Soil fungal and bacterial communities in southern boreal forests of the Greater Khingan Mountains and their relationship with soil properties

Soil microbial communities drive key ecological and biogeochemical processes in terrestrial ecosystems, including organic matter turnover, pollutant breakdown, nutrient mineralization, nitrogen fixation, and mycorrhiza formation. Through secretion of hydrolases, microbes decompose plant litter, returning nutrients to the soil and thereby influencing soil properties such as nutrient pools and enzyme activities. Conversely, soil properties (nutrient availability, pH, moisture) regulate microbial community structure and activity, indicating a two-way interaction. Prior studies have shown pH and moisture shape total and active microbial communities, phosphorus additions can increase respiration and biomass, and carbon and nitrogen availability influence microbial structure. Boreal forests (taiga) are extensive biomes dominated by a limited set of genera and are important to the global carbon budget. The Greater Khingan Mountains in northeast China host the southernmost boreal forests, with distinct broadleaf (birch, aspen) and coniferous (larch, pine) stands. While various aspects of boreal forests have been studied, relationships between soil microbial communities and soil properties in these systems remain underexplored. Given evidence that stable microbial communities and soil C and N stocks generally develop 30–50 years after afforestation, the study focused on mature stands: birch and aspen (~50 years) and larch and pine (~96 years). The authors hypothesized that soil fungal and bacterial communities are directly associated with soil properties (total and available nutrients, hydrolase activities, moisture, pH) in southern boreal forests of the Greater Khingan Mountains and that tree species drive differences in microbial communities and soil properties.

The paper reviews evidence for bidirectional links between soil microbial communities and soil properties: pH and moisture regulate microbial communities in forests; phosphorus additions can stimulate microbial respiration and biomass; carbon and nitrogen availability affect community structure. In boreal contexts, vegetation composition has been shown to determine microbial activities, and microbial biomass/activity may be independent of litter diversity where litter is present. Across reclaimed and natural boreal soils, bacterial abundance has been linearly related to pH, total nitrogen, and C/N ratio. Tree species effects on soil C and N stocks and microbial properties have been documented in temperate and boreal systems, with bacterial and fungal communities varying by tree species and forest type (temperate deciduous, subtropical evergreen/deciduous). The review highlights that despite such findings, explicit relationships between microbial communities and soil properties in boreal forests—particularly in China’s southern boreal region—are insufficiently characterized, motivating the present study.

Study site: Mohe Forest Ecosystem National Positioning Observation and Research Station (53°17′–53°30′ N, 122°06′–122°27′ E) in the Greater Khingan Mountains, northeast China. Mean annual temperature −5.5 °C, precipitation 425 mm concentrated in July–August; long frost period leading to permafrost. Four natural, single-dominant-species forests were selected: birch (Betula platyphylla), aspen (Populus davidiana), larch (Larix gmelinii), and pine (Pinus sylvestris var. mongolica). Canopy density 0.8. Stand ages: larch 96±5 yr, pine 96±7 yr, birch 50±5 yr, aspen 50±5 yr. To avoid confounding by age, comparisons were made within forest types: birch vs aspen (broadleaf) and larch vs pine (coniferous). Sampling: In each forest, three sampling areas (replicates) were randomly selected (20 m × 30 m). From each area, five topsoil cores (17 cm diameter, 5 cm height) were collected post-litter removal using a five-diagonal scheme. Soils were cooled, sieved to 2 mm, and pooled per area to form composite samples for microbial and soil analyses. Fresh 2 mm soil was used for microbial community, dissolved organic C, NH4+-N, NO3−-N, and moisture; air-dried 2 mm soil for available K, available P, pH, and enzyme activities; air-dried 0.149 mm soil for total organic C, total N, total K, and total P. Soil properties: Total N, NH4+-N, NO3−-N by AutoAnalyzer; total and dissolved organic C by elemental analyzer; total and available K by flame photometry; total and available P by spectrophotometry. Enzyme activities by colorimetric assays: urease (NH3 produced mg g−1 d−1), protease (amino acids mg g−1 d−1), sucrase (reducing sugars mg g−1 h−1), cellulase (glucose μg g−1 h−1). Soil pH and moisture followed standard methods. DNA extraction and sequencing: DNA extracted with FastDNA SPIN Kit for Soil; quality checked by NanoDrop and agarose gel. Bacteria: 16S rRNA gene V3–V4 (338F/806R); fungi: ITS1–ITS2 (ITS1F/ITS2R). PCR with TransStart FastPfu, purification (AxyPrep), quantification (QuantiFluor-ST), library prep (Illumina TruSeq) and sequencing per manufacturer protocols. Bioinformatics: Quality filtering with Trimmomatic v0.39, merging with FLASH v1.2.11; reads truncated at average Q<20 over 50 bp window; up to two primer mismatches; ambiguous bases removed; overlaps >10 bp merged. OTUs clustered/classified at 97% similarity using RDP classifier v11.5. Filtering: reads with abundance <5 in all three replicates removed; per-replicate read counts normalized to the minimum depth. Analyses: Rarefaction (Mothur v1.30.2). Venn analyses (VennDiagram). Relative abundance bar plots (ggplot2). Beta diversity with PCoA (Bray–Curtis) and PERMANOVA (vegan; 999 permutations). Soil properties compared by Student’s t-test, PCA (prcomp), PERMANOVA (Bray–Curtis). Relationships between microbial communities and soil properties assessed with RDA/CCA (vegan; anova), grouping variables due to sample size constraints: (a) dissolved organic C, NH4+-N, NO3−-N, available K, available P; (b) total organic C, total N, total K, total P, pH; (c) urease, protease, sucrase, cellulase, moisture. Variance partitioning analysis (VPA) quantified contributions of highlighted soil variables. Functional annotation: fungi via FUNGuild v1.0; bacteria via PICRUSt v1.1.0 (COG categories). Functional differences evaluated by PERMANOVA; individual guild/category differences by t-tests. Sequence data deposited in NCBI BioProject PRJNA624797.

Sequencing depth and diversity: Fungal ITS (ITS1–ITS2) yielded ~805,820 clean reads (avg length 245 bp); bacterial 16S V3–V4 yielded ~641,184 reads (avg length 416 bp). Rarefaction indicated sufficient depth. Fungal reads clustered into 386 OTUs (238 species, 165 genera, 103 families, 58 orders, 28 classes, 9 phyla). Bacterial reads clustered into 1460 OTUs (674 species, 329 genera, 222 families, 147 orders, 63 classes, 26 phyla). In broadleaf forests, 281 fungal OTUs and 1232 bacterial OTUs detected; in coniferous forests, 287 fungal OTUs and 1431 bacterial OTUs detected. OTU overlap and dominance: Broadleaf (birch vs aspen): 159 (56.58%) fungal OTUs and 914 (74.19%) bacterial OTUs shared; 122 (43.42%) fungal and 318 (25.81%) bacterial OTUs were forest-specific. Coniferous (larch vs pine): 71 fungal and 1195 bacterial OTUs shared; 216 (75.26%) fungal and 236 (16.49%) bacterial OTUs were forest-specific. Dominant OTUs: fungi—birch OTU117 (Russula sp.), aspen OTU750 (Piloderma sp.), larch OTU502 (Archaeorhizomyces sp.), pine OTU1268 (Mortierella elongata); bacteria—birch and larch OTU2595 (Bradyrhizobium sp.), aspen OTU2417 (unclassified, class AD3), pine OTU3214 (unclassified, order Acidobacteriales). Community differences: PCoA showed discrete clustering; PERMANOVA indicated significant differences in fungal and bacterial beta diversity between birch vs aspen and larch vs pine (p=0.001; r2 fungi 0.909; bacteria 0.812). Functional composition: Fungal functional composition differed overall between forests (PERMANOVA p=0.001). No individual fungal guild differed between birch and aspen; five guilds differed between larch and pine (p<0.05). Guild G2 (Endophyte–Litter/Soil/Undefined Saprotroph) most abundant and significantly higher in pine (78.68%) than larch (23.27%). Guilds G4 (Soil Saprotroph) and G5 (Ectomycorrhizal–Orchid Mycorrhizal–Root Associated Biotroph) >10% in larch (22.50% and 10.87%) but <1% in pine; G3 (Undefined Saprotroph) 15.97% in larch vs 3.74% in pine; G8 (Arbuscular Mycorrhizal) 0.01% in larch vs 13.8% in pine. Bacterial functional categories (COG) also differed overall (p=0.001); six categories differed between birch and aspen, and six between larch and pine (p<0.05). Only H (Coenzyme transport and metabolism) differed in both comparisons; maximum absolute differences among categories were small (≤0.33%). Soil properties: PCA showed clear separation (axes explained 83.77% total variance); PERMANOVA p=0.001. Birch vs aspen: birch had higher total organic C and total N (p<0.05); lower total and available P, available K, protease activity, and pH (p<0.05). Larch vs pine: larch had higher pH; pine had higher NH4+-N, NO3−-N, dissolved organic C, total and available P, total and available K, and protease activity (all p<0.05). Urease, sucrase, and moisture showed no significant differences in either comparison. Microbe–soil property associations (RDA/CCA): Broadleaf fungi associated with NH4+-N (r=0.892, p=0.038), total P (r=0.975, p=0.044), total K (r2=0.961, p=0.006), and protease (r2=0.990, p=0.039). Coniferous fungi associated with available K (r=0.993, p=0.038), total organic C (r=0.926, p=0.007), pH (r2=0.998, p=0.022), and protease (r=0.892, p=0.036). Broadleaf bacteria associated with NH4+-N (r=0.951, p=0.007), dissolved organic C (r=0.849, p=0.031), available K (r2=0.900, p=0.036), total N (r=0.858, p=0.039), total P (r=0.876, p=0.003), pH (r=0.793, p=0.033), and cellulase (r2=0.822, p=0.022). Coniferous bacteria associated with available K (r2=0.995, p=0.018). NO3−-N, available P, urease, sucrase, and moisture showed no strong associations (p>0.05). Variance partitioning: For fungal communities, variance explained by K nutrition 65.11%, NH4+-N 37.20%, total P 38.34%, protease 39.34%; cumulative explained variance 73.78%. For bacterial communities, explained by K 61.39%, NH4+-N 38.82%, total P 54.52%, protease 39.93%; cumulative 52.79%. Synthesis: Protease activity consistently associated with fungal (but not bacterial) communities across forest types. NH4+-N and total P associated with both fungi and bacteria in broadleaf forests only. Potassium (total/available) consistently associated with both fungi and bacteria across forest types.

The study demonstrates that tree species in southern boreal forests are associated with distinct soil microbial community compositions, functional profiles, and soil properties, reinforcing previous findings that vegetation influences soil C and N stocks and microbial characteristics. Protease activity emerged as a key correlate of fungal communities across both broadleaf and coniferous forests, aligning with the role of fungal saprotrophs in initiating protein degradation and nitrogen mineralization. The differential associations observed between broadleaf and coniferous forests—particularly for NH4+-N and total P—indicate biome-specific or litter-driven controls on microbial communities and suggest that vegetation type modulates microbe–soil property linkages. The consistent importance of soil potassium (both total and available) to both fungal and bacterial communities across forest types highlights K as an often underappreciated driver in boreal systems, with implications for plant health, pest and disease resistance, and forest productivity. These findings contrast with some subtropical studies where K has limited relevance to microbial activity, possibly due to differences in sampling depth, forest type, or latitude. The authors propose that differences in plant litter inputs among forest types likely underpin observed divergences in soil properties and microbial communities, consistent with the known two-way interactions between soil communities and soil environment.

Southern boreal forests of the Greater Khingan Mountains exhibit distinct soil fungal and bacterial community structures, functional attributes, and soil physicochemical properties between broadleaf (birch, aspen) and coniferous (larch, pine) stands. Key relationships include: (a) protease activity is strongly correlated with fungal communities in both broadleaf and coniferous forests, but not with bacterial communities; (b) NH4+-N and total P correlate with both fungal and bacterial communities in broadleaf forests only; (c) potassium availability/content shows strong correlations with both fungal and bacterial communities across both forest types. These results underscore tree species effects on soil communities and properties and highlight K as an important factor for soil–microbe dynamics in boreal forests. Future research should test the generality of these relationships across broader boreal regions and explicitly evaluate the role of litter composition in shaping soil–microbiota interactions.

The study is limited to one boreal region (Greater Khingan Mountains) with three replicates per forest type, which may constrain generalizability. Stand ages differed between broadleaf (~50 years) and coniferous (~96 years) forests; to mitigate confounding, comparisons were restricted within forest types, but cross-type age effects cannot be fully excluded. The authors note limited comparable data from other boreal regions, making it uncertain whether the observed microbe–soil relationships generalize broadly. Additionally, some soil properties (e.g., NO3−-N, available P, urease, sucrase, moisture) showed no significant associations in this dataset, which may reflect sample size or site-specific conditions.