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.