Soil microorganisms, particularly Actinobacteria, play pivotal roles in maintaining terrestrial ecosystem functioning through organic matter decomposition, nutrient cycling, and symbiotic plant interactions [8]. Among these, the phylum Actinobacteria—notably the genus Streptomyces—has garnered global attention due to its unparalleled metabolic versatility, including the production of bioactive compounds (e.g., antibiotics, enzymes) and contributions to soil structure stabilization [18]. Despite their ecological significance, the distribution and adaptive mechanisms of Actinobacteria in extreme environments, such as saline-alkaline soils, remain understudied, particularly in arid and semi-arid regions like North Africa. Ecological and Agricultural Relevance
Actinobacteria thrive in diverse soil habitats but exhibit pronounced dominance in rhizospheres due to root exudate-mediated interactions [13]. Their ability to decompose complex organic polymers (e.g., cellulose, chitin) and solubilize phosphates makes them critical agents of soil fertility [27]. Furthermore, Streptomyces spp. Produce over 70% of clinically used antibiotics [6], highlighting their biotechnological potential. However, their resilience to abiotic stressors—especially salinity and pH fluctuations—is poorly characterized in coastal agroecosystems, where such factors critically limit microbial activity.RegionalContextandKnowledgeGapsLibya’s Mediterranean coastal soils (e.g., Al-Haniya region) face escalating salinity due to seawater intrusion and unsustainable irrigation [4]. While global studies have explored Actinobacteria in temperate soils [10]; [28], data from North Africa’s saline-alkaline environments are sparse. Existing research in similar climates suggests Actinobacteria may mitigate salt stress in crops but no systematic studies have validated this in Libyan contexts. Additionally, the interactive effects of temperature, pH, and salinity on Actinobacteria communities remain unresolved, limiting predictive models for soil health management[16]. This study addresses these gaps byquantifying the spatial (vertical/horizontal) distribution of Actinobacteria in Al-Haniya’s rhizospheric versus bulk soils. Evaluating the impacts of key abiotic factors (temperature: 25–40°C; pH: 4–8; salinity: CaCl₂/NaCl/MgCl₂) on Actinobacteria growth dynamics. Identifying dominant genera and correlating their abundance with soil physicochemical properties. We hypothesize that: Actinobacteria populations will be significantly higher in rhizospheric soils due to root-derived carbon inputs. Streptomyces will dominate across all soil layers, exhibiting optimal growth at near-neutral pH (6–7.5) and moderate salinity (1–5 g/L). Soil organic carbon and clay content will positively correlate with Actinobacteria abundance. By integrating field surveys with controlled experiments, this work provides the first comprehensive assessment of Actinobacteria ecology in Libyan coastal soils, offering insights into sustainable agriculture and saline soil rehabilitation.

Materials and Methods

Study Area and Sampling Design

The study was conducted in Al-Haniya, a coastal agricultural region in northeastern Libya (32°50′N, 21°29′E), characterized by Mediterranean climate (mean annual rainfall: 250 mm) and saline-alkaline soils.

Sampling Strategy

A stratified random sampling approach was employed across 150 subplots (15 m × 10 m grid) in a Vicia faba (faba bean) cultivated field.

Soil Depths

Rhizosphere soil (RS): Collected from root surfaces (0–2 mm adhering soil).Bulk soil (BS): Sampled from 0–2.5 cm, 2.5–5 cm, and 5–10 cm depths using a sterile auger.Replicates: Five composite samples per depth/location (n = 15 plots × 4 depths × 5 replicates = 300 samples total). Samples were stored at 4°C for ≤24 h before processing.

Soil Physicochemical Analysis

Physical Properties

Texture: Determined via hydrometer method [10], after organic matter removal (H₂O₂) and dispersion (sodium hexametaphosphate). Moisture Content: Oven-dried at 105°C for 24 h [8].

Chemical Properties

PH: Measured in 1:1 soil: water suspension (Jenway 3310 pH meter). Electrical Conductivity (EC): 1:1 soil: water extract (dS/m at 25°C). Organic Carbon: Walkley-Black wet oxidation method (Khalil et al., 2024) Cation Exchange Capacity (CEC): Ammonium acetate (1 M, pH 7.0) displacement [26].Nutrients: TotalNitrogen: Kjeldahl digestion [12]. Phosphorus: Olsen’s extraction [28].

Microbial Analysis

Culture-Dependent EnumerationTotal Bacteria: Serial dilutions (10⁻¹–10⁻⁷) plated on Nutrient Agar (NA), incubated at 30°C for 48 h.Actinobacteria: Selective isolation using Starch-Casein Agar (SCA) supplemented with cycloheximide (50 µg/mL) to inhibit fungi [33]. Plates incubated at 30°C for 7 days.CFU CountingColonies counted at 30–300 CFU/plate threshold; expressed as CFU/g dry soil[23].Morphological Identification: Macroscopic: Colony morphology (color, pigmentation, texture). Microscopic: Gram staining and light microscopy (1000×) for hyphal structure [25].

Experimental Treatments

Temperature Gradient: SCA plates inoculated with soil suspensions (10⁻⁵ dilution) incubated at 25°C, 30°C, 35°C, and 40°C for 7 days.pH Tolerance:SCA media adjusted to pH 4, 5, 6, 7, 7.4 (control), and 8 using HCl/NaOH.Salinity Stress: SCA supplemented with CaCl₂, NaCl, or MgCl₂ at 0.1 g, 1 g, and 5 g/L. [20]. Statistical AnalysisSoftware: SPSS v26.0. Tests:ANOVA with post-hoc LSD (p < 0.05) for treatment effects.Pearson correlations between Actinobacteria abundance and soil variables.Data Presentation: Means ± standard error (SE); significance denoted by lowercase letters (e.g., 35ᵃ vs. 40ᵇ).

Results

Soil Physicochemical Characteristics

The study area exhibited distinct soil properties critical for Actinobacteria ecology (Table 1): Texture: Clay-dominated (45.2 ± 3.1% clay, 36.8 ± 2.4% silt, 18.0 ± 1.8% sand)pH: Strongly alkaline (7.9 ± 0.4 in bulk soil; 8.1 ± 0.3 in rhizosphere)Salinity (EC): Higher in rhizosphere (6.2 ± 0.8 dS/m) vs bulk soil (4.7 ± 0.6 dS/m) (p < 0.05)Organic Carbon: Rhizosphere (2.1 ± 0.3%) > bulk soil (1.6 ± 0.2%) (p = 0.012).

Vertical populations decreased with depth 0-2.5 cm: 7.8 × 10⁷ ± 0.9 CFU/g 5-10 cm: 5.2 × 10⁷ ± 0.7 CFU/g (p = 0.003). Horizontal Rhizosphere (9.1 × 10⁷ ± 1.1 CFU/g) showed 32% higher counts than bulk soil (6.9 × 10⁷ ± 0.8 CFU/g) (p = 0.001).Abiotic Factor Responses Temperature Affects Optimal growth at 30°C (48.3 × 10⁷ ± 3.2 CFU/g). Significant reduction at 40°C (29.7 × 10⁷ ± 2.1 CFU/g; p = 0.008) pH Tolerance: No growth below pH 5.0 Peak activity at pH 7.0 (102 ± 8 CFU/g) vs pH 8.0 (64 ± 6 CFU/g) (p = 0.015). Salinity Stress CaCl₂: Stimulated growth up to 5 g/L (51 × 10⁷ ± 4.1 CFU/g) NaCl: Inhibitory above 1 g/L (38% reduction at 5 g/L vs control) MgCl₂: Intermediate tolerance (44 × 10⁷ ± 3.8 CFU/g at 1 g/L).[14].

Actinobacteria Abundance and Distribution

Genus-Level Composition:Streptomyces dominated (88.6 ± 4.2% of isolates)Minor constituents: Nocardia (7.3 ± 1.8%), Micromonospora (4.1 ± 1.2%)

Correlation Analysis

Strong positive correlations:Actinobacteria vs organic carbon (r = 0.82, p < 0.001)Streptomyces vs clay content (r = 0.71, p = 0.003)Negative relationships:Total counts vs EC (r = -0.63, p = 0.012)Nocardia vs depth (r = -0.58, p = 0.021)

Discussion

Ecological Significance of Actinobacteria Distribution

Our study reveals two key ecological patterns in Al-Haniya’s soils:Rhizosphere Preferencethe 32% higher Actinobacteria abundance in rhizospheric soils (p = 0.001) aligns with global observations [18],butcontrasts with arid-region studies where salinity suppresses microbial activity [19]. This suggests Libyan coastal Streptomyces strains may have evolved unique adaptations to simultaneous root exudate stimulation and salt stress. DepthDecline: The 33% reduction in Actinobacteria from the surface (0-2.5 cm) to subsurface (5-10 cm) layers (p = 0.003) mirrors nutrient gradients in clay-rich soils [15]. However, the persistence of Nocardia at depth (r = -0.58) implies niche partitioning – likely due to its documented oligotrophic capabilities [1].

Mechanisms of Abiotic Stress Tolerance

Salinity Adaptation the CaCl₂-enhanced growth (51 × 10⁷ CFU/g at 5 g/L) challenges the paradigm that NaCl is universally inhibitory [9]. We proposecalcium may stabilize membranes under ionic stress (Elhafiet al., 2024).Streptomyces EPS production could mitigate osmotic shock [31]. pHconstraints
The complete growth inhibition below pH 5.0 supports the “alkaliphile specialization” hypothesis for arid soil Actinobacteria [22]. Notably, the pH 7.0 optimum matches Libyan crop rhizosphere pH ranges [4], suggesting co-evolution with local flora.

Agricultural and Biotechnological Implications

SalineSoilRehabilitation: The strong correlation between Streptomyces and organic carbon (r = 0.82) indicates manure amendments could boost their populations for soil structuring – critical for Libya’s 38% salt-affected farmland [17].Bioinoculant Development: Strain-specific tolerance to MgCl₂ (44 × 10⁷ CFU/g at 1 g/L) suggests potential as seed coatings for magnesium-rich soils. This aligns with successful Streptomyces-based biofertilizer trials in Egypt [2].Antibiotic Discovery the 88.6% Streptomyces dominance highlights untapped biosynthetic potential. Libyan coastal strains may yield novel compounds, as marine-derived Streptomyces show 23% higher antibiotic diversity than terrestrial isolates [27].

Conclusion

Actinobacteria, especially Streptomyces, are vital to Al-Haniya’s soil ecosystem, thriving in saline-alkaline conditions and contributing to nutrient cycling. Future work should explore their field-scale applications and genomic potential.

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