2.1 Experimental design
To investigate the effects of microorganisms on N. tabacumgrowth, all plant-growth experiments were conducted in a greenhouse under natural light and ambient conditions. The daytime temperature was 16-33°C, the night-time temperature was 11-20°C, and the relative humidity was 40-85% during the treatment period. The experiments were conducted at the Pu’an Flue-cured Tobacco Scientific Research Base, in Guangyuan, Sichuan Province, China (32.004N, 105.493E). Briefly, after homogenization, natural field-sampled soil was divided into two parts in March 2017. The properties of the soil were as follows (based on kg-1dry soil): pH 7.6, organic carbon 9.68 g, total nitrogen 1.62 g, total phosphorus 0.58 g, and total potassium 18.72 g. One part was used to cultivate N. tabacum for two consecutive seasons from April 2017 to September 2018, which was defined as continuous cropping soil (CCS). The other part remained uncultivated and was defined as natural fallow soil (FS). In 2019, half of each conditioned soil was sterilized. This procedure results in four different types of conditioned soil: (i) Continuous cropping soil without sterilization treatment (C_NS); (ii) continuous cropping soil with sterilization treatment (C_S); (iii) natural fallow soil without sterilization treatment (F_NS); and (iv) natural fallow soil with sterilization treatment (F_S). Therefore, the experimental layout was completely randomized with two factors (soil type and sterilization treatment). Three replicates with three plants each were used to account for sampling errors. N. tabacum seeds were first imbibed in sterile water for 24 h, and then surface-sterilized in 75% ethanol for 30 s, followed by a brief wash with 10% NaClO (30 min) and five subsequent washes with sterile water. In the end, N. tabacumseeds were sown onto the surface of each conditioned soil type. Nitrogen fertilizer (105 kg ha-1) was applied to ensure good N. tabacumgrowth.