2.1. Materials
The material was collected during the expedition of the Vietnam-Russian Tropical Research and Technological Centre from 2016 to 2018 in Xuan Son National Park, Phu Tho Province; Pu Hoat Nature Reserve and Pu Mat National Park, Nghe An Province; Kon Plong Protected Forest, Kon Tum province; and Kon Chu Rang Nature Reserve and Kon Ka Kinh National Park, Gia Lai Province. All samples were taken once at the beginning of the wet season. Samples were taken according to the method generally accepted for soil microbiological studies, uniformly in all the studied parks and reserves. Sample plots of 400 sq·m (20 × 20 m) were established for sampling in typical park landscapes. For each site, a description was made, and then 10 point samples were randomly selected. From these samples, a thoroughly mixed composite sample was made in the laboratory. Samples were collected according to the following scheme: plant litter, soil at a depth of 0–5 cm and 5–20 cm (
Table 1). In the forests of Pu Hoat Nature Reserve, Kon Ka Kinh National Park and Kon Chu Rang Nature Reserve, we collected suspended soils forming in the baskets of epiphytes of the genera
Asplenium (
Aspleniaceae) and
Drynaria (
Polypodiaceae).
2.1.1. Xuan Son National Park
The national park is located in the Phu Tho province in the northern part of Vietnam; its area is about 15 thousand hectares. The climate is characterized by hot summers and cool winters, with an average annual temperature of 22–23 °C. The studied substrates in this territory were collected at 2 sites. Site 1 is a tropical low-mountain polydominant broadleaf karst forest with a predominance of representatives of the families
Elaeocarpaceae,
Lauraceae,
Moraceae,
Sabiaceae and
Anacardiaceae on karst rocks in the valley of a temporary watercourse (21.121648° N, 104.945771° E, 580 m a.s.l.). The soil is dark brown ferrallitic on limestone. The litter is abundant, forming a thick layer consisting of leaves at various stages of decomposition (
Figure 1). Site 2 is located on the Mount Ten peak; it is a tropical mid-mountain polydominant broadleaf forest on granite with a predominance of representatives of the families
Fabaceae,
Lauraceae and
Magnoliaceae (21.115354° N, 104.934785° E, 1200 m a.s.l.). The soil is mountainous dark brown ferrallitic, covered with litter from banana trees and fallen trunks lying on the ground. The litter is abundant, forming a thick layer, represented by leaves at different stages of decomposition.
2.1.2. Pu Hoat Nature Reserve
The Pu Hoat Nature Reserve is located in the Nghe An province. In this territory, the samples were collected at 2 sites: in a river valley and on a ridge slope. Site 1 is a tropical valley broadleaf polydominant tall-stemmed forest with a predominance of
Terminalia sp. and
Aglaiagigantean trees with a vertical structure of medium complexity on waterlogged alluvial soils on granite in the valley of the Zut Suoi River (19.762038° N, 104.802386° E, 845 m a.s.l.). The soil is alluvial brown with fragments of granite. Partially fragmented leaf litter with a thickness of 3–5 cm is at different stages of decomposition of plant material. At this site, we collected suspended soils from baskets of epiphytes of the
Aspleniaceae and
Polypodiaceae family [
28]. Site 2 is a tropical tall-stemmed mountain forest with a predominance of
Cunninghamia lanceolate (
Cupressaceae) trees on granite on a steep ridge slope (19.775998° N, 104.803729° E, 1370 m a.s.l.). The soil is mountainous red-yellow humus–ferrallitic. Coniferous litter from twigs and needles of
Cunninghamia with a thickness of 10–15 cm is fragmented at different stages of decomposition of plant material.
2.1.3. Pu Mat National Park
Pu Mat National Park is located in the northern part of Central Vietnam, in the Nghe An province; it occupies 194 thousand hectares. The samples were collected at a site in a tropical valley polydominant tall-stemmed permanently humid forest with the presence of Dracontomelondao, Bischofia javanica, etc., in the valley of the Khe Choang River on its shales (18.955816° N, 104.685032° E, 200 m a.s.l.). The soil is red-yellow ferrallitic. Leaf litter with a thickness of 3–5 cm is partially fragmented at different stages of decomposition of plant material.
2.1.4. Kon Chu Rang Nature Reserve
Kon Chu Rang Nature Reserve is located on a mountain plateau in the Gia Lai province in Central Vietnam (14°26′–14°35′ N, 108°30′–108°39′ E); its area is 15,900 hectares. In the territory of this reserve, sampling was carried out at 4 sites. Site 1 is a tropical low-mountain broadleaf polydominant tall-stemmed forest with a predominance of trees from the families
Lauraceae,
Burseraceae,
Myrtaceae and
Hamamelidaceae on a permanently wet gentle slope on fragmented basalts (14.51795° N, 108.54593° E, 1000 m a.s.l.). The soil is mountainous red-yellow humus–ferrallitic. The litter with a thickness of 1–2 cm does not completely cover the soil surface; it consists of partially fragmented leaves at different stages of decomposition (
Figure 2). Site 2 is a tropical low-mountain mixed forest with a predominance of trees from the family
Podocarpaceae (
Dacrydium elatum), as well as less-represented
Hamamelidaceae (
Simingtonia),
Rhodoliaceae (
Rhodolia),
Fagaceae and
Sterculiaceae (
Scaphium) with a vertical structure of medium complexity on short-profile soils on a basalt slab (14.48856° N, 108.56924° E, 1050 m a.s.l.). The soil is mountainous red-yellow humus–ferrallitic. The litter covers the soil surface in a thick layer of 10–15 cm; it consists of twigs and needles of
Dacrydium elatum with individual leaves at different stages of decomposition. Site 3 is a tropical low-mountain broadleaf forest with a predominance of trees from the families
Dipterocarpaceae,
Clusiaceae,
Ebenaceae,
Fabaceae, etc., on the edge of the plateau on short-profile soil on basalt (14.514043° N, 108.571246° E, 1025 m a.s.l.). The soil is mountainous red-yellow humus–ferrallitic. Leaf litter of a thickness of 1–2 cm is partially fragmented with plant material at different stages of decomposition. In this territory, we collected suspended soil from the baskets of epiphytes of the
Dryopteridaceae family. Site 4 is a low-mountain mixed forest with a predominance of trees from the
Podocarpaceae family, as well as less-represented
Hamamelidaceae,
Rhodoleiaceae,
Myrtaceae, etc., with a vertical structure of medium complexity on waterlogged short-profile soils on a basalt slab (14.468823° N, 108.562208° E, 995 m a.s.l.). The soil is red-yellow humus–ferrallitic. Deciduous–coniferous litter consisting of needles and twigs of
Dacrydium elatum with a thickness of 10–15 cm is partially fragmented with plant material at different stages of decomposition.
2.1.5. Kon Plong Protected Forest
The Kon Plong Protected Forest located in the Kon Tum province of Central Vietnam is characterized by a tropical monsoon climate with a wet season from May to October and a dry season from November to April. Soil and litter samples were collected at a site in a tropical mid-mountain mixed polydominant tall-stemmed permanently humid forest with a predominance of trees from the families Podocarpaceae (Dacrycarpus imbricatus), Magnoliaceae (Michelia, Mangletia, Kmeria), Myrtaceae (Syzygium), Calophyllaceae (Calophyllum), Elaeocarpaceae (Slonea) and Betulaceae (Betula) on a gentle slope with outcrops of granite fragments (14.753985° N, 108.297858° E, 1400 m a.s.l.). The soil is mountainous red-yellow humus–ferrallitic. The litter with a thickness of 1–2 cm does not completely cover the soil surface; it consists of partially fragmented leaves at different stages of decomposition.
2.1.6. Kon Ka Kinh National Park
Kon Ka Kinh National Park is located on the Kon Tum Plateau in the Gia Lai province of Central Vietnam (14°09′–14°30′ N, 108°16′–108°28′ E); its area is 41,780 hectares. The terrain is mountainous, with altitudes varying from 570 m in the river valleys to 1748 m at the top of the Kon Ka Kinh mountain. The area has a contrasting, tropical monsoon climate with a wet season from May to November and a dry season from December to April. In this territory, samples were collected at 6 sites, the first two in the eastern part of the park, the rest in the southwestern. Site 1 is a forest on a slope (14.296336° N, 108.445607° E, 700 m a.s.l.) in the eastern part of the Kon Ka Kinh National Park. The soil is brown tropical thin sandy loam. Leaf litter is partially fragmented at different stages of decomposition. Sampling was carried out on suspended soil from epiphyte baskets. Site 2 is a forest on a ridge (14.320337° N, 108.444608° E, 900 m a.s.l.) in the eastern part of the national park. The soil is mountainous red-yellow humus–ferrallitic. Leaf litter is represented by individual leaves and sprigs of needles. Site 3 is a tropical low-mountain valley polydominant permanently wet tall-stemmed forest with a predominance of trees from the families Euphorbiaceae, Myrtaceae, Moraceae, Duabangaceae, Lauraceae, Fagaceae and Meliaceae in the valley of the A Yun River in the southwestern part of the national park (14.21937° N, 108.31765° E, 1000 m a.s.l.). The soil is hydromorphic dark humus–ferrallitic with traces of gleying. The litter lies in a thick layer, represented by leaves at different stages of decomposition that have fallen at different times. Site 4 is a tropical mid-mountain polydominant tall-stemmed forest with a predominance of trees from the families Juglandaceae, Fagaceae, Elaeocarpaceae and Magnoliaceae on a wide crest of the ridge (14.22287° N, 108.331880° E, 1500 m a.s.l.). The soil is mountainous red-yellow humus–ferrallitic. The litter completely covers the soil surface in a layer of 2–5 cm (1–2 leaves); it is very dry and consists of partially fragmented leaves at different stages of decomposition. Site 5 is a light low-stemmed valley forest in the southwestern part of the Kon Ka Kinh National Park (14.217081° N, 108.283478° E, 860 m a.s.l.). It is a tropical flooded polydominant forest on light soils, with a predominance of Shorea siamensis Miq., Shorea roxburghii G. Don (Dipterocarpaceae), Schima (Theaceae), Irvingia (Irvingiaceae), Streblus asper Lour. Ficus spp. (Moraceae) and Syzygium (Myrtaceae). It is located in a wide flooded river valley and remains waterlogged for a long time. The soil is sandy loamy alluvial on shales and clays. The litter consists of large leaves, partially washed away by water flows. Site 6 is a mixed forest in the southwestern part of the national park (14.193672° N, 108.323651° E, 1160 m a.s.l.). It is a tropical mid-mountain tall-stemmed forest with a predominance of Pinus dalatensis (Pinaceae), Elaeocarpus (Elaeocarpaceae), Schima (Theaceae), Podocarpus neriifolius (Podocarpaceae) and Rhodoleia (Hamamelidaceae). It grows in a wide ridge and on a gentle slope of a ridge composed of slates. The soil is brown forest and well-drained. The litter forms a thick, dense layer (15–20 cm), composed of leaves, needles and twigs, differentiated into layers according to the degree of decomposition.
2.2. Research Methods
Soil pH was determined in the filtered supernatant of water suspensions of soil samples (soil:water = 1:2.5). To determine the acidity of the litter, we used the filtrate of an equilibrium solution prepared in the proportion of litter:water = 1:25.
The total abundance of actinomycetes in the substrates was determined by inoculation of suspensions at serial dilutions of 1:1000 and 1:10,000 on the Gause-1 solid nutrient medium (mineral agar 1) (g/L): K
2HPO
4—0.5; MgSO
4—0.5; KNO
3—1; NaCl—0.5; FeSO
4—traces; starch—20; agar—20; pH 7.2–7.4 [
29]. To prepare a suspension, 1 g of soil or litter was taken and placed in a flask with 100 mL of sterile water. To desorb mycelium from soil particles, the suspension was treated with a Bandelin Sonopuls HD 2070 ultrasonic disperser (Germany) for 2 min at a power of 50%. Each dilution was cultured in three replications. Plates were incubated in a thermostat at 28 °C for 7–10 days, and then the total number of grown colonies was counted. The number of cultivated actinomycetes per 1 g of soil (CFU/g) was calculated according to the formula
, where a is the average number of colonies per dish; n is the dilution from which the inoculation was made; b is the volume of plated drop of suspension, mL; and c is the soil sample, g.
The length of the actinomycete mycelium was measured on a luminescent microscope by staining the preparations of the soil suspension with acridine orange dye [
30]. We used the same suspensions as for the inoculation method. For one sample, 6 preparations were made on two defatted glass slides. Then, 0.01 mL of the suspension was applied to each preparation with a micropipette and distributed over an area of 4 cm
2. Then, the preparation was dried in air, fixed over a burner flame and stained with an aqueous solution of acridine orange (the working dye solution was used at a concentration of 1:10,000). The exposure time of preparations in the dye was 3 min, then rinsing in tap water was 2 times for 5 min. After staining, the preparations were dried at room temperature and examined under a LUMAM-IZ microscope (Russia). Light filters ZhS-19, ZhS-18, objective lens (×90 L) and eyepieces (×4 or ×5) were used. On each preparation, in 50 fields of view, the length of fragments of actinomycete mycelium was measured using an ocular ruler. Based on all measurements, the average length of the actinomycete mycelium in the field of view was determined. The length of actinomycete mycelium in 1 g of soil (N, m/g) was calculated by the formula:
, where S1 is the preparation area, μm
2; a is the average length of actinomycete mycelium in the field of view, µm; n is the suspension dilution index, mL; v is the volume of a drop applied to glass, mL; S2 is the microscope field of view area, µm
2; and c is the soil sample, g.
Pure cultures of actinomycetes were isolated by successive subculturing using Gause-1 medium (mineral agar 1); inoculations were incubated at 28 °C.
The antibiotic activity of actinomycete strains was determined by the agar block method, incubating the inoculations for 20–24 h at a temperature favorable for the development of test organisms (
Bacillus subtilis,
Aspergillus niger and
Candida albicans) [
31]. Joint cultivation of test organisms and actinomycetes was carried out on glucose–peptone–yeast agar (g/L) [
29]: glucose—1; peptone—2; yeast extract—1; casein hydrolyzate—1; CaCO
3—1; agar—20; glycerin—10 mL; pH 7.2.
The ability to degrade cellulose in the isolated strains of actinomycetes was identified by the presence of growth on Getchinson’s medium with filter paper (g/L): K2HPO4—1; CaCl2—0.1; MgSO4—0.3; NaCl—0.1; FeCl3—traces; NaNO3—2.5; agar—20; pH 7.2.
To determine the temperature range and optimal growth temperature, pure cultures of actinomycetes were cultivated at temperatures of 4, 10, 15, 22, 30, 37, 41, 45, 49.5 and 54 °C for 5 days in a liquid PC medium (g/L): NaCl—0.5; tryptone—5; yeast extract—2.5; glucose—1. The optical density of strains in the liquid medium was measured at a wavelength of λ = 660 nm (OD660).
To identify acid-tolerant and acidophilic strains of actinomycetes, pure cultures were sown on Gause-1 solid media with pH values of 5.0 and 4.0. The media were prepared using a phosphate–citrate buffer mixture (0.2 M Na2HPO4, 0.1 M citric acid).
DNA isolation, amplification and sequencing of the 16S rRNA gene. Identification of pure cultures of actinomycetes was carried out by sequencing of the 16S rRNA gene. The isolated DNA of pure cultures was PCR-amplified with primers that are universal for representatives of the
Bacteria phyla: 8-27f [5′-AGAGTTTGATCCTGGCTCAG-3′] and 1492r [5′-GGTTACCTTGTTACGACTT-3′]. PCR was carried out in a reaction mixture (25 µL) containing 10–50 ng of DNA template using an iCycler thermal cycler (BioRad, Hercules, CA, USA) in the following mode: 3 min at 94 °C, followed by 30 cycles (0.5 min at 94 °C, 0.5 min at 50 °C, 0.5 min at 72 °C), then 7 min at 72 °C. The length of the resulting fragments was assessed on a 1.0% agarose gel with ethidium bromide. DNA sequencing was performed using the ABI PRISM
® BigDye™ Terminator v. 3.1 Kit and an ABI 3730 DNA Analyzer (Applied Biosystems, Foster City, CA, USA) in accordance with the manufacturer’s recommendations. Preliminary analysis of the resulting nucleotide sequences was performed using the BLAST search against the NCBI GenBank database. The resulting sequences were compared with those of reference-type organisms. Sequence editing was performed using the BioEdit program [
32,
33].
High-throughput sequencing of 16S rRNA gene fragments. To assess the taxonomic diversity of actinomycete complexes, we used the method of high-throughput pyrosequencing of the variable region of the 16S rRNA gene in total soil DNA. The method is based on the identification of an evolutionarily conserved gene. During the study, an analysis of the V3–V4 hypervariable region of the 16S rRNA gene was performed for each of the microorganisms in the sample. The study was carried out by the next generation sequencing (NGS) method using an Illumina MiSeq platform with subsequent bioinformatics processing of the obtained data. Sample preparation was carried out using the two-stage polymerase chain reaction (PCR) technique. In the first stage, amplification of the hypervariable V3–V4 region of the 16S rRNA gene was performed using the primers universal for all prokaryotes. In the second stage, the PCR product obtained in the first stage was amplified with the purpose of barcoding the library. The resulting amplicons after purification on magnetic particles and measurement of concentration by the fluorometric method were ready-made DNA libraries suitable for multiplex sequencing on the Illumina platform. Subsequently, DNA analysis was performed on a new generation Illumina MiSeq sequencer using the paired end reads (2 × 300 bp), generating at least 10,000 paired reads per sample. Processing of sequencing data was carried out using the QIIME 1.9.1 automated algorithm, which includes combining forward and reverse reads, removing technical sequences, filtering sequences with low reliability of reads for individual nucleotides (quality less than Q20), filtering chimeric sequences, aligning reads to the 16S rRNA reference sequence and distributing sequences by taxonomic units using the Silva database version 132. The classification algorithm of operational taxonomic units (OTUs) with an open reference (Open Reference OTU picking) was used; the classification threshold was 97%.
Bioinformatics analysis. The functional characteristics of bacterial communities were predicted using the Local Mapper module of the iVikodak software package [
34] and the KEGG database [
35]. Heat maps were built using the ClustVis Internet resource (
http://biit.cs.ut.ee/clustvis accessed on 11 July 2022). The Venn diagram was built using the Venny 2.1 online resource (
https://bioinfogp.cnb.csic.es/tools/venny/ accessed on 14 July 2022). The sequences of the 16S rRNA gene of microbial communities were deposited to GenBank under the accession number PRJNA861932.