Hoagland s no 2 basal salt mixture

Plants were inoculated with bacteria as described above. Twenty-four hours from the inoculation, the seedlings were introduced in test tubes (12 mL of volume) with semisolid (0.4% agar) Hoagland’s No.2 basal salt mixture (Sigma-Aldrich) supplemented, when required, with 50 µM CdCl₂. Plants were watered every 5 days with sterilized Hoagland’s No.2 basal salt mixture. When required, 50 µM CdCl₂ and bacterial suspensions (A₆₀₀ = 0.6) were added to the irrigation solution. The growing plants were kept in a greenhouse at 30 °C for 15 days. After 15 days, root and shoot samples of 50–100 mg each were taken for metal extractions. The determined dry weights of these samples were used to calculate average root and leaf Cd concentrations per g dry weight. Each experiment was performed in triplicate and the number of plants used was 5.

Maize seeds were sown in 42 pots of 1.5 L each (4 seed per pot), filled with the same soil used for the acute phytotoxicity test, placed inside the “walk-in” chamber facility (2.5 m × 3.9 m × 3 m h) of the Department of Environmental Biology, Sapienza University of Rome (Salvatori et al. 2013 (link)). Inside the chamber, environmental parameters were maintained as follows: photosynthetic active radiation = 700 μmol m⁻² s⁻¹; photoperiod = 12 h; air temperature = 25.0 ± 2 °C; relative humidity = 60 ± 5%. During the whole experimental period, pots were randomly relocated in the chamber every day to prevent position effects. Starting from the 9th day after sowing (DAS), each pot was provided once a week with 30 mL of Hoagland’s No. 2 Basal Salt Mixture (Sigma-Aldrich Co) at ¼ of strength. At DAS 12, germinated plants were thinned to one per pot. At DAS 26, when plants had an average of 3.4 ± 0.5 leaves, the SLES treatment was applied. Pots were randomly divided into 3 experimental sets, of 14 pots each: C, control, not treated; T360 mg kg⁻¹, treated with 360 mg kg⁻¹ SLES; T1200 mg kg⁻¹, treated with 1200 mg kg⁻¹ SLES. These SLES concentrations were chosen on the basis of the results of the acute phytotoxicity test. SLES treatment was provided in 100 mL of deionized water per pot; concurrently, control plants were irrigated with 100 mL of deionized water.

Once 50% of plants reached the third true‐leaf stage, a bulk nutrient solution was prepared such that 40 mL of solution could be allocated to each plant in 50‐mL Falcon tubes. Nutrient solution in 50‐mL tubes consisted of 0.4 g/L Hoagland's No. 2 basal salt mixture (Sigma Aldrich Chemical Co. St. Louis, MO, USA) supplemented with ~0.5 g/L Professional General Purpose 20‐20‐20 fertilizer up to an EC of 1200 mS/cm. This bulk nutrient solution was then split into two, and mannitol was added into one of the solutions to a concentration of 10 mM. pH of both control and mannitol‐supplemented solutions was then recorded. Fifty mL tubes were then filled with 40 mL of either control or mannitol‐supplemented nutrient solution. Tubes were wrapped in aluminum foil to prevent algal growth. Tubes with solution, but no caps, were then weighed. Caps containing seedlings were then brought into the lab and were firmly screwed onto individual tubes. Tubes with seedlings screwed on were then weighed to determine initial seedling weight. If the roots of any sample did not reach the nutrient solution when fully closed into the cap, additional solution was added and the tube (with and without cap) was reweighed. Samples were evenly spaced into 50‐mL tube racks and then returned to the greenhouse.