Frontiers in Plant Science - Soil salinity is an increasingly serious problem worldwide that reduces agricultural output potential. Selected beneficial soil bacteria can promote plant growth and augment tolerance to biotic and abiotic stresses.
Bacterial Suspension CultureBacillus subtilis strain GB03 was streaked onto LB agar plates and incubated at 28°C without light for 24 h (The bacterium strain was presented by Professor Paul W. Paré at Texas Tech University, USA). Bacterial cells were then harvested from LB agar plates, transferred into liquid LB media and cultured at 28°C with 250 rpm rotation to yield 109 colony forming units (CFU) mL−1, as determined by optical density and serial dilutions (Zhang et al., 2008a).
Plant Growth and Treatments
White clover (Trifolium repens L. cultivar Huia) seeds were surface sterilized with 2% NaClO for 1 min followed by 70% ethanol for 10 min, and rinsed with sterile water five times (The white clover seeds was presented by Dr. Wanhai Zhou at Gansu Agricultural University, China). Seeds were then sown in presterilized plastic pots (diameter 20 cm, depth 22 cm, 10 seeds/pot with eight replicates) containing 600 g of heat-sterilized (95°C, 48 h) vermiculite and soil mix (1:1) watered with half strength Hoagland's nutrient solution (5 mM KNO3, 1 mM NH4H2PO4, 0.5 mM Ca(NO3)2, 0.5 mM MgSO4, 60 μM Fe-citrate, 92 μM H3BO3, 18 μM MnCl2·4H2O, 1.6 μM ZnSO4·7H2O, 0.6 μM CuSO4·5H2O, and 0.7 μM (NH4)6Mo7O24·4H2O). Each pot was watered with nutrient solution (400 mL) once per week. After germination, six uniform seedlings per pot were selected for continued growth and inoculated directly to the soil with 1 mL bacterial suspension culture as inoculation treatment or 1 mL liquid LB medium as a control. For salt stress treatments, seedlings were watered with 0, 50, 100, and 150 mM NaCl present in the nutrient solution. Each treatment contained eight pots as replications. Plants were grown in a glasshouse at the temperature regulated to around 28°C during the day and 22°C at night. Relative humidity averaged 65 and 75% for day and night periods, respectively, with natural photoperiod and light intensity.