In Vitro Propagation and NaCl Tolerance of the Multipurpose Medicinal Halophyte Limoniastrum monopetalum

Limoniastrum monopetalum is an evergreen perennial shrub native to Mediterranean coastal sands and salt marshes. It has adapted to a variety of environmental stresses and is used in traditional medicine and as an ornamental plant. In the present study, an efficient micropropagation protocol for this species was developed to facilitate the production of selected genotypes and promote its wider use. Research has focused on the effects of various cytokinin types [benzyladenine (BA), zeatin, 6-furfurylaminopurine (kinetin) or 6-γ-γ-dimethylallilopurine (2iP)] and concentrations (0.0–4.0 mg·L−1) and various NaCl concentrations (0.0–20 g·L−1) during all stages of in vitro culture. For in vitro establishment, Murashige and Skoog (MS) medium supplemented with 0.5 mg·L−1 BA and 0.0 or 5.0 g·L−1 NaCl was most appropriate (100% explant response, 3–4 shoots per explant, 2 cm shoot length). The best results for shoot multiplication (100% response, 9 shoots per explant, 0.8–1.0 cm shoot length) were obtained with low (0.5 mg·L−1) BA or relatively high (2.0 mg·L−1) kinetin concentrations in the medium; however, 0.5 mg·L−1 kinetin should be preferred in the case of production of multiple rooted microshoots during one stage. The addition of NaCl at relatively low concentrations (2.5 or 5.0 g·L−1) in a medium supplemented with 0.5 mg·L−1 BA doubled shoot multiplication but did not improve shoot elongation (100% explant response, 16 shoots per explant, 0.8 cm shoot length). For in vitro rooting, half-strength MS medium supplemented with 1.0 mg·L−1 IBA was most appropriate (97% rooting, 9.4 roots per microshoot, 1.2 cm root length). Regarding the effects of NaCl on in vitro rooting, microshoots were relatively tolerant to NaCl concentrations up to 10.0 g·L−1. The effects of NaCl depend on the micropropagation stage; they are synergistic during shoot multiplication and tolerant during rooting. However, explants responded satisfactorily in its absence, indicating that NaCl was not necessary as a medium component. Ex vitro acclimatization and establishment of plantlets was 100% successful in a mixture of peat:perlite 1:1 or 2:1 (v/v).

Screening of the High-Rhizosphere Competent Limoniastrum monopetalum’ Culturable Endophyte Microbiota Allows the Recovery of Multifaceted and Versatile Biocontrol Agents

Halophyte Limoniastrum monopetalum, an evergreen shrub inhabiting the Mediterranean region, has well-documented phytoremediation potential for metal removal from polluted sites. It is also considered to be a medicinal halophyte with potent activity against plant pathogens. Therefore, L. monopetalum may be a suitable candidate for isolating endophytic microbiota members that provide plant growth promotion (PGP) and resistance to abiotic stresses. Selected for biocontrol abilities, these endophytes may represent multifaceted and versatile biocontrol agents, combining pathogen biocontrol in addition to PGP and plant protection against abiotic stresses. In this study 117 root culturable bacterial endophytes, including Gram-positive (Bacillus and Brevibacillus), Gram-negative (Proteus, Providencia, Serratia, Pantoea, Klebsiella, Enterobacter and Pectobacterium) and actinomycete Nocardiopsis genera have been recovered from L. monopetalum. The collection exhibited high levels of biocontrol abilities against bacterial (Agrobacterium tumefaciens MAT2 and Pectobacterium carotovorum MAT3) and fungal (Alternaria alternata XSZJY-1, Rhizoctonia bataticola MAT1 and Fusarium oxysporum f. sp. radicis lycopersici FORL) pathogens. Several bacteria also showed PGP capacity and resistance to antibiotics and metals. A highly promising candidate Bacillus licheniformis LMRE 36 with high PGP, biocontrol, metal and antibiotic, resistance was subsequently tested in planta (potato and olive trees) for biocontrol of a collection of 14 highly damaging Fusarium species. LMRE 36 proved very effective against the collection in both species and against an emerging Fusarium sp. threatening olive trees culture in nurseries. These findings provide a demonstration of our pyramiding strategy. Our strategy was effective in combining desirable traits in biocontrol agents towards broad-spectrum resistance against pathogens and protection of crops from abiotic stresses. Stacking multiple desirable traits into a single biocontrol agent is achieved by first, careful selection of a host for endophytic microbiota recovery; second, stringent in vitro selection of candidates from the collection; and third, application of the selected biocontrol agents in planta experiments. That pyramiding strategy could be successfully used to mitigate effects of diverse biotic and abiotic stresses on plant growth and productivity. It is anticipated that the strategy will provide a new generation of biocontrol agents by targeting the microbiota of plants in hostile environments.