskidy-4_Vrancea *<0.001 *<0.001 *<0.001
6_Suceava-5_Neamt 1 1 1
7_Bardejov-5_Neamt 0.682 0.990 1
8_Beskidy-5_Neamt *<0.001 *<0.001 *0.001
7_Bardejov-6_Suceava 0.758 0.978 0.996
8_Beskidy-6_Suceava *<0.001 *<0.001 *<0.001
8_Beskidy-7_Bardejov *0.010 *0.004 *0.007
Table 2: Results of the one-way ANOVA test for statistical 
significance (expressed as p-values) of selected variables 
(region, sample, type, and stand type) in explaining alpha di-
versity index values
Preglednica 2: Rezultati enosmernega testa ANOVA za prever-
janje statistične značilnosti učinka izbranih spremenljivk (reg-
ije vzorčenja, tipa vzorčenja in vrste sestoja) na vrednosti in-
deksov alfa diverzitete
 Richness Shannon Evenness
Region *<0.001 *<0.001 *<0.001
Sample type 0.876 0.701 0.579
Stand type 0.146 0.096 0.095
	 Acta	Silvae	et	Ligni	135	(2024),	53–63
63
Table 3: Results of PERMANOVA test on the explanatory ef-
fect of the selected variables on bacterial species composi-
tion at the given locations. The tested variables were region, 
sample, type, stand type, and a space variable consisting of 
three PCNM vectors.
Preglednica 3: Rezultati testa PERMANOVA za ocenjevanje 
učinka izbranih spremenljivk pri pojasnjevanju sestave bakter-
ijske združbe na vzorčnih lokacijah. Testirane spremenljivke 
vključujejo: regije vzorčenja, tipa vzorčenja, vrste sestoja ter 
prostorsko spremenljivko, sestavljeno iz treh PCNM-vektorjev.
 Degrees of freedom Sum of squares R2 F-statistic p-value
Region
Model 7 4.6856 0.27008 2.1144 *0.001
Residual 40 12.6631 0.72992  
Total 47 17.3487 1   
Sample type
Model 1 0.7694 0.04435 2.1348 *0.001
Residual 46 16.5793 0.95565  
Total 47 17.3487 1   
Stand type
Model 2 0.6464 0.03726 0.8707 0.925
Residual 45 16.7023 0.96274  
Total 47 17.3487 1  
Space
Model 3 2.2928 0.13216 2.2336 *0.001
Residual 44 15.0559 0.86784  
Total 47 17.3487 1   
64
Suban	N.,	Maksimović	O.,	Šibanc	N.,	Martinović	T.,	Darenova	E.,	Čater	M.,	Grebenc	T.:	The	effect	of	forest	gaps	on	the	...