**Background:** Halophytes are plants adapted to saline environments, and their associated microbial communities—particularly endophytic bacteria—are thought to play a key role in host adaptation to salt stress. Halotolerant plant growth-promoting endophytic bacteria (HPGPE) can support plant growth through mechanisms such as production of phytohormones (e.g., IAA), ACC deaminase activity (which lowers stress ethylene), nutrient solubilization, and biofilm formation. This study aimed to characterize the endophytic root microbiota of three succulent halophytes—Salicornia europaea, Suaeda maritima, and Camphorosma annua—collected from the Slano Kopovo salt marsh in Serbia, an internationally recognized Ramsar site.
**Methods:** Root samples from three individual plants per species were collected. A common rhizosphere sample was prepared per species. For culture-independent analysis, DNA was extracted from macerated root tissue and bacterial 16S rRNA gene sequencing was performed using the Illumina MiSeq platform with ZymoBIOMICS custom primers. OTUs were clustered at 94% and 97% identity. For culture-dependent analysis, serial dilutions of macerated root tissue were plated on Nutrient agar supplemented with 5%, 10%, and 15% NaCl and root tissue extract. ACC deaminase-producing bacteria were enriched using DF salts minimal medium with ACC as the sole nitrogen source. All isolates were screened for salt tolerance up to 25% NaCl. PGP traits (IAA production, ACC deaminase activity, ammonia production, siderophore production, EPS production, biofilm formation, and P/Zn/K solubilization) were assessed at 0%, 3%, and 7% NaCl. Selected isolates were identified by 16S rRNA gene sequencing.
**Key Results:** Culture-independent analysis revealed that Proteobacteria (72% in S. maritima and C. annua; 47% in S. europaea) and Actinobacteria (14%, 12%, and 33%, respectively) were the dominant phyla. Pseudomonadaceae were the most abundant family in S. maritima (16.64%) and C. annua (12.02%), while S. europaea showed a more even distribution of families. Alpha diversity varied: 374, 400, and 264 unique reads per 16,230 reads for S. maritima, C. annua, and S. europaea, respectively. Only 19.09% of genera were shared across all three plants. A total of 32 endophytes were isolated, and 20 were selected for further characterization. These belonged to seven genera: Acinetobacter (2), Kushneria (4), Pseudomonas (3), Halomonas (7), Halobacillus (2), Planococcus (1), and Klebsiella (1). Halomonas was the predominant genus. Halobacillus strains grew at up to 25% NaCl; Kushneria and Halomonas up to 18%; Pseudomonas, Acinetobacter, and Klebsiella only up to 3%. Five ACC deaminase-positive strains were identified (Acinetobacter, Klebsiella, Pseudomonas). Nine isolates produced IAA (range 2.10–4.62 µg/ml), with Halomonas sp. 14KX2 producing the highest amount (4.62 µg/ml at 0% NaCl). 90% of isolates produced ammonia at 0% and 3% NaCl; 22% lost this ability at 7% NaCl. 75% produced siderophores at 0% NaCl; three Kushneria and two Halomonas strains retained this at all salt concentrations. 95% produced EPS at 0% NaCl; 47% retained this at elevated salt. All isolates formed biofilms at 0% NaCl (55% weak, 40% moderate); at 7% NaCl, 26.6% lost this ability. Five ACC deaminase producers (14A2, 14A5, 15A1, 15A2, 16A1) solubilized K, P, and Zn at 0% and 3% NaCl. Klebsiella 14A5 showed the highest solubilization indices (K: 8.64 at 0%, 5.12 at 3%; P: 4.53 at 0%, 3.29 at 3%; Zn: 5.05 at 0%, 5.55 at 3%). Only Kushneria 16.1 solubilized phosphorus at 7% NaCl.
**Clinical Implications:** This study demonstrates that the root endosphere of indigenous succulent halophytes harbors a diverse and cultivable community of halotolerant bacteria with multiple PGP traits that are retained under saline conditions. These bacteria represent a valuable bioresource for developing microbial inoculants aimed at improving crop growth and salt tolerance in saline agricultural soils. The findings also underscore the importance of plant species in shaping endophyte community composition and the potential of habitat-adapted bacteria for sustainable agriculture in salt-affected regions.