Substrates and materials
In October of 2017, approximately 3000 kg of surface soil, 0 to 30 cm deep, was collected from the pear garden site of the Lvyuan Fruit Industry Co., Ltd., Jianning County, Sanming City, Fujian Province, China (34°04' N, 108° 10' E). The soil type is red loam and the texture is clay, the field water holding capacity is 33.8%, soil organic matter content is 15.70 g kg-1, total nitrogen content is 2.08 g kg-1, available potassium content is 184.5 mg kg-1, available phosphorus content is 45.8 mg kg. -1, and the pH is 5.33. The collected soil was naturally air-dried and passed through a 5 mm sieve before use. From January to November 2018, we carried out a potted split-root test at the Nanjing Agricultural University Test Base (31°36' N, 119°10' E), Baima Town, Lishui District, Nanjing, Jiangsu Province. The climate type at test site is the transition zone from north subtropical to mid-subtropical. The climate is mild and humid, the annual average for temperature is 15.4 °C, sunshine is 2240 h, and rainfall is 1087.4 mm, the frost-free period is 237 d. For the monthly climatic conditions during the test period see Fig. 1.
Split-root boxes and experimental design
Brown plastic planting boxes, 39 x 39 x 45 cm (l x w x h), were used for the split-root test, each box was divided into two 39 x 19 x 45 cm (l x w x h) chambers, the planting depth was 40 cm. The base plate (39 x 39 x 3 cm) of each box was black plastic, with a 10 mm diameter water outlet on each of the four sides. Four hollow poles (21 cm x 2 mm) were seeded into the base plate and four black mesh water-repellent plastic plates with many 2 mm diameter filter holes were slid into place between the poles, forming the sides of the box, each seam was then sealed with strips of black plastic. The box was then divided into two equal chambers with a white hard plastic plate 39 x 38 x 0.3 cm, (l x w x h), and marked with L (left) and R (right). The interface between the partition and the panel was sealed with waterproof 580 glass glue, and was allowed to dry naturally. In order to facilitate the fixation of the root systems, a 3 x 3 cm2 notch was cut in the middle of the top of the white plate (Fig. S2).
On January 25, 2018 approximately 25 kg of air-dried soil (bulk density 0.84 g cm-3) was loaded into the compartments of each planting box and irrigated until the soil water content was about 30% (vol). 1-year old dormant ‘Cuiguan’ pear trees with two fork-shaped primary roots, in this experiment, according to the root order nomenclature by Barczi et al. [27], we treated them as the 1st order roots, that were nearly identical in length were selected and planted. Each rootstock was Pyrus betulaefolia and the height above the base was approximately 60 cm. Roots were distributed as evenly as possible into the two chambers, then each box was wrapped in black plastic grass cloth to prevent moisture evaporation and weed growth. The following six treatments were set up for the split-root test: no fertilizer in either chamber (NF-NF), no fertilizer in the L chamber-chemical fertilizer in the R chamber (NF-CF), no fertilizer in the L chamber-bio-organic fertilizer in the R chamber (NF-BIO), chemical fertilizer in both chambers (CF-CF), bio-organic fertilizer in both chambers (BIO-BIO), bio-organic fertilizer in the L chamber and chemical fertilizer in the R chamber (BIO-CF); there were six replicates per treatment. The bio-organic fertilizer was purchased from Jiangsu Xinli Bio-fertilizer Engineering Center Co., Ltd. This product was a mix of solid decomposed organic fertilizer and agricultural amino acids (6:4 dry weight) inoculated with Bacillus amyloliquefaciens SQR9. B. amyloliquefaciens SQR9 colonies per gram of fertilizer were 1 × 109 CFU g−1 dry weight. The composition of this fertilizer was N 4.77%, P2O5 2.26%, and K2O 1.00%. Each BIO chamber received 500 g of fertilizer as a one-time base treatment, this amounted to 2% (w/w) to soil. Chemical fertilizer consisted of analytically pure urea (N 46%), potassium sulfate (K2O 50%) and superphosphate (P2O5 15%). 70% of the urea and potassium chloride and 100% of the superphosphate were added to the CF chambers as a base fertilizer on Jan 25, 2018. The remaining of urea and potassium chloride was applied as a top dressing on June 20, 2018. Each CF chamber received equal amounts of N, P, and K (23.8 g N, 13.3 g P2O5, 23.8 g K2O).
These fertilizers were granular solids which were fully dissolved in 2 L water before application, and were uniformly applied to the chambers in combination with irrigation. The trial period began on March 20 (germination stage) and ended on November 10 (abscission period). During this time an automatic drip irrigation system was used to supply the water to the trees, the flow rate was 12.5 ml min-1. The weekly irrigation schedule was 18:00 ~ 20:00 on Wednesdays and Saturdays, except for July to August, when it was 18:00 ~ 20:00 on Tuesdays, Thursdays, and Saturdays.
Sampling and measurements
210 days after budding (DAB), plant heights from the base of the trees to the highest apical bud were measured with a tapeline, and trunk girths were measured with a Vernier caliper 15 cm from the base of the trees. From each treatment group, three plant boxes were randomly selected, and the root systems were phenotyped using Shovelomics [28]. A black marking pen was used to mark on the “L” side of each trunk, about 50 cm from the base, to distinguish the root chambers. Roots were gently extracted from the box chambers and were cleaned of soil by gentle shaking, sterile tweezers, then slow agitation while immersed in a 60 cm diameter plastic bucket of water. Finally, the root systems were gently rinsed with tap water until no soil remained.
The number of second order lateral roots were counted manually, then three second order lateral roots (2nd LR) about 20 cm in length, were randomly selected from each side (L and R) of each sample, and cut away from the parent roots using a sterilized garden shear. These roots were placed horizontally on a plastic plate covered with a black photography cloth, the entangled and overlapping roots were separated using a sterile tweezer then photographed with a Canon EOS 750D Digital SLR camera (EF - S 18 - 135 mm f / 3.5 - 5.6 IS STM lens, 24.2 million) fixed at a vertical height of 60 cm above the samples. All images were saved for subsequent RSA analysis. After photo documentation, all roots were separately placed in a bubble envelope and dried at 70 °C for 72 h and then weighed. The specific root length (SRL m g−1, the ratio of root length to dry mass of fine roots), root tissue density (RTD g cm−3, root dry mass divided by fresh root volume), and the ratio of root surface area to root dry biomass (RSAB, cm2 g-1) were calculated. Fine roots (diameter < 2 mm) were smashed in a SPEX 8000-D hybrid grinder (SPEX, Edison, NJ, USA) to analyze the root carbon concentration (RCC) and root nitrogen concentration (RNC). Ten fourth lateral roots (4th LR) were also randomly selected from each side of each sample, cut into 1 cm segments starting from the root apex, then fixed for 48 h in FAA fixative solution (90 ml of 50% ethanol, 5 ml of 100% glacial acetic acid and 5 ml of 37% formaldehyde) for anatomical structure analysis.
Root morphology
Semi-automatic SmartRoot (https://smartroot.github.io) [29, 30] combined with ImageJ 1.46R (http://imagej.nih.gov/ij/download.html) were used to analyze the lateral root images for total lateral root length (TLRL), total lateral root surface area (TLRA), total lateral root volume (TLRV), total number of lateral roots (TLRN), mean lateral root diameter (MRD), mean branching angle (MRA), and mean inter-branch density (MID), maximum order of lateral roots (MOLR), and branching intensity (BI).
Root anatomy
The FAA fixed root segments were stained with 2% saffron green then dehydrated in a series of increasing ethanol concentrations 75%, 85%, 90%, 95%, 100%, then alcohol/benzene, xylene, and then embedded in 65 °C paraffin. From each sample 3 to 5 root sections, about 8 μm thick, were cut with a RM2016 microtome (Shanghai Leica Instrument Co., Ltd.) then embedded in Technovit 7100 resin (Heraeus Kulzer, Germany). From each sample, three slides were randomly chosen for observation. Slides were photographed using a DM21-J1200 optical microscope and ScopeImage 9.0 software. ImageJ 1.46R was used to measure cork layer thickness, xylem thickness, phloem thickness, vessel diameter, stele radius, number of vessels in each stele, total cross-sectional area of the xylem (CSAX), and the ratio of stele diameter to root diameter (SDRD). Representative cross-sectional images of these 4th lateral roots are shown in Fig. S5.
Root chemical analysis and activity
Root carbon and nitrogen concentrations were measured using a Macro Elemental Analyzer (vario MACRO, Elementar Co., Hanau, Germany). Root activity was measured using the TTC (triphenyl tetrazolium chloride) method described in Chen [31], activity was expressed as the deoxidization ability (mg g−1 FW h−1).
Soil sampling and chemical analysis
Soil samples from roots were collected 210 days after budding (DAB). Specifically, approximately 100 g of soil was shaken from the roots in each chamber, air dried, and then passed through a 2 mm sieve to remove impurities such as stones, glass, and residual roots. An acidometer (MT-5000, Shanghai, China) was used to measure soil pH. A flow injection auto-analyzer (AA3, Seal Co., Germany) was used to determine soil ammonium (NH4+-N) and nitrate (NO3−-N) concentrations. Total soil N concentrations were determined using an elemental analyzer (Vario Macro, Elementar Analysensysteme, Germany). Soil organic matter content (SOM) was determined by the chromic acid titration method according to Bao [32]. Ammonium acetate was used to extract soil available K (AK), which was then quantified using flame photometry. Sodium bicarbonate was used to extract soil available P (AP), which was then quantified using the molybdenum blue method Bao [32].
Statistical analyses
One-way ANOVA was used to identify the effects of chemical and bio-organic fertilizer on root morphology, anatomy, and chemical composition. The difference in significance between treatments was determined by Fisher's LSD analysis (P < 0.05). Pearson’s correlation was used to determine the relationships among RSA parameters, anatomical characteristics, roots chemical concentrations, and soil physicochemical traits using R v.3.1.3 software. Linear regression analysis was used to describe the relationship between the key RSA parameters of pear trees. Root traits and environmental factors were completed with the CANOCO 4.5 software package using R v.3.1.3 software [33]. Redundancy analyses (RDA) were used to test the interrelationships of root traits, and their responses to soil nutrient following the different fertilization. All statistical analyses were performed using the IBM SPSS 20.0 software. Visualization of all data was done in the Origin 2019b software.