Genome-based classification of Streptomyces pinistramenti sp. nov., a novel actinomycete isolated from a pine forest soil in Poland with a focus on its biotechnological and ecological properties

A genomic-based polyphasic study was undertaken to establish the taxonomic status and biotechnological and ecological potential of a Streptomyces strain, isolate SF28T, that was recovered from the litter layer in a Polish Pinus sylvestris forest. The isolate had morphological characteristics and chemotaxonomic properties consistent with its classification in the genus Streptomyces. It formed long straight chains of spores with smooth surfaces, contained LL-diaminopimelic acid, glucose and ribose in whole-organism hydrolysates, produced major proportions of straight, iso- and anteiso- fatty acids, hexa- and octa-hydrogenated menaquinones with nine isoprene units and had a polar lipid pattern composed of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmethylethanolamine, glycophospholipids and three uncharacterized components. Phylogenetic trees prepared using 16S rRNA gene and multilocus gene sequences of conserved housekeeping genes showed that the isolate formed a branch that was loosely associated with the type strains of several validly published Streptomyces species. A draft genome generated for the isolate was rich in natural product-biosynthetic gene clusters with the potential to produce new specialised metabolites, notably antibiotics, and stress related genes which provide an insight into how it may have become adapted to the harsh conditions that prevail in acidic forest soils. A phylogenomic tree based on the genomes of the isolate and its phylogenetic neighbours confirmed that it formed a distinct lineage well separated from its closest evolutionary relatives. The isolate shared low average nucleotide identity and digital DNA:DNA hybridization values with its phylogenomic neighbours and was also distinguished from them using a combination of cultural and micromorphological properties. Given this wealth of taxonomic data it is proposed that isolate SF28T (= DSM 113360 T = PCM 3163 T) be classified in the genus Streptomyces as Streptomyces pinistramenti sp. nov. The isolate showed pronounced antimicrobial activity, especially against fungal plant pathogens.

phosphatidylmethylethanolamine, glycophospholipids and three uncharacterized components. Phylogenetic trees prepared using 16S rRNA gene and multilocus gene sequences of conserved housekeeping genes showed that the isolate formed a branch that was loosely associated with the type strains of several validly published Streptomyces species. A draft genome generated for the isolate was rich in natural product-biosynthetic gene clusters with the potential to produce new specialised metabolites, notably antibiotics, and stress related genes which provide an insight into how it may have become adapted to the harsh conditions that prevail in acidic forest soils. A phylogenomic tree based on the genomes of the isolate and its phylogenetic neighbours confirmed that it formed a distinct lineage well separated from its closest evolutionary relatives. The isolate shared low average nucleotide identity and digital DNA:DNA hybridization values with its phylogenomic neighbours and was also distinguished from them using a combination of cultural and micromorphological properties. Given this wealth of taxonomic data it is proposed that isolate SF28 T (= DSM 113360 T = PCM 3163 T ) be classified in the genus Streptomyces as Streptomyces pinistramenti sp. nov. The isolate showed pronounced antimicrobial activity, especially against fungal plant pathogens.

Introduction
The genus Streptomyces was proposed by Waksman and Henrici (1943) for aerobic, filamentous, spore-forming actinomycetes and its formal description subsequently emended by Kämpfer (2012). The genus currently includes 685 validly published species (http:// www. bacte rio. net/ strep tomyc es. html) but remains underspeciated (Sivalingam et al. 2019). Multilocus sequence analyses of concatenated, protein coding, conserved house-keeping genes and associated phenotypic properties provide a more reliable way of recognizing novel Streptomyces species than corresponding studies based on 16S rRNA gene sequences (Labeda et al. 2012Zhuang et al. 2020). It is also clear that genomic-based classifications are accelerating progress in streptomycete systematics as they provide greater resolution between closely related Streptomyces species than corresponding trees based on single and concatenated sequences of conserved genes Nouioui et al. 2018;Kusuma et al. 2021). In addition, improved metrics, such as pairwise average nucleotide identity (ANI) and in silico DNA:DNA hybridization (DDH) values, facilitate the recognition of species boundaries (Chun et al. 2018).
Streptomycetes are a unique source of antibiotics which include many used in agriculture, medicine and veterinary practice (Chater, 2016;Qi et al. 2021). Members of the genus are considered to be gifted (Baltz 2017) as they have large genomes (≥ 8.0 Mbp) rich in natural product-biosynthetic gene clusters (NP-BGCs) with the potential to encode for novel and uncharacterized antibiotics of potential therapeutic value, as exemplified by Streptomyces leeuwenhoekii strains isolated from an extreme hyper-arid Atacama desert soil (Busarakam et al. 2014;Castro et al., 2018). Novel streptomycetes from extreme biomes are proving to be a potential rich source of new bioactive molecules Sivalingam et al. 2019;Sivakala et al., 2021) thereby underpinning the premise that abiotic conditions in extreme biomes select for strains with the capacity to synthesize novel specialised metabolites (Bull 2011). However, little attention has been focused on the delineation of Streptomyces species isolated from coniferous forest soils (Golińska et al. 2022), exceptions include the recognition of Streptomyces abietis (Fujii et al. 2013), Streptomyces pini (Madhaiyan et al. 2016) and Streptomyces piniterrae (Zhuang et al. 2020). Streptomycetes from pine forest soils are also known to be antagonistic towards fungal pathogens of pine seedlings (Golińska and Dahm, 2013). It is becoming increasingly apparent that whole-genomes of actinomycetes isolated from extreme habitats contain stress-related genes that can provide an insight into how they adapt to harsh abiotic conditions that prevail therein (Abdel-Mageed et al. 2020;Golińska et al. 2022).
The present study, a continuation of earlier work on the diversity of filamentous actinomycetes in coniferous litter and soil (Golińska et al. 2022), was designed to establish the taxonomic provenance of a Streptomyces strain, isolate SF28 T , recovered from pine forest litter and to determine its ability to inhibit the growth of fungal pathogens. The isolate and its closest phylogenomic neighbours were the subject of a polyphasic study that included information drawn from whole-genome sequences. The resultant data show that the strain inhibits the growth of diverse fungal phytopathogens and belongs to a new Streptomyces species, designated Streptomyces pinistramenti sp. nov.

Materials and methods
Isolation, maintenance and cultural conditions Strain SF28 T was isolated from partially decomposed needles (F-horizon) under Pinus sylvestris trees growing on the southern slope of an inland sand dune in the Torun Basin, Poland (52°55′37''N, 18°42′11''E) in October 2013, using a standard dilution plating procedure (Goodfellow et al. 1967) and starch-casein agar (Küster and Williams 1964) adjusted to pH 4.5 using 1 M HCl. Details of the sampling site and the selective isolation procedure have been described previously (Golińska et al. 2016). The isolate was maintained on starch-casein agar slopes (pH 5.5) at room temperature and as suspensions of mycelial fragments and spores in 20% (v/v) glycerol at − 80 °C. Biomass for most of the chemotaxonomic and molecular systematic studies was prepared by growing the strain in flasks of yeast extract-malt extract broth (ISP 2; International Streptomyces Project medium 2; Shirling and Gottlieb 1966), adjusted to pH 5.5, and shaken at 150 rpm for 3 weeks at 28 °C. Cells were harvested by centrifugation and washed three times in sterile distilled water; biomass for the chemotaxonomic analyses was freeze-dried and that for the molecular systematic studies stored at − 20 °C.

Phylogenetic analyses
Genomic DNA was extracted from the isolate using a GenElute™ Bacterial Genomic Kit (Sigma-Aldrich, Germany) and a 16S rRNA gene amplified by PCR following procedures described by . The PCR product was purified using a purification kit (Qiagen, Germany), according to the manufacturer's instructions, and a quality check made using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). The purified PCR product was sequenced at the Institute of Biochemistry and Biophysics (IBB) of the Polish Academy of Sciences in Warsaw, Poland, using an ABI 3730xl Genetic Analyzer (Applied Biosystems, Thermo Fisher Scientific, USA).
The almost complete 16S rRNA gene sequence of the isolate was compared with corresponding sequences of type strains of closely related species using the EzBioCloud server (https:// www. ezbio cloud. net; Yoon et al. 2017). A maximum-likelihood (ML) and maximum-parsimony (MP) phylogenetic tree was generated using the Single-Gene Trees Phylogeny online tool (https:// www. dsmz. de/ servi ces/ online-tools/ single-gene-phylo genies; Meier-Kolthoff et al. 2013a) adapted for single genes. Multiple sequence alignments were generated using MUSCLE software (Edgar 2004) and a ML tree was inferred from alignments with RAxML (Stamatakis 2014) using rapid bootstrapping together with the auto maximal-relative-error (MRE) bootstrapping criterion (Pattengale et al. 2010). Similarly, a MP tree was constructed from alignments with the Tree Analysis New Technology (TNT) program (Goloboff et al. 2008) using 1000 bootstraps together with tree bisection and reconnection branch swapping and ten random sequence replicates. The sequences were checked for compositional bias using the X 2 test, as implemented in PAUP* (Swofford 2002). The neighbour-joining algorithm (Saitou and Nei 1987) and the MEGA7 software package (Kumar et al. 2016) were used to generate a phylogenetic tree and evolutionary distances calculated using the Kimura-2-parameter model (Kimura 1980) with 1000 bootstrap repetitions (Felsenstein 1985). The root position of the trees were determined using a 16S rRNA gene sequence taken from the genome of Kitasatospora setae DSM 43861 T (NC_016109.1) using the SEED viewer (Aziz et al. 2012). A multilocus genome analysis based on 16S rRNA, atpD, gyrB, recA and rpoB gene sequences was carried out using an established procedure (Carro et al. 2012) and a MLSA tree generated from nearly 4000 nt using the ML algorithm. Sequence data from all of these genes for each of the tested strains have been deposited in GenBank with the accession numbers shown in Table S1.

Cultural, morphological and phenotypic properties
The growth and cultural properties of the isolate were recorded from tryptone-yeast extract, yeast extractmalt extract, oatmeal, inorganic salts-starch, glucoseasparagine, peptone-yeast extract-iron and tyrosine agars (ISP media 1-7; Shirling and Gottlieb 1966) and from Bennett's and modified Bennett's agar (Jones 1949), HSA5 agar (Busti et al. 2014), nutrient (Becton Dickinson, USA) and 100-fold diluted nutrient agar (Becton Dickinson, USA) and tap water (Harris 1986) agar after 4 weeks incubation at 28 °C. The colour of aerial and substrate mycelia and diffusible pigments were determined by comparison against NBS/IBCC Colour Charts (Kelly 1958). Hyphal and spore chain features of the isolate were recorded on acidified ISP 2 agar plates (pH 5.5), after 4 weeks at 28 °C, using the coverslip technique of Kawato and Shinobu (1959). Spore arrangement and spore surface ornamentation were established by examining goldcoated dehydrated preparations with growth taken from ISP 2 agar plates (pH 5.5), using the procedure described by O'Donnell et al. (1993) and a scanning electron microscope (Model 1430 V P, LEO Electron Microscopy Ltd, Cambridge, England).
The isolate was also examined for a combination of phenotypic properties. Its ability to grow over a range of pH values (pH 4-13 at single unit intervals), temperatures (4, 10, 15, 20, 25, 30, 35, 40 °C) and in the presence of various NaCl concentrations (1-15 at single unit intervals) were determined using acidified ISP 2 agar (Shirling and Gottlieb 1966) as the basal medium; the pH levels were achieved using KH 2 PO 4 / HCl (pH range 4-5), KH 2 PO 4 /K 2 HPO 4 (pH range 6-8) and K 2 HPO 4 /NaOH (pH range 9-13) buffer systems. Standard biochemical, degradative and physiological properties were examined using media and methods described by Williams et al. (1983), albeit with media adjusted to pH 5.5. All of the tests were carried out, in triplicate, using 12-well plates that were inoculated using a standard inoculum corresponding to 5 on the McFarland scale (Murray et al. 1999) and a multipoint inoculator (Mast Uri®Dot, Mast Group Ltd., Merseyside, UK); the inoculated plates were incubated for 3 weeks at 28 °C (apart from the temperature tests). The enzymic activities of the isolate were determined, in duplicate, using API-ZYM kits (BioMerieux, France), according to the manufacturer's instructions.

Chemotaxonomy
Isolate SF28 T was examined for the presence of chemical markers using standard chromatographic methods with appropriate controls. Thin-layer chromatography (TLC) was used to determine isomers of diaminopimelic acid following Staneck and Roberts (1974) and whole-organism sugars according to Hasegawa et al. (1983). Isoprenoid quinones and polar lipids were extracted from freeze-dried cells, as described by Minnikin et al. (1984) and separated using high performed liquid chromatography (HPLC; Kroppenstedt 1985) and two-dimensional TLC (Minnikin et al. 1984), respectively. Cellular fatty acids were extracted, methylated after Miller (1982) with minor modifications from Kuykendall et al. (1988), analysed using the protocol of the Sherlock Microbial Identification (MIDI) system, version 5 (Sasser 1990) and the resultant peaks identified using the ACTIN1 3.80 database.
Whole genome sequencing and phylogenomic analyses Genomic DNA was extracted from biomass of isolate SF28 T following growth in ISP 2 broth for 7 days at 28 °C using the protocol provided by MicrobesNG (Birmingham, UK; http:// www. micro besng. uk) and sequenced on a MiSeq instrument (Illumina, San Diego, USA). Genomic DNA libraries were prepared at MicrobesNG using Nextera XT library preparation kits. The purity and concentration of the extracted genomic DNA was measured using the Microlab STAR handling system (Hamilton, Birmingham, UK) and libraries generated using Kapa Biosystems library quantification kits designed for Illumina instruments on a LightCycler 96 real time PCR instrument (Roche, West Sussex, UK). The libraries were sequenced following the 2 × 250 bp pairedend protocol (MicrobesNG). Reads were trimmed using Trimmomatic software version 0.30 (Bolger et al. 2014) and their quality assessed using in-house scripts from MicrobesNG; those under 1000 bp were discarded. The resultant reads were assembled into contigs using Spades 3.7 software (Bankevich et al. 2012), annotated with Prokka 1.11 (Seemann 2014) and analysed using the SEED Viewer (Aziz et al. 2012). The genome sequence of isolate SF28 T was deposited in the GenBank database under accession number JAJCXB000000000.
The genome sequences of isolate SF28 T , the type strains of closely related Streptomyces species and Kitasatospora setae DSM 43861 T were uploaded onto the Type (Strain) Genome Server (TYGS; Meier-Kolthoff and Göker 2019) and compared using the MASH algorithm which allows a fast approximation of intergenomic relatedness between strains (Ondov et al. 2016). A phylogenomic tree was inferred with FastME 2.1.4 (Lefort et al. 2015) from GBDP distances calculated from the genome sequences and branch lengths scaled using the GBDP distance formula d 5 (Meier-Kolthoff et al. 2013a); GBDP pseudo-bootstrap support values above branches on the tree were based on 100 replications. The tree was rooted at the midpoint (Farris 1972). Average nucleotide identity (ANI; Rodriguez and Konstantinidis 2016) and digital DNA:DNA hybridization (dDDH; Meier-Kolthoff et al. 2013b) values between genomes of the isolate and its closest phylogenomic neighbours were determined using the online resource from the Rodriguez and Konstantinidis group (http:// enveomics. gatach. edu/) and the GGDC web server (http:// ggdc. dsmz. de/ ggdc), respectively.

Genomic analysis
The presence of BGCs in the genomes of the isolate and its phylogenomic neighbours was investigated using anti-SMASH 6.0 software with "strict" detection criteria and extra features, including Known-ClusterBlast, ClusterBlast, SubClusterBlast, MIBiG cluster comparison, ActiveSiteFinder, RREFinder, Cluster Pfam analyses, Pfam-based GO term annotation and TIGRFam analysis (Blin et al. 2021). The distribution of functional gene classes and the presence of stress response genes in the genome of isolate SF28 T were analyzed using the RAST-SEED webserver at https:// rast. nmpdr. org/ Antimicrobial screening The isolate was tested for its ability to inhibit the growth of Bacillus subtilis PCM 2021, Escherichia coli ATCC 25,922, Klebsiella pneumoniae ATCC 700,603, Micrococcus luteus ATCC 10,240, Pseudomonas aeruginosa ATCC 10,145 and Staphylococcus aureus ATCC 25,923 using a standard agar plug assay (Fiedler 2004). It was grown on ISP 2 agar (Shirling and Gottlieb 1966) for 3 weeks at 28 °C when agar plugs (ø = 5 mm) were taken from the plates and placed in square Petri dishes (Sterilin, UK). Overnight cultures of the reference strains (50 µL) grown at 37 °C were used to seed 25 mL of Luria Bertani broth (LB, Becton Dickinson, USA) to an optical density (OD) of 0.6 prior to diluting them to an OD of 0.0125 with 100 mL of LB broth and the same volume of nutrient agar (Becton Dickinson, USA). The final concentration of bacterial cells in the preparations was 1.5-2 × 10 6 CFU mL −1 . The resultant preparations were thoroughly mixed and poured into the square Petri dishes containing the plugs and incubated for 24 h at 37 °C; inhibition zones around the agar plugs were recorded in millimetres. All of the tests were carried out in triplicate.
A co-culture method described by Świecimska et al. (2021) was used to determine the ability of the isolate to inhibit the growth of fungal and fungallike plant and human pathogens. Briefly, the isolate was streaked as lines across one side of Potato Dextrose Agar (PDA, Becton Dickinson, USA) plates which were incubated for 14 days at 28 °C. The discs (ø = 8 mm) of pathogens grown on PDA Petri plates for 7-14 days were placed on the opposite side of the plates inoculated with the isolate and the preparations incubated for 7 days in the case of Alternaria alternata IOR 1783, Fusarium culmorum and Fusarium oxysporum (isolated from pine roots), Fusarium culmorum D and Fusarium graminearum D (isolated from wheat), Phytophthora plurivora (isolated from the rhizosphere of oak), Rhizoctonia solani (isolated from a pine root) and Sclerotina sclerotiorum IOR 2242, for 14 days for Fusarium poae A and Fusarium tricinctum A (isolated from wheat), Botritis cinerea IOR 1873 (isolated from tomato), Colletotrichum acutatum IOR 2153 (isolated from blueberry), Fusarium culmorum IOR 2333 (isolated from pine), Fusarium oxysporum IOR 342 (isolated from pine), Phytophtora cactorum IOR 1925 (isolated from strawberry), and for 21 days for Fusarium graminearum A and Fusarium oxysporum D (isolated from wheat), Fusarium solani IOR 825 (isolated from parsley), Phytophthora cryptogea IOR 2080 (isolated from Lawson cypress), Phytophthora megasperma IOR 404 (isolated from raspberry) and Phoma lingam IOR 2284 (isolated from rape). The human pathogens, Trichophyton erinacei DSM 25374 T and Trichophyton thuringense DSM 25373 T were incubated for 14 days. All tests were carried out in triplicate at 28 °C; the negative controls were cultures of the pathogens grown under the same incubation conditions. Inhibition (I) of pathogen growth was calculated using the formula: I (%) = (C-T/C) × 100, where C is the diameter of pathogen growth in the control sample and T the diameter of the pathogen growth in each of the co-culture samples.
The isolate grew well on ISP 2, ISP 6, Bennett's and modified Bennett's agar, moderately well on ISP 1 and 3 and nutrient agar but poorly or not at all on the remaining media, as shown in Table S3.

Phylogeny
The almost complete 16S rRNA gene sequence generated for isolate SF28 T (1414 nt) was deposited in Genbank (accession number: OK576049). The isolate formed a branch in a well-supported subclade in the ML/MP tree which included the type strains of 22 Streptomyces species few of which were closely related based on low bootstrap values. Isolate SF28 T was most closely related to the type strains of Streptomyces kronopolitis (Liu et al. 2016), Streptomyces chattanoogensis (Burns and Holtman 1959) and Streptomyces lydicus (De Boer et al. 1956) showing 16S rRNA gene sequence similarities with them of 99.3% (10 nt differences), 99.2% (12 nt differences) and 99.2% (12 nt differences), respectively (Fig. S3). The corresponding sequences between the isolate and the type strains of the remaining Streptomyces species ranged from 98.0 (28 nt differences) to 98.9% (15 nt differences; Table S4). The isolate and the S. kronopolitis strain formed a wellsupported branch in the neighbour-joining tree that was loosely associated with the other Streptomyces strains (Fig. S4).
Multilocus sequence analyses of single copies of conserved housekeeping genes provide greater resolution between closely related streptomycetes than corresponding 16S rRNA gene sequence studies as they are based on comparisons of many more nucleotide sequences (Labeda et al. 2012. In the present study, the isolate was assigned to a subcluster that was supported by a 64% bootstrap value (Fig. 1). This taxon encompassed the type strains of 10 Streptomyces species, eight of which featured in the subclade defined in the 16S rRNA gene tree. Isolate SF28 T was most closely related to the type strains of S. chattanoogensis, Streptomyces inhibens (Jin et al. 2019), S. kronopolitis and S. lydicus sharing nucleotide sequence similarities with them of 96.2%, 96.0%, 95.9% and 95.8% (Table S5). The MLSA evolutionary distances between the isolate and the type strains of the most closely related Streptomyces species ranged from 0.039 to 0.124 (Table S6), values well above the species threshold of 0.007 used to distinguish between closely related strains, including streptomycetes (Rong and Huang 2014). Based on these data isolate SF28 T was not closely related to the S. chattanoogensis, S. inhibens and S. kronopolitis strains as it showed evolutionary distance values with them of 0.039, 0.040 and 0.042, respectively. A corresponding distance score of 0.43 was recorded between the isolate and the type strains of Streptomyces celluloflavus (Nishimura and Kimura 1953;Madhaiyan et al. 2020), and S. lyidicus.
Greater confidence can be placed in the topology of phylogenomic trees than in corresponding 16S rRNA and MLSA trees as they are based on millions not thousands of unit characters . It is evident from the phylogenomic tree (Fig. 2) that the sequence generated for isolate SF28 T and corresponding data available on the type strains of its phylogenetic relatives showed that the isolate formed a singleton in a well-supported subclade which included the type strains of 14 Streptomyces species, including those representing S. chattanoogensis, S. kronopolitis and S. lyidicus.

Comparison of genomes
The draft genome sequence of isolate SF28 T has been deposited in GenBank (accession number JAJCXB000000000). It is composed of 133 contigs, has 6594 protein coding genes, 74 RNA genes and L50 and N50 scores of 21 and 117,612, respectively. The total genome size was found to be 7.85 Mbp and the digital (d) G + C content 71.5%. The major classes of functional gene clusters in the genome of the isolate were associated with amino acids and derivatives (441), carbohydrates (297) and protein (237) metabolism, as shown in Figure S5. In general, the genome of the isolate was of a similar size to those of its evolutionary neighbours, as exemplified by S. chattanoogensis NRRL ISP-5002 T  The dDDH relatedness values between the isolate and its evolutionary relatives fell within the range 21.7 to 28.7% (Table S7), which is well below the 70% threshold for the assignment of bacterial strains to the same species (Wayne et al. 1987). It can also be seen from this Table that the corresponding ANI similarities ranged from 80.5 to 85.1%, similarities  Lee et al. 2016). These results indicated that isolate SF28 T represents a new Streptomyces species which is only loosely associated with its evolutionary relatives. It seems likely that the position of the isolate and its closest phylogenetic/phylogenenomic relatives will only be settled by the addition of new species to this unstable part of Streptomyces gene trees. Other Streptomyces species found to form distinct lineages in Streptomyces gene trees include Streptomyces adelaidensis (Kaewkla et al. 2021), Streptomyces leeuwenhoekii (Busarakam et al. 2014) and Streptomyces tardus (Králová et al. 2021).

Phenotypic properties
Closely related species can usually be distinguished using a broad-range of phenotypic properties (Komaki and Tamura, 2019;Kusuma et al. 2021). Cultural and morphological properties have been shown to be particularly predictive in this respect, as exemplified in extensive phylogenetic analyses of streptomyces species (Labeda et al. 2012. In the present study, isolate SF28 T was examined for cultural and key morphological features, and for its ability to metabolize a broad range of carbon and nitrogen sources, enzymes and growth characteristics. It is particularly encouraging that identical results were recorded for the duplicated and triplicated cultures. Comparison of some of these properties with corresponding data acquired for S. decoyicus NRRL 2666 T , its closest phylogenomic neighbour based on dDDH similarity, showed that the strains can be distinguished readily using cultural and morphological properties, as well as by associated phenotypic properties that were also recorded using media and methods described by Williams et al. (1983). It is significant that these strains showed different properties when grown on oatmeal agar and exhibit markedly different spore chain morphologies (Table 1). In addition, only the isolate grew at 10 °C and at pH 4.0, and 5.0; it also showed more activity than S. decoyicus NRRL 2666 T when grown on the sole carbon sources.

Fig. 2 Phylogenomic tree showing relationships between isolate SF28 T and the type strains of the most closely related
Streptomyces species constructed using the TYGS server. The numbers above the branches are GBDP pseudo-bootstrap sup-port values greater than 60% from 100 replications with an average branch support of 95.4% The tree was rooted at the midpoint (Farris 1972) The isolate was distinguished from the type strains of related Streptomyces species listed in Table S7 as they showed different cultural features on oatmeal agar and formed spiral chains of spores, albeit ones with smooth surfaces (Kämpfer, 2012;Liu et al. 2016;Komaki and Tamura 2019;Madhaiyan et al. 2020). It was also be separated from the S. chattanoogensis, S. hygroscopicus subsp. glebosus, S. inhibens and S. piniterrae strains as these strains formed spiny, ridged or wrinkled spores in spiral chains (Kämpfer 2012;Jin et al. 2019;Zhuang et al. 2020).

Antimicrobial activity
Good congruence was found between antimicrobial screens that were either duplicated or triplicated. Isolate SF28 T formed zones of inhibition against the B. subtilis (9.7 ± 1.2 mm), E. coli (3.0 ± 1.0 mm), K. pneumoniae (2.0 ± 0.0 mm), M. luteus (3.0 ± 0.1 mm), P. aeruginosa (3.0 ± 0.1 mm) and S. aureus (6.2 ± 0.6 mm) strains in the agar plug assays (Fig. S6). Similar results have been recorded by members of novel Streptomyces species isolated from natural habitats, including extreme hyper-arid Atacama Desert soils (Sharma et al. 2014;Goodfellow et al. 2017;Abdelkader et al. 2018;Le Roes-Hill et al. 2018). Similarly, the isolate inhibited the growth of diverse fungal pathogens, including representatives of several Fusarium and Phythophthora species, as shown in Table 2 and Figure S7. These results provided further evidence that novel Streptomyces species from different habitats (Sharma et al. 2014;le Roes Hill et al. 2018), including extreme hyperand Atacama Desert soils (Goodfellow et al. 2017;Abdelkoder et al. 2018) produce new natural products, especially antifungal antibiotics that inhibit the growth of phytopathogens (Zhao et al. 2017;Singh and Dubey 2018;Qi et al. 2019;Peng et al. 2021) Kumar and Goodfellow (2010). DNA G + C content and genome size are from GenBank. Both strains formed spores with smooth surfaces, degraded adenine, gelatin, casein, hypoxanthine, L-tyrosine and uric acid, used D-arabitol, D-cellobiose, dextrin, D-fructose, D-galactose, glycogen, D-glucose, glycerol, myo-inositol, D-maltose, D-melezitose, D-ribose, sucrose and D-trehalose as sole carbon sources, and L-alanine, L-glutamic acid, L-histidine, L-isoleucine, DL-methionine, L-phenylalanine, L-threonine and L-valine as sole nitrogen sources, and grew at pH 9 and 10 and 30 °C. Neither of the strains hydrolysed allantoin, degraded starch or used L-arabinose as a sole carbon source and which thereby show promise as biocontrol agents (Cao et al. 2020). It is also interesting that the isolate showed pronounced activity against representatives of the two Trichophyton species (Table 2, Fig. S7).

Genome mining
AntiSMASH predicts BGCs and potential products based on the percentage of genes from the closest known bioclusters showing significant BLAST hits against corresponding clusters in the genomes of strains under consideration (Blin et al. 2021).
The genome of isolate SF28 T was found to contain 29 BGCs, notably ones predicted to encode for druggable molecules such as non-ribosomal peptide synthetases (Table 3) which may be involved in the results of the antimicrobial screening studies considered above. Eleven of the bioclusters showed at least 50% gene identity with known compounds, as exemplified by those associated with the synthesis of anantin C (75% gene identity), a peptide antagonist of the atrial natriuretic factor (Tietz et al. 2017), desferrioxamine E (100% gene identity), a siderophore which forms stable hexadentate complexes with ferric ions (Barona-Gómez et al. 2004), ectoine (100% gene identity), which protects against osmotic stress and desiccation (Prabhu et al. 2004), ethylenediaminesuccinic acid hydroxyarginine (EDHA) (100% gene identity), a second line siderophore (Spohn et al. 2018), and lugdunomycin (74% gene identity), a novel aromatic polyketide with antibacterial activity (Wu et al. 2019). Similarly, several bioclusters were predicted to encode for a range of products, as exemplified by those involved in the synthesis of alkylresorcinol (100% gene identity) which has multiple biological functions (Funabashi et al. 2008;Nakano et al. 2012), a heat-stable antifungal factor (75% gene identity) that inhibits the growth of diverse fungi (Yu et al. 2007) and the tallysomycins (TLMs) (60% gene identity), which are antitumor antibiotics (Tao et al. 2007). Other bioclusters found in the genomes of the isolate were associated with the synthesis of 3,7-dihydroxytropolone (33% gene identity) which shows antimicrobial, anticancer and antiviral activity (Chen et al. 2018), lankacidin C (26% gene identity) that is known to have antibacterial and antitumor properties (Ayoub et al. 2019) and stenothricin (13% gene identity), a cyclic depsipeptide that exhibits antibacterial activity (Liu et al. 2014). Other BGCs encoded for antibacterial compounds such as dutomycin and lactonamycin showed 4 and 3% gene identity, respectively, with known compounds (Matsumoto et al. 1999;Sun et al. 2016). Moreover, the genome of isolate SF 28 T included 7 bioclusters that were predicted to encode for unknown compounds. The SEED analyses (Aziz et al. 2012) showed that the genome of isolate SF28 T contained 61 putative stress related genes, notably those linked to cold and heat shock responses, and DNA repair and oxidative stress (Table S8, Fig. S5). This complement of genes included cspC and cspE which express for cold shock proteins (Etchegaray and Inouye 1999) and chaperone genes such as clpB, clpC, clpX and hrcA that are involved in responses to heat shock (Li et al. 2011). In turn, genes such as betA, betB and proU are involved in the uptake of betaine and choline, metabolites, Table 2 Antimicrobial activity of isolate SF28 T against fungal and fungal-like organisms evaluated using the co-cultured method I; % inhibition of fungal growth that was calculated using the formula: I (%) = (C-T/C) × 100, where C is the diameter of pathogen growth in the control sample and T the diameter of the pathogen growth in each of the co-culture samples  which have a role in responses to osmotic stress (Boncompagni et al. 1999;Nau-Wagner et al. 2012), as are enzymes expressed by genes katE and soxR (Normand et al. 2012;Golińska et al. 2020), and the products of genes trx and trxR (Kim et al. 2008). The genome of the isolate also included genes such as RecF, RecO and RecR, UvrD which are associated with DNA repair and stabilizing (Hickson 2003;Kang and Blaser 2006). The detection of a CoxG gene, which encodes for a subunit of carbon monoxide dehydrogenase suggests that the isolate may be able to adapt to a chemolitotrophic lifestyle by using carbon monoxide as a carbon and energy source (Lorite et al. 2000), as is the case with the type strains of Streptomyces thermocarboxydovorans and Streptomyces thermocarboxydus (Kim et al. 1998). The genome of the isolate is also rich in genes that express for DNA polymerase Sigma factors, as shown in Table S8. Some of these genes, such as sigB, which is involved in osmotic stress, are upregulated under acidic conditions (Kim et al. 2008). These authors also showed that this applies to heat shock genes, including those found in the genome of the isolate, notably ones supressing proteins belonging to the DnaK family and chaperones, such as GroEL2. It is also interesting that the genome of the isolate contained gene atpA, which is involved in a transmembrane protein transport system and is known to enhance survival of bacteria under acidic conditions (Guan and Liu 2020). These results provide further evidence that pH is a major factor governing the survival and distribution of streptomycetes in acidic soils (Williams et al. 1971;.

Conclusions
Isolate SF28 T showed antimicrobial activity, notably against fungal pathogens, has a large genome rich in BGCs predicted to encode for a broad range of specialised metabolites, especially putatively new antibiotics, and stress related genes, notably ones associated with adaptation to acidic conditions. It is also evident from the sequence data that isolate SF28 T forms a distinct lineage within the evolutionary radiation occupied by Streptomyces species. It is only loosely associated with its closest phylogenetic/ phylogenomic neighbours, a point underlined by corresponding low ANI and dDDH similarities. It can also be distinguished from these organisms using key cultural and micromorphological properties. Consequently, the isolate is considered to represents a novel Streptomyces species for which the name Streptomyces pinistramenti sp. nov. is proposed.
Description of Streptomyces pinistramenti sp. nov.
The type strain SF28 T (= DSM 113360 T = PCM 3163 T ) was isolated from partially decomposed needles under Pinus sylvestris trees growing on the southern slope of an inland sand dune in the Toruń Basin, Poland.
Novel streptomycetes isolated from extreme habitats are a rich source of new specialised metabolites, including antibiotics Sivalingam et al. 2019;Sivakala et al. 2021). It is, therefore, surprising that streptomycetes known to be common in coniferous forest soils (Golińska et al. 2022) have received little attention, especially since they have been shown to be antagonistic to fungal pathogens (Golińska and Dahm 2013;Cao et al. 2020) and can form mutualistic associations with the pine beetle, Dendroctonus frontalis (Strzelczyk and Szpotański 1989;Scott et al. 2008). It is also interesting that S. piniterrae jys28 T , an isolate from the rhizosphere soil of Pinus yunnanensis, contains a putative gene cluster that encodes for the synthesis of heliquinomycins which belong to the rubromycin family of compounds (Zhuang et al. 2020). Consequently, the discovery that S. pinistramenti SF28 T shows pronounced activity against diverse fungal plant pathogens provides further evidence that novel Streptomyces species isolated from coniferous forest soils merit greater attention as a source of new bioactive metabolites. It is also interesting that S. pinistramenti SF28 T and S. piniterrae jys28 T are associated with strains that synthesise novel antibiotics, as exemplified by S. decoyicus NRRL ISP-5087 T which produces psicofuranine, a purine nucleoside antibiotic that shows antibacterial and antitumor activity (Eble et al. 1959;Vavra et al 1959).