A bare soil patch left by coal mining has yielded a candidate for a completely new Streptomyces species—with unusual chemistry and thousands of predicted plant-growth-promoting genes. Bare soils developing after open cast mining are one of the unexplored ecosystems of anthropogenic origin.

These soils provide a unique opportunity to study ecological succession and microbial colonization in environments drastically altered by human activity. In the Sokolov brown coal mining area, Miocene clays were brought to the surface and then used for reclamation or left for primary succession.

Those specialized clays harbor taxonomically distinct and diverse microorganisms and can serve as reservoirs for unexplored microbial diversity. Strain BCCO 10 0861T was initially isolated for bioprospecting purposes, and its aerial mycelium coloration and soluble pigment production distinguished it from other isolates.

Whole-genome sequencing and comprehensive comparative genomic analyses then confirmed its distinct phylogenetic position. The strain was characterized against four closest phylogenetic relatives: Streptomyces geranii, Streptomyces lacrimifluminis, Streptomyces turgidiscabies, and Streptomyces reticuliscabiei.

Based on robust polyphasic evidence, the study proposes a novel species represented by this single isolate. The strain came from bare soil on Miocene lacustrine clay at the top of reclaimed mine heaps in the early successional stage of the Sokolov brown coal district.

The soil had a pH of 7.8, and the strain was isolated on Reasoner’s 2A agar from clay deposited after brown coal mining in 2008. Cultures were maintained on yeast extract–malt extract agar and as cell suspensions in fifteen percent glycerol stocks at minus seventy-six degrees Celsius for long-term preservation.

For identification, genomic DNA was extracted from a three-day-old culture, and the almost complete 16S rRNA gene was amplified by PCR, sequenced, assembled, and compared with type species in EzBioCloud. The closest sequence similarity was ninety-nine percent to Streptomyces turgidiscabies, with lower similarities to the other listed strains.

The evolutionary history of BCCO 10 0861T was constructed using multiple tree-building algorithms to ensure topological robustness. The sequence alignments were aligned with CLUSTALW on MEGA eleven, and a Maximum-Likelihood tree was inferred. The Maximum-Likelihood topology and the corresponding neighbor-joining tree revealed a highly stable, distinct monophyletic lineage for BCCO 10 0861T.

Both algorithms independently confirmed Streptomyces geranii, Streptomyces lacrimifluminis, Streptomyces turgidiscabies, and Streptomyces reticuliscabiei as the closest type strains. Node stability was supported by bootstrap values from seventy-one to ninety-nine percent, with critical nodes shared across both analyses marked by an asterisk.

In panel B, the annotation columns to the right of the genome tree provide a second layer of evidence: species-cluster labels, genome size, G+C content, and protein count are displayed for each strain. BCCO 10 0861T appears with a genome size of about nine megabases, roughly seventy percent G+C, and nearly twelve thousand protein-coding genes, alongside closely related strains with visibly similar profiles.

The BCCO 10 0861T genome contained eleven thousand eight hundred twenty-four protein-encoding genes and had a G plus C content of seventy point zero five percent. To determine its genomic taxonomic position, the whole-genome assembly was compared with eighteen Streptomyces type strains using the Type Strain Genome Server.

The comparison used Thermobispora bispora as an outgroup and reference genome sequences from the NCBI genome database and TYGS genome databases. The Genome BLAST Distance Phylogeny method confirmed the same four closest organisms: Streptomyces lacrimifluminis, Streptomyces reticuliscabiei, Streptomyces turgidiscabies, and Streptomyces geranii.

Looking across Table One, the BCCO ten-oh-eight-six-one-T column has eleven contigs, while the neighboring genomes range from one hundred four to nine hundred eighty-six. Its contig N-fifty is one hundred eighty-two point seven kilobases, and the table also lists four thousand eight hundred eleven plant-growth-promoting-trait genes.

Genome-wide comparisons using TYGS and JSpeciesWS confirmed that BCCO 10 0861T is distinct from its closest phylogenetic neighbors. Its digital DNA-DNA hybridization values ranged from thirty-four point seven to thirty-seven point five percent, significantly below the seventy-percent threshold for species delimitation.

Average Nucleotide Identity values ranged from eighty-six point two six to eighty-seven point two four percent, below the ninety-five to ninety-six percent species boundary. Together, these results support classifying BCCO 10 0861T as a novel species within the genus Streptomyces.

Cultural characteristics were observed after growth at twenty-eight degrees Celsius for two weeks on several ISP media and nutrient agar. The strain developed a white aerial spore mass well on ISP2 and ISP4, and moderately on ISP3. Its vegetative mycelium was pink-orange-brown, and it produced a soluble pink-orange pigment on ISP2, ISP3, ISP4, and ISP7.

Cell morphology was examined by scanning electron microscopy and transmission electron microscopy after twenty-one days of incubation at twenty-eight degrees Celsius on ISP2. The strain developed rectus flexibilis sporophores, with spores averaging one point four six four micrometers by zero point six eight two micrometers.

In panel A, the SEM view shows a dense network of elongated, textured hyphae, with a distinct curved sporophore emerging among them. Panel B resolves chains of individual spores along branching filaments, while panel C shows the corresponding cream-white colony with several smaller, pigmented colonies at the plate’s edge.

BCCO 10 0861T exhibited a mesophilic growth range from four to thirty degrees Celsius, neutrophilic pH tolerance from six to eight, and moderate halotolerance from zero to two percent sodium chloride. To maintain stability across pH four point zero to twelve point zero, the study implemented a multi-buffer system.

Citrate-phosphate, phosphate, and carbonate-bicarbonate solutions were used across the pH ranges, with pH measured at intervals of one and temperatures tested from four to fifty-five degrees Celsius. Sodium chloride tolerance was examined from zero to ten percent in intervals of one using ISP2 medium.

Some biosynthetic gene clusters usually found in the majority of streptomycetes were absent from BCCO 10 0861T, including desferrioxamine B and E, diisonitrile antibiotic SF2768, and epsilon-poly-L-lysine clusters. Humidimycin, inthomycin B, triacsin C, and EDHA biosynthetic gene clusters were present only in the genome of BCCO 10 0861T.

Phenazines and scabichelin are specialized metabolites that can act as biocontrol agents or virulence factors, with positive or negative effects on plants. In the strain rows, the colored blocks show that the core-metabolome pattern is broadly shared, while the specific-metabolite columns contain the clearest row-to-row differences.

The right-hand summary also varies across strains, with the most pronounced contrast appearing in the polyketide category, where the displayed counts range from three to fifteen. The genome of BCCO 10 0861T contained a biosynthetic gene cluster for putative virulence factors, but only their concomitant and synergistic action may be associated with plant virulence and pathogenicity.

The complete list of biosynthetic gene clusters identified in all investigated strains is provided in Supplementary Table five. Pan-genome analysis found that BCCO 10 0861T possessed the highest number of orthologous genes, with ten thousand eight hundred twenty-seven according to the Database of Clusters of Orthologous Genes.

The compared strains shared four thousand one hundred ninety identical genes, while BCCO 10 0861T had four thousand two hundred eighty-seven unique genes. For the inhibition assay, Streptomyces strains were inoculated on Mueller-Hinton agar and cultivated at twenty-eight degrees Celsius, while overnight indicator cultures were added in a T-shape and co-cultivated until the next day.

BCCO 10 0861T showed a different pattern of inhibitory activities compared with the other species. It weakly inhibited Bacillus subtilis and Moraxella catarrhalis, inhibited Candida albicans, and did not affect Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, or Pseudomonas aeruginosa.

Plant growth-promoting traits were detected with PlaBA-db. BCCO 10 0861T had the most abundant plant-growth-promoting-trait gene count, at four thousand eight hundred eleven. The compared genomes collectively shared eight hundred eighty-five identical plant-growth-promoting-trait genes, while BCCO 10 0861T contained two thousand two hundred fifty-five distinct genes.

BCCO 10 0861T did not produce siderophores in the assay, despite genomic predictions suggesting the potential for scabichelin-type siderophore production, which was not experimentally validated. In contrast, the strain demonstrated the ability to produce soluble proteases, IAA, and solubilized phosphates from tricalcium phosphate.

Notably, BCCO 10 0861T was the only strain that produced hydrocyanic acid. The proposed strain is a Gram-positive bacterium with pink to brown substrate mycelium and dense white to gray aerial mycelium on several ISP media.

It produces pink-orange soluble pigment, does not produce melanin on ISP6 or ISP7, and has rectus flexibilis sporophores with rod-shaped spores. It grows from four to thirty degrees Celsius, with optimal growth at twenty-eight degrees Celsius, and grows at pH six to eight.

The strain can easily utilize glycerol, D-mannose, and D-xylose, but cannot utilize cellulose, D-fructose, or D-ribose. The genome of BCCO 10 0861T showed the highest number of unique plant-growth-promoting-trait genes, at two thousand two hundred fifty-five.

No association with potato pathogenicity was predicted in its genome. Digital DNA-DNA hybridization values from thirty-four point seven to thirty-seven point five percent, Average Nucleotide Identity values from eighty-six point two six to eighty-seven point two four percent, and phenotype distinguish the strain from closely related strains.

Based on the polyphasic approach and genomic content, the strain represents a novel Streptomyces species named Streptomyces chodovensis. Streptomyces chodovensis is supported as a novel species by genomic, phylogenetic, chemical, and phenotypic evidence, while its plant-associated traits remain promising predictions that still need experimental validation.