Field site description
Pot experiments were conducted from August 2019 to October 2019 at a net house of the Chinese Academy of Agricultural Sciences (39°57'37'' N, 116°20'0.9'' E), Beijing, China. Sandy loam potting soil was collected from the 0–20 cm surface layer of cultivated soil from the International High-tech Industrial Park, Chinese Academy of Agricultural Sciences (39°36'53'' N, 116°36'89'' E), Langfang, Hebei Province. The soil bulk density was 1.43 g cm3, the pH was 8.25, the organic matter content was 10.06 g kg− 1, the available N content was 58.4 mg kg− 1, the P content was 20.4 mg kg− 1, and the K content was 112.4 mg kg− 1.
Experimental design
Four treatments were designed: three SSM treatments (S3, S2, and S1) and a control FSM treatment (F). Manual irrigation with upper and lower limits (70–90% field capacity) was used for treatment F. Different NPI water pressures of − 3, −6, and − 9 kPa were used to achieve SSM conditions for S3, S2, and S1, respectively. The three SSM treatments were designed to generate a water content gradient that matched the range of soil moisture content in treatment F and explore the trends among different soil moisture content levels under SSM. Three replicates were performed per treatment, where each pot was regarded as a replicate, with a total of 12 pots in the experiment.
The NPI device was consists of three parts: a negative pressure generator, a water bucket (inner diameter of 13.1 cm and height of 80 cm), and a capillary water emitter (length 250 mm, outer diameter 19 mm, and inner diameter 10 mm); each part was connected via silica gel pipes (Fig. 1A). Absorption of water by crops reduces the soil water pressure (SWP). Thus, once the SWP is lower than the negative water pressure set, the soil actively absorbs water from the water bucket to prevent a further decline in SWP, thereby maintaining soil moisture stability [16].
In the present study, Lactuca sativa L. var. longifolia, which is commonly referred to as Meilijian lettuce, was used as the experimental romaine lettuce. The sowing of seeds in each pot began on August 20, 2019, and six holes per pot and five seeds per hole were used. Before sowing, 8.35 g of urea, 4.57 g of calcium super-phosphate, and 2.56 g and potassium sulphate powder were mixed and applied to each pot. Each pot contained 23 kg of soil and received 5 L of water to fully wet the soil. The initial volumetric soil moisture content (θv) was 28.3%. Once seedling grow four leaves, the seedlings were thinned to one per hole, with six plants per pot. The NPI system was used to control pot water from the time at which most plants had grown four leaves (September 16) until harvest (October 12).
Plant and soil sampling
Plant and pot soil samples were collected when the lettuce was mature. The whole plant (including roots) was removed, and the above-ground and below-ground parts were isolated to keep the roots attached to the soil. Soil removal began with gentle shaking of large clods until a 1–2 mm soil layer was left on the roots; soil that fell off was regarded as bulk soil. The remaining soil was placed into a centrifuge tube filled with phosphate buffered saline and vibrated until the rhizosphere soil fell off. Roots were removed from the centrifuge tube, and the centrifuge tube was placed in a high-speed centrifuge at 4 ℃ and 10,000 rpm for 10 min; any sediment remaining in the tube was classed as rhizosphere soil [4].
DNA extraction, PCR amplification, and sequencing
DNA was extracted from 0.5-g samples of rhizosphere and bulk soil using a Fast DNA SPIN Kit (MP Biomedicals, USA). The concentration and quality of DNA were determined using NanoDrop 2000 (Thermo Scientific, USA) and 1% agarose gel electrophoresis, respectively. The v3–v4 region of the bacterial 16S rRNA gene was amplified by PCR using the upstream primers 338F and downstream primer 806R. The v5–v7 region of the fungal ITS gene was amplified by PCR using the upstream primer ITS1F and the downstream primer ITS2R. Each DNA sample was amplified by PCR with three replications, and PCR-amplified products were tested by agarose gel electrophoresis (2%). The PCR products were purified using an Axyprep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) and a Quantus™ Fluorometer (Promega, USA) for quantitative analysis. Purified PCR products were sequenced using high-throughput Illumina MiSeq (Illumina, USA). The obtained sequences were submitted to the NCBI database (accession numbers PRJNA744013 and PRJNA744028 for bacterial and fungal sequences, respectively).
Measurements
Plant growth and yield Plant growth parameters were measured every 10 days in terms of plant height, number of leaves, and the largest leaf length and width. The above-ground yield was measured after harvest.
Water parameters An AZS-100 soil moisture meter (Beijing Aozuo Ecology Instrumentation Ltd.) was used to measure the soil volumetric moisture content every two days during the irrigation period. Soil moisture stability was evaluated using the coefficient of variation (CV) of soil moisture, which was calculated according to [17]:
Cv = σ / µ (1)
where σ and µ represent the standard deviation and average value of the soil moisture content, respectively. A weak temporal variation in soil moisture exists when Cv ≤ 0.1, a medium temporal variation exists at 0.1 < Cv < 1, and strong variation exists when Cv ≥ 1.
The coefficient of fluctuation (Cf) of the soil moisture was calculated according to [16]:
Cf = ∑ [|θi - θi−1| / ( θi + θi−1 ) / 2] / ( n-1) (2)
where θi is the observed soil moisture content at time i, θi−1 is the observed soil moisture content at the previous moment of time i, and n is the number of observations. The coefficient of fluctuation reflects the stability of soil moisture, with smaller values representing a more SSM content.
The water consumption of lettuce was calculated according to [16]:
ETk = Mk − ΔW = Mk − (θmk - θmk−1) × ms/ρw, (3)
where ETk is the amount of water consumed in the Kth period, Mk is the amount of irrigation in the Kth period, ΔW is the change in soil water storage, θmk is the soil mass moisture content at the Kth moment, θmk−1 is the soil mass moisture content at the previous Kth moment, ms is the weight of soil in the pot, and ρw is the density of water (1 g cm− 3).
The WUE of a single plant was calculated as:
WUE = Y / I (4)
where Y is the plant yield (g), and I is the irrigation volume (L) per plant (irrigation volume per pot divided by six).
Statistical analyses
Raw data of the high-throughput sequences were processed using fastp 0.20.0, flash 1.2.7, uparse 7.1, RDP classifier 2.2, and mothur1.30.2. All other data were processed using Microsoft Excel 2016. All data were analysed via SPSS 17.0 and plotted using origin8.5 and R v3.6.6.