Strain HUAS 11-8^T was isolated from the rhizosphere soil of Cynara scolymus, which was collected in Changde city of Hunan Province, China (29.20201^° N 111.98113^° E), as described by Mo et al. (2017). The purified strain HUAS 11-8^T was prepared for short-term preservation on Reasonerʼ2A (Reasoner and Geldreich, 1985) slopes at 4°C and suspended in a sterile 30% (w/v) glycerol solution for long-term conservation at −80°C. The type strain Amycolatopsis rhizosphaerae JCM 32589^T was purchased from the Japan Collection of Microorganisms (JCM). Strains HUAS 11-8^T and A. rhizosphaerae JCM 32589^T were tested under the same conditions.

The genome sequencing of strain HUAS 11-8^T was completed by using a Nanopore PromethION sequencing system at Wuhan Benagen Technology Co., Ltd. (Hubei, China). Genomic DNA extraction and PCR conditions of the 16S rRNA gene were carried out using the method described by Mo et al. (2023). The 16S rRNA gene of strain HUAS 11-8^T was amplified using universal primers (27F and 1492R) (Lane, 1991). The 16S rRNA gene sequence of strain HUAS 11-8^T was compared with the EzBioCloud database² (Yoon et al., 2017). Closely related reference strains were downloaded and used for constructing phylogenetic trees using neighbor-joining (NJ) (Saitou and Nei, 1987), maximum-likelihood (ML) (Felsenstein, 1981), and maximum-parsimony (MP) (Kluge and Farris, 1969) with 1,000 bootstrap replications in MEGA 11 (Tamura et al., 2021). According to the result of 16S rRNA gene sequence analysis, the genome sequences of type strains that were closely related to strain HUAS 11-8^T were selected for reconstructing the phylogenomic tree using the Type (Strain) Genome Server. The ANIm and dDDH values between the genomes of strain HUAS 11-8^T and its relatives were calculated according to the aforementioned description. The gene prediction analysis and functional annotation of the genome of strain HUAS 11-8^T were performed by the NCBI Prokaryotic Genome Annotation Pipeline v4.4 and Rapid Annotation using Subsystem Technology v.2.0 (RAST³) (Aziz et al., 2008). The secondary metabolism biosynthetic gene clusters of strain HUAS 11-8^T and antibiotic resistance genes were analyzed using antiSMASH version 6.0.1⁴ and the Comprehensive Antibiotic Resistance Database (CARD⁵), respectively (Blin et al., 2011; Alcock et al., 2020). Clustered regularly interspaced short palindromic repeat sequences (CRISPRs) of strain HUAS 11-8^T were identified by CRISPR-case Finder⁶ (Makarova et al., 2019), and then the genomic islands were predicted by the Island Viewer 4 webserver (Claire et al., 2017).⁷

Spore features of HUAS 11-8^T were observed by a light microscope (NE620, Ningbo Yongxin Optics) and scanning electron microscope (FEI-Quanta 450, America), using cultures grown on Reasonerʼ 2A after incubation for 21 days at 28°C. The cultural features of strains HUAS 11-8^T and A. rhizosphaerae JCM 32589^T were observed on various media, including Gauseʼs synthetic No. 1 medium (Atlas, 1993), Reasonerʼ 2A and ISP 2–7 media (Shirling and Gottlieb, 1966) for 21 days at 28°C. Color determinations, such as the aerial mycelium, the substrate mycelium, and diffusible soluble pigments, were delineated according to the methods of Shirling and Gottlieb (1966). Growth was carried out on tryptic soy broth (TSB) medium for 14 days at temperatures (4, 10, 15, 20, 22, 25, 27, 30, 35, 37, 40, 45, 50, and 55°C), pH (2.0–12.0, at intervals of 1.0 pH unit), and concentrations of NaCl (0–15%, w/v, at an interval of 1% w/v). The following tests, i.e., carbon and nitrogen source utilization, starch hydrolysis, gelatin liquefaction, nitrate reduction, and Tweens (20, 40, 60, and 80) degradation, were performed according to the methods described by Xu et al. (2007).

Biomass for chemotaxonomic analysis was collected by centrifugation after culturing for 5–7 days at 28°C in TSB in shake flasks. The cellular fatty acids of strains HUAS 11-8^T and A. rhizosphaerae JCM 32589^T were detected as described by Sherlock MIDI protocol (Sherlock Microbial Identification System, version 6.0B) (MIDI, 2005), which were carried out by the Marine Culture Collection of China (MCCC). The diaminopimelic acid isomers in the cell wall peptidoglycan were separated by thin-layer chromatography and analyzed using a solution of ninhydrin in acetone (Hasegawa et al., 1983). The whole-cell sugars were analyzed as described by Lechevalier and Lechevalier (1970). Polar lipids and menaquinones were extracted and analyzed using the method described by Ruan and Huang (2011).

The genome sequence size of strain HUAS 11-8^T is 7,474,574 bp with a DNA G + C content of 70.3%. A total of 7,267 genes (7,092 coding genes, 74 RNA genes, and 101 pseudogenes) and 7,193 CDSs (7,092 CDSs with protein and 101 CDSs without protein) were predicted. The analysis of the genome of strain HUAS 11-8^T by the RAST Server revealed 316 subsystems that could be classified into 23 categories, and the subsystem coverage was 19%. The represented subsystem features identified were “Amino Acids and Derivatives” (378 CDSs), “Carbohydrates” (368 CDSs), “Fatty Acids, Lipids, and Isoprenoids” (214 CDSs), “Cofactors, Vitamins, Prosthetic Groups, Pigments” (213 CDSs), “Protein Metabolism” (173 CDSs), “DNA Metabolism” (121 CDSs), “Respiration” (109 CDSs), “Nucleosides and Nucleotides” (92 CDSs), “Metabolism of Aromatic Compounds” (91 CDSs), “Virulence, Disease and Defense” (63 CDSs), “Miscellaneous” (51 CDSs), “RNA Metabolism” (51 CDSs), “Stress Response” (44 CDSs), “Cell Wall and Capsule” (39 CDSs), “Membrane Transport” (35 CDSs), “Nitrogen Metabolism” (35 CDSs), “Phosphorus Metabolism” (32 CDSs), “Regulation and Cell signaling” (22 CDSs), “Sulfur Metabolism” (11 CDSs), “Iron acquisition and metabolism” (8 CDSs), “Dormancy and Sporulation” (7 CDSs), “Potassium metabolism” (6 CDSs), and “Secondary Metabolism” (3 CDSs). RAST revealed that strain HUAS 11-8^T comprised lots of putative genes known to be associated with the abilities of dealing with harsh environmental conditions found in plant-associated environments, such as osmotic stress, oxidative stress, detoxification, stress response-no subcategory (SigmaB stress response regulation, dimethylarginine metabolism, and bacterial hemoglobins), and periplasmic stress. Three genes (Streptomyces venezuelae rox, vanR gene in vanO cluster, and Mycobacterium tuberculosis folC with mutation conferring resistance to para-aminosalicylic acid) of strain HUAS 11-8^T related to antibiotic resistance were recognized by CRAD analysis, which might confer resistance to rifamycin antibiotic, glycopeptide antibiotic, and salicylic acid antibiotic. The potential secondary metabolite biosynthetic gene clusters in strain HUAS 11-8^T were analyzed by antiSMASH, and 16 gene clusters were annotated. The three main biosynthetic gene clusters were aryl polyene, non-ribosomal peptide synthetase (NRPS), and type I polyketide synthase (PKS) [T1PKS]. The two T1PKS gene clusters had 96 and 8% similarities to macrotermycins and A54145, respectively. Meanwhile, nine CRISPR repeats of strain HUAS 11-8^T were identified in the genome. In total, 10 genetic islands with a size range from 4,724 to 30,712 bp were identified in the genome of strain HUAS 11-8^T.

Based on EzBioCloud perform alignment, it was found that strain HUAS11-8^T had 16S rRNA gene similarities of 99.78% with A. rhizosphaerae JCM 32589^T, 97.8% with A. dongchuanensis YIM 75904^T, and < 97.8% sequence similarities to other Amycolatopsis species. An ML phylogenetic tree based on 16S rRNA gene sequence demonstrated that strain HUAS 11-8^T was most closely related to A. rhizosphaerae JCM 32589^T (Figure 2). The relationship between HUAS 11-8^T and A. rhizosphaerae JCM 32589^T also appeared in the NJ and MP trees (Supplementary Figures S2, S3). This topological structure was further supported by the results of phylogenomic analysis (Figure 3). Nevertheless, the ANIm and dDDH values between strains HUAS 11-8^T and A. rhizosphaerae JCM 32589^T were 96.3 and 68.5%, respectively, lower than the 96.6 and 70% thresholds recommended for the delineation of a novel Amycolatopsis species. Furthermore, this result was further confirmed by phenotypic and chemotaxonomic differences between strains HUAS 11-8^T and A. rhizosphaerae JCM 32589^T (Table 1). For example, the spore chains of strain HUAS11-8^T are branched, and the spores are oval, spherical, and short-rod. While A. rhizosphaerae JCM 32589^T produces long spore chains and spherical spores. In addition, the dominant menaquinones are MK-9 (H₄), MK-9 (H₂), and MK-9 (H₆). Galactose, ribose, and xylose were detected as the whole-cell reducing sugars in strain HUAS11-8^T. The dominant menaquinone of strain A. rhizosphaerae JCM 32589^T is MK-9 (H₄, ₆, ₈) and MK-10 (H₂, ₆). Galactose and arabinose were detected as the whole-cell reducing sugars in strain A. rhizosphaerae JCM 32589^T.

Morphologically, strain HUAS 11-8^T produced white substrate hyphae and aerial mycelia that differentiated into spore chains on Reasonerʼ2A medium after incubation for 21 days at 28°C. Spore chains were branched, and spore surfaces were smoothed (Figure 4). This strain was observed to grow well on Gauseʼs synthetic No. 1 medium, Reasonerʼ2A, and ISP 2–7 serial media (Supplementary Table S3). Growth was observed at 20–35°C (optimum, 30°C), at pH 6.0–9.0 (optimum, pH 7.0), and in the presence of 0–3% of NaCl (optimum, 0–1%). Detailed physiological and biochemical characteristics are provided in the species description. The dominant menaquinones were MK-9(H₄) (75.2%), MK-9(H₂) (20.6%), and MK-9(H₆) (2.1%). The strain was found to contain meso-diaminopimelic acid as the cell wall amino acid. Galactose, ribose, and xylose were detected as whole-cell reducing sugars. The major fatty acids (≥10%) were iso-C_16:0 (30.5%), C_16:0 (10.8%), and C_17:1 ω6c (10.0%). The detailed fatty acid composition is shown in Supplementary Table S4. The polar lipid pattern consisted of diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylinositol (Supplementary Figure S4). All these morphological and chemotaxonomic data were consistent with the assignment of strain HUAS 11-8^T to the genus Amycolatopsis.

In conclusion, based on phenotypic, chemotaxonomic, and genotypic data, strain HUAS 11-8^T could be distinguished from A. rhizosphaerae JCM 32589^T. Therefore, it is concluded that strain HUAS 11-8^T represents a novel species of the genus Amycolatopsis, for which the name Amycolatopsis cynarae sp. nov. is proposed.