Halophyte and medicinal plants as a source of Plant Growth Promoting Bacteria and natural extracts for sustainable applications

Introduction and aims. Soil salinization affects over 20% of global cultivated land, with projections reaching 50% by 2050. The Mediterranean basin particularly faces increasing challenges from irregular rainfall and advancing salinity, expanding marginal lands characterized by low fertility and multiple environmental stressors. Apulia's coastal environments host diverse halophytic communities and associated microbiomes representing unexplored reservoirs of beneficial microorganisms. Halophytes display specialized salt adaptations and harbor salt-tolerant rhizosphere/endosphere bacteria that could enhance plant resilience through nutrient solubilization, phytohormone synthesis, and stress resistance induction. Additionally, halophytes and medicinal plants from marginal environments produce bioactive compounds with potential antimicrobial and biostimulant properties. Despite growing interest in these biological resources, major knowledge gaps persist regarding microbial community dynamics in Mediterranean halophytes, the biostimulant potential of essential oils in horticulture, and integrated approaches combining Plant Growth-Promoting Bacteria (PGPB) isolation with natural product applications. On this basis, this thesis explored halophytes and medicinal plants as sources of PGPB and natural extracts for sustainable agriculture and food preservation, targeting: (1) halotolerant PGPB characterization from Cakile maritima (sea rocket) and Arthrocaulon macrostachyum (glasswort); (2) Lavandula angustifolia essential oil (LEO) antimicrobial and biostimulant evaluation; (3) naturally preserved ready-to-eat Salicornia products. Materials and methods. Plants were collected from coastal sites in Apulian marginal areas to isolate and select cultivable bacteria (180 from C. maritima, 110 from A. macrostachyum) that underwent comprehensive PGPB screening: phosphate/silicon solubilization, IAA/siderophore production, salt tolerance (5-17.5% NaCl), and drought tolerance. RAPD-PCR differentiation and 16S rRNA sequencing enabled identification, and Principal Component Analysis (PCA) guided PGPB candidate selection. Soil samples also underwent shotgun metagenomic sequencing with bioinformatic analysis for taxonomic classification and species abundance quantification. Lavandula angustifolia flowers were harvested from an agrivoltaic system in San Severo (Apulia, Italy) and subjected to hydrodistillation to obtain LEO (Lavender Essential Oil), whose chemical composition was determined by GC-MS analysis. Its antimicrobial activity was tested against six microbial targets (Staphylococcus aureus, Bacillus spp., Escherichia coli, Pseudomonas spp., Candida albicans, Pichia membranifaciens) at 2.0-8.0% concentrations. LEO biostimulant effects on radish (Raphanus sativus) were assessed at 0.5-4.0% LEO across two greenhouse cycles, monitoring biometric, physiological, and mineral parameters. Salicornia europaea was characterized for phenolics, flavonoids, antioxidants, and anthocyanins. Microbiological challenge tests were conducted by inoculating ready-to-eat Salicornia products with Pseudomonas fluorescens and Enterobacter agglomerans. Natural antimicrobials (thyme oil and grapefruit seed extract) were applied in biodegradable packaging under air and modified atmosphere (MAP: 65% N₂, 30% CO₂, 5% O₂) at 4°C and 15°C. Results. Metagenomics revealed bacterial dominance (92-94%) with Actinomycetota (50-52%) and Pseudomonadota (35-36%) predominant. C. maritima isolates demonstrated nutrient solubilization (35-36%), IAA production (14%), and salt tolerance >10% NaCl (46%, with 20 strains at 17.5%). A. macrostachyum showed similar functional diversity with phosphate solubilization (16%), silicon solubilization (23%), siderophore production (31%), and salt tolerance >10% NaCl (45%, with 28 strains at 17.5%). Three complementary candidates were selected per species: Pantoea agglomerans, Pantoea sp., and Bacillus sp. from C. maritima; Pseudomonas sp., Bacillus safensis, and Peribacillus frigotolerans from A. macrostachyum. LEO composition was dominated by linalool (36.81%) and linalyl acetate (41.0%). Antimicrobial assays demonstrated dose-dependent inhibition, with S. aureus showing the highest susceptibility (89% inhibition at 8.0% LEO) and E. coli greatest resistance (15% inhibition at 8.0%). MIC values clustered around 150-160 mg/mL across species. Biostimulant trials revealed modest improvements at 0.5-2.0% LEO, with severe phytotoxicity at 4.0% suggesting herbicidal potential. Nutritional characterization of S. europaea revealed substantial bioactive content: 250±75 mg GAE/100g phenolics, 95±30 mg CE/100g flavonoids, 12.5±5.0 μmol TE/g antioxidants. Under refrigeration (4°C, air), GFSE extended P. fluorescens lag phase 8.6-fold (from 0.76 to 6.51 days) and reduced maximum load by 2 log CFU/g. MAP at 4°C achieved 2-3 log reductions (4.37-5.62 log CFU/g maximum) with extended lag phases (7.38-9.78 days). MAP and GFSE combination provided optimal preservation (4.54 log CFU/g P. fluorescens) with maintained pH stability and color quality throughout 90-day storage. Conclusions. Mediterranean halophytes harbor diverse, salt-adapted bacterial communities. Selected PGPB strains combine multiple beneficial traits with exceptional salt tolerance (17.5% NaCl), supporting consortium-based biofertilizer development. LEO demonstrated potent antimicrobial activity but limited biostimulant efficacy at crop-tolerable concentrations. The 4.0% phytotoxicity warrants natural herbicide investigation. Ready-to-eat Salicornia products preserved with GFSE and MAP demonstrated commercial viability, offering sustainable alternatives to synthetic preservatives. This thesis provides actionable knowledge for developing biological inputs compatible with organic and low-input farming systems, addressing multiple United Nations Sustainable Development Goals (Zero Hunger, Climate Action, Life on Land) while supporting the European Green Deal objectives for sustainable food systems. The demonstrated potential of halophyte-associated microbiomes and bioactive compounds represents a significant step toward climate-resilient agriculture in marginal environments, though realization of this potential requires continued interdisciplinary research integrating microbial ecology, plant physiology, molecular biology, and agricultural engineering.