Ethos plus

For ICP‐MS analyses of Na⁺, K⁺ and Ca²⁺, roots and shoots were harvested from salt stressed plants including HM053 inoculated salt stressed plants. Tissues were dried in an oven, weighed and ground up by hand using a mortar and pestle. The samples were digested in 3.0 ml of concentrated nitric acid at 190°C using a Milestone Ethos Plus (Milestone SRL) microwave digestion system, then were diluted to 50 ml with ultrapure water followed by gravimetric dilution by a factor of 10 with 0.45 M nitric acid. Samples were analyzed via Perkin‐Elmer NexION ICP‐MS in Kinetic Energy Discrimination mode. Reference materials included NIST SRM 1570 spinach leaves and NIST SRM 1573 tomato leaves prepared as samples and analyzed in the same way. Internal standards at known concentrations were prepared from stock solutions (High Purity Standards) and used to calibrate instrument response.

For ICP-MS analyses, roots and shoots were dried in an oven, weighed into digestion vessels and digested in 3.0 mL of concentrated nitric acid at 190 °C in a Milestone Ethos Plus (Milestone SRL, Sorisole, Italy) microwave digestion system. Digestants were diluted to 50 mL with ultrapure water and gravimetrically diluted by a factor of 10 with 0.45 N nitric acid. Samples were analyzed via Perkin-Elmer NexION ICP-MS in Kinetic Energy Discrimination mode. Total elemental ion counts were measured of ⁵⁶Fe and normalized to ¹²C counts. Reference materials included NIST SRM 1570 spinach leaves and NIST SRM 1573 tomato leaves prepared as samples were. Internal Calibration standards of Sc, In, and Tl at known concentrations were used from stock solutions (High Purity Standards, Charleston, SC 29418, USA).
For Laser Ablation-ICP-MS, roots were removed from shoots and a 3-cm section of primary lateral root was further excised above the apical meristem. Roots were sectioned in OCT embedding media to 100 μm thickness (Fisher Scientific Inc., Hampton, NH 03801, USA) and placed on quartz microscope slides for freeze drying in a FreezeZone 1 dryer (Labconco Corp., Kansas City, MO, USA) before ablation.

To determine the “pseudo-total” element content of the soil, the Hungarian Standard MSZ 21470-50 2006 was followed with slight modifications. The sample preparation procedure and the microwave digestion of 0.5 g of soil (<0.1 mm) in cc. HNO₃ and in cc. H₂O₂ (3:1 v v⁻¹) solutions are described in detail elsewhere [87 (link),88 (link)].
To determine the soluble (“plant-available”) element content of the soil, the MSZ 20135 [93 ] was followed. The sample preparation procedure and the extraction of 0.5 g soil (<0.1 mm) with Lakanen–Erviö (LE) solution (0.02 M H₄-EDTA in 0.5 M ammonium acetate buffer and 0.5 M acetic acid, pH 4.65; Lakanen and Erviö, 1971) is described in published articles [87 (link),88 (link)].
From the prepared (dried and ground to particles < 0.1 mm) plant samples, 0.5 g was loaded into the pressure-proof bombs of the microwave digester (Milestone Ethos Plus, Sorisole BG, Italy). To all samples, 5 mL of distilled cc. HNO₃ and 3 mL 30% (v v⁻¹) H₂O₂ (Scharlau, Barcelona, Spain) were added [87 (link)].
Elemental analysis of all soil or plant samples was conducted with the inductively coupled plasma optical emission spectrometry (ICP-OES) technique, applied on an iCAP 7000 spectrophotometer (Thermo Fischer Scientific, Waltham, MA, USA). For the calibration, a multielement standard solution (n = 2) was applied. All element analyses were performed with 4 replicates.