Plant and soil samples were collected on June 20, 2023, from a healthy, continuously cropped Lavender field in Huocheng County, Xinjiang, China (80°40′44″ E, 43°39′08″ N, altitude ~780 m). The weather was overcast during sampling, with an air temperature of 20-31°C (data sourced from the China Meteorological Administration). LLV and LAM were selected as the study subjects. 5 sampling points were established for each variety. At each point, 3 plants with uniform and vigorous growth were selected. Root, stem, and leaf tissues were sampled concurrently. For rhizosphere soil collection, roots at approximately 30 cm below the soil surface were carefully excavated, and the soil adhering to the root surface was gently brushed off using a sterile brush. The rhizosphere soil samples collected from multiple sampling points for each lavender variety were thoroughly mixed to form a composite sample, which was then evenly divided into three replicates. One for metagenomic sequencing, one for soil physicochemical property analysis, and one for microbial isolation. All samples were immediately placed on ice and transported to the laboratory. Soil aliquots designated for DNA extraction were stored at -80°C, while those intended for physicochemical analysis and microbial isolation were air-dried and stored for subsequent use. Following this procedure, a total of 6 rhizosphere soil samples were obtained-3 for LLV and 3 for LAM.

Rhizosphere soil samples were sent to the Eco-Environment Analysis and Testing Center of the Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, for the determination of the following indicators: total salt content, pH, organic matter, total nitrogen, available nitrogen, and available phosphorus.

Actinobacteria were isolated using a modified Gauze’s No. 1 medium. Soil samples were subjected to a 10-fold serial dilution, spread onto the agar plates, and incubated at 37c°C for 7–14 days. Distinct colonies were selected and repeatedly streaked for purification. Preliminary identification was based on colony morphology, Gram staining, and spore structure.

Genomic DNA was extracted using the Chelex-100 method (reagent purchased from Bio-Rad, USA). The nearly full-length 16S rRNA gene was amplified with universal primers 27F and 1492R. The PCR products were sequenced by Sangon Biotech (Shanghai, China). The resulting sequences were compared against the EzBioCloud database for taxonomic assignment, and a phylogenetic tree was constructed using MEGA software (version 12.0).

Note: Throughout this manuscript, the term “actinobacteria” refers to the bacterial isolates that were preliminarily selected based on colony and mycelial morphology characteristic of filamentous actinomycetes, and taxonomically confirmed by 16S rRNA gene sequencing to belong to the phylum Actinobacteria (synonym Actinomycetota).

Salt and Alkali Tolerance: Bacterial growth was assessed across media containing 2% to 25% (w/v) NaCl and adjusted to pH levels ranging from 5 to 12 to evaluate their salt and alkali tolerance, respectively.

Nitrogenase (NITS) Activity: NITS activity was measured using an ELISA kit (Jiangsu Enzyme Immunity Industrial Co., Ltd., China) according to the manufacturer’s instructions. Activity was calculated based on a standard curve (0–120 U/L) and expressed in units per liter (U/L).

ACC Deaminase Activity: ACC deaminase activity was determined using an ELISA kit (Jiangsu Enzyme Immunity Industrial Co., Ltd., China) as per the protocol. Activity was calculated using a standard curve (0–48 U/L) and reported in U/L.

Siderophore Production: Siderophore production was preliminarily evaluated using the Chrome Azurol S (CAS) plate assay and further quantified via the liquid CAS assay after cultivation, with results expressed as the A/Ar ratio. The CAS medium used was purchased from Thermo Fisher Scientific (China).

Phosphate Solubilization: The phosphate-solubilizing ability was initially screened on solid NBRIP medium by measuring the dissolution zone after 7 days of incubation. Quantitative analysis was performed in liquid culture using the molybdenum blue colorimetric method to determine the concentration of soluble phosphorus. All chemical reagents used, including potassium hydroxide, sodium molybdate, concentrated sulfuric acid, and phosphorus standard solution (analytical grade), were purchased from Zhiyuan Chemical Reagent Co., Ltd. (Tianjin, China).

IAA Production: The ability to produce indole-3-acetic acid (IAA) was assessed by growing strains in LB broth supplemented with L-tryptophan. The culture supernatant was mixed with Salkowski reagent, and the absorbance was measured at 530 nm. IAA concentration was quantified using a standard curve.

For inoculum preparation, each strain preserved on agar slants (containing glucose 4 g/L, yeast extract 4 g/L, malt extract 5 g/L, peptone 4 g/L, NaCl 10 g/L, and agar 15 g/L, pH 7.5) was first activated on fresh solid medium. After incubation at 37 °C for 24-48 h, a single colony was picked with a sterile loop and inoculated into a 50 mL flask containing 10 mL of liquid seed medium. The culture was then incubated at 37 °C with shaking at 180 rpm until the optical density at 600 nm (OD₆₀₀) reached approximately 0.6. This seed culture was used to inoculate (1 % v/v) 300 mL of production medium in a 500 mL flask, which was incubated under the same conditions until the late-exponential phase. After cultivation, cells were harvested by centrifugation at 8000 × g for 10 min at 4 °C using a centrifuge (LEGEND MICRO 17, Thermo Fisher Scientific, China). The pellet was then washed twice gently with sterile saline or phosphate buffer to remove residual medium components. The suspension was adjusted to a final OD₆₀₀ of 0.5 ± 0.04, which served as the single-strain inoculum. For co-inoculation treatments (e.g., Microbacterium algeriense C4 + Streptomyces sp. A1), equal volumes of the respective single-strain suspensions were mixed thoroughly. All inoculants were prepared freshly and used within 4 h of preparation.

Four strains (Streptomyces sp. A1, Streptomyces marianii B3, Streptomyces marianiiC3, Microbacterium algeriense C4) showing prominent plant growth−promoting traits were selected for single-strain and co-inoculation (C4 + A1) treatments. A. thaliana (ecotype Col-0) seeds were surface-sterilized and sown on solid MS medium. After stratification at 4 °C for 2 days, plates were transferred to a growth chamber set at 22 °C with a 16 h light/8 h dark photoperiod for germination and early seedling growth. At the four-leaf stage, seedlings were transplanted into pots (10.5 cm in diameter × 11 cm in height) filled with a mixed substrate consisting of vermiculite, perlite, and native lavender rhizosphere soil (3:1:1, v/v/v). After a 3-day acclimatization period, plants were inoculated by applying 1 mL of bacterial suspension (OD₆₀₀ = 0.5 ± 0.04) evenly around the root zone; control plants received the same volume of sterile water. Following inoculation, all plants were cultivated in the same growth chamber. After 60 days, plants were harvested to measure growth and physiological parameters, including plant height, root length, biomass, leaf number, and chlorophyll content.

After 60 days of cultivation, 10–15 seedlings were randomly selected from each treatment group. The seedlings were rinsed with distilled water and gently blotted dry before weighing to record the fresh weight (g). The roots were separately weighed to record the root fresh weight (g). Subsequently, the seedlings were placed in a drying oven at 75°C for 24 hours and then weighed again to determine the dry weight (g).

For other randomly selected seedlings from different treatment groups, plant height (cm) and root length (cm) were measured using a vernier caliper. Leaf area was determined using a leaf area meter.

To determine chlorophyll content, 0.2 g of fresh leaf sample from different treatment groups was placed in a stoppered graduated test tube. Then, 20 ml of a 1:1 (v/v) acetone and absolute ethanol extraction solution was added. After light-proof treatment, the mixture was kept in the dark for extraction. When the sample in the tube turned completely white (after approximately 24 hours), the extract was filtered into a brown volumetric flask. The absorbance of the extract was measured at wavelengths of 645 nm, 663 nm, and 652 nm using a UV-Vis spectrophotometer, with the acetone-ethanol mixture serving as the blank control.

The contents of chlorophyll a, chlorophyll b, and total chlorophyll (mg·L^-1) were calculated using the following formulas:

All experiments were performed with at least three independent replicates. Data are expressed as the mean ± standard deviation. Statistical analysis was conducted using SPSS software (version 26.0). Differences between treatments were assessed by one-way analysis of variance (ANOVA), followed by Duncan’s multiple range test for post-hoc comparisons. Statistical significance was defined at ^*p^*< 0.05, and different lowercase letters in figures/tables indicate significant differences between groups. Graphs were generated using GraphPad Prism (version 9).

To characterize the structure and diversity of endophytic microbial communities, high-throughput sequencing targeting the bacterial 16S rRNA gene and fungal ITS region was performed on root, stem, and leaf tissues of LLV and LAM. The rarefaction curves for all samples plateaued, confirming adequate sequencing coverage for comprehensive microbial analysis (Figures 1a; 2a).

Analysis of the rhizosphere soil microbial community composition in lavender revealed that at the phylum level, Actinomycetota was the absolutely dominant phylum, followed by Pseudomonadota and Bacillota (Figure 3a). At the genus level, the relative abundances of Streptomyces and Nocardioides were significantly higher than those of other taxa, indicating their core status in the lavender rhizosphere micro-ecosystem (Figure 3b).

Functional annotation results showed that in the KEGG Level 1 classification, microbial gene functions were primarily enriched in major categories such as “Metabolism”, “Genetic Information Processing”, and “Environmental Information Processing” (Figure 3c). Further subdivision of the “Metabolism” pathway at Level 2 revealed high enrichment of functions closely related to plant nutrient uptake and stress response, including “Carbohydrate metabolism”, “Amino acid metabolism”, and “Metabolism of cofactors and vitamins” (Figure 3f). Notably, the “Metabolism of terpenoids and polyketides” pathway also exhibited relatively high abundance, suggesting that rhizosphere microorganisms may be involved in the synthesis or regulation of plant secondary metabolites, thereby indirectly enhancing plant stress resistance.

Annotation results from the CAZy database indicated that Glycoside Hydrolases (GHs) and Glycosyl Transferases (GTs) were the two most abundant carbohydrate-active enzyme families, implying that this microbial community plays a significant role in the decomposition of complex carbohydrates like cellulose and hemicellulose, thereby facilitating soil organic matter degradation and nutrient cycling.

CAZy database annotation revealed that Glycoside Hydrolases (GHs) and Glycosyl Transferases (GTs) were the two most abundant carbohydrate-active enzyme (CAZyme) families. Based on this genetic potential, we speculate that this microbial community may play an important role in decomposing complex carbohydrates, such as cellulose and hemicellulose, thereby potentially contributing to soil organic matter degradation and nutrient cycling (Figures 3d, e).

COG functional classification analysis further revealed significant enrichment of genes associated with stress response and competitive adaptation, primarily including “Transcription”, “Replication, recombination and repair”, “Defense mechanisms”, and “Secondary metabolites biosynthesis, transport and catabolism” (Figure 3g). These genomic features suggest that the microbial community may possess the genetic underpinnings to adapt to the saline-alkaline stress environment in Xinjiang and to potentially synthesize antimicrobial and stress-resistance compounds.

Redundancy analysis (RDA) results demonstrated significant correlations (p< 0.05) between key soil physicochemical factors (e.g., pH, available phosphorus AP, soil organic matter SOM) and the distribution of various COG functional categories (e.g., amino acid transport and metabolism, carbohydrate transport and metabolism) as well as KEGG Level 2 metabolic pathways (e.g., nitrogen metabolism, sulfur metabolism, ABC transporters). This finding suggests that the unique soil environment in the lavender rhizosphere may have selected for and shaped a functionally active microbial community. This community, through mechanisms such as efficient nutrient cycling, stress response, and secondary metabolite synthesis, could potentially contribute to improving the rhizosphere microecology and enhancing plant stress tolerance (Figures 3h–k).

A total of 168 actinobacterial strains were isolated from 6 lavender rhizosphere soil samples using 4 different isolation media: Gauze’s No. 1 agar medium, inorganic phosphate-solubilizing bacterium medium, organic phosphate-solubilizing bacterium medium, and nitrogen-fixing bacterium medium. Based on colony morphology (size, shape, color, margin features) and microscopic observation of mycelial structure, 30 representative strains were initially selected. Phylogenetic analysis of their 16S rDNA sequences revealed that these strains belonged to 4 genera: Streptomyces, Microbacterium, Micrococcus, and Nocardiopsis. Among them, Streptomyces was the dominant genus, comprising 22 strains, followed by Microbacterium (4 strains), while Micrococcus and Nocardiopsis each represented by 2 strains.

Further screening based on enzymatic activity assays was performed on these 30 strains, from which 10 strains exhibiting superior activity were selected for subsequent experiments. The phylogenetic tree of these 10 strains is shown in Figure 4. Morphological observations of the 10 representative strains were carried out on ISP2, ISP3, and ISP5 media (Table 1, Figure 5) using an inverted microscope (Leica DMI8, Leica Microsystems (Shanghai) Co., Ltd., China). The majority of colonies appeared white to grey-white, powdery, with regular edges and an opaque appearance. Abundant aerial hyphae with branching structures and rich sporulation were observed. All strains were identified as Gram-positive using a Gram staining kit (HuanKai Microbial Technology Co., Ltd., Guangdong, China).

In this study, a systematic evaluation of salt-alkali tolerance and multiple plant growth-promoting (PGP) traits was conducted on ten actinobacterial strains isolated from the lavender rhizosphere soil. The salt and alkali tolerance tests revealed that all strains were capable of growing across a broad range of NaCl concentrations (2%-25%) and pH levels (5-12), demonstrating extensive adaptability to saline and alkaline conditions. Among them, strains A3, B2, and C4 exhibited optimal growth under 5% NaCl, indicating relatively strong salt tolerance (Figure 6a). Under highly alkaline conditions (pH 11-12), strains A3, C1, and C4 maintained good growth, suggesting significant alkali tolerance potential (Figure 6b).

All tested strains grew in Ashby’s nitrogen-free medium. Growth curves showed that they entered the logarithmic phase at 7 h, reached the peak growth at 24 h, and entered the decline phase after 43 h, preliminarily confirming their nitrogen-fixing potential (Figure 6c). Further assessment of indole-3-acetic acid (IAA) production revealed that all ten strains could secrete IAA. After 60 hours of cultivation, the IAA yields ranged between 19.933 and 55.0 mg/L, with strain C4 producing the highest amount, indicating strong phytohormone synthesis capability (Figure 6d). Quantitative evaluation of siderophore production showed that strain C4 had the lowest A/Ar value (0.637), indicating the highest relative siderophore yield, whereas strain A1 had the highest A/Ar value (1.887), indicating a relatively lower yield (Figure 6e). Measurements of NITS and ACC deaminase activities confirmed that all strains possessed both enzymatic activities. The activity ranges were 22.803-25.409 U/L for NITS and 61.317-122.515 U/L for ACC deaminase. Specifically, the NITS activity of strains B2, B3, and C3 exceeded 110 U/L, and the ACC deaminase activity of strains A1, C3, and C4 exceeded 40 U/L (Figures 6f, g). In the evaluation of phosphate solubilization, five strains (A1, B3, C1, C3, and C4) formed solubilization halos on inorganic phosphorus medium. Strain C4 produced the largest halo, and its soluble phosphorus content in liquid culture reached 131.867 mg/L (Figure 6h).

These results indicate the successful isolation of a collection of actinobacterial resources from the lavender rhizosphere with broad-spectrum plant growth-promoting potential. Several strains simultaneously exhibited multiple biological functions, including salt-alkali tolerance, enzyme production, nitrogen fixation, phosphate solubilization, siderophore production, and IAA synthesis, demonstrating their potential to alleviate environmental stress and promote plant growth, thereby laying a foundation for their future application.

To verify the plant growth-promoting potential of Actinobacteria derived from the lavender rhizosphere, four strains (C4, C3, A1, B3) that exhibited excellent performance in prior functional screenings were selected. These strains were prepared as individual inoculants and specific bacterial consortia (C4+A1, C3+B3, A1+B3) and applied to A. thaliana seedlings via root irrigation. The growth-promoting effects on the plants were systematically evaluated, with the growth status of A. thaliana seedlings from each treatment group shown in Figure 7a.

Among the single-strain treatments, strain C4 demonstrated the most prominent effect. The C4-inoculated plants showed an increase of 9 leaves, a plant height increase of 1.5 cm, fresh and dry weight increases of 0.279 g and 0.224 g respectively, a root length increase of 1.023 cm, and a chlorophyll content increase of 0.366 mg/g wt. The other strains (C3, A1, and B3) also exhibited varying degrees of growth-promoting effects. Notably, the bacterial consortium treatments generally outperformed the single-strain treatments, with the C4+A1 combination showing the most significant effects: plant height increased by 7.587 cm, fresh and dry weights increased by 0.631 g and 0.383 g respectively, and chlorophyll content increased by 0.708 mg/g fresh weight. All these parameters were significantly higher than those of the control group and most single-strain treatments (Figures 7b–g).

Additionally, although some parameters in the pure medium treatment group were slightly higher than those in the control (CK), its growth-promoting effect remained significantly lower than all bacterial suspension treatments. This indicates that the observed plant growth promotion primarily resulted from the biological activity of the inoculated Actinobacteria, rather than interference from medium components.

In summary, this study demonstrates that Actinobacteria originating from the lavender rhizosphere, particularly strains C4 and A1 and their consortium, can effectively promote the growth of A. thaliana seedlings. They significantly enhance biomass accumulation, root development, and photosynthetic pigment content, demonstrating substantial potential for application as plant growth-promoting bacteria.