A split supply of bio-organic and chemical fertilizer synergistically affects root system architecture and improves above-ground growth in pear tree (Pyrus pyrifolia Nakai) CURRENT STATUS:

Background: Root system architecture (RSA) is highly plastic, responding to nutrient availability and the heterogeneity of the soil environment. However, the linkage of root morphology to anatomy and root nutrient, and its implication for root function at the heterogeneous application of bio-organic and chemical fertilizer have not yet been defined, especially for pear trees. Results: In this study, a split-root experiment was conducted using 1-year old ‘Cuiguan’ trees. No fertilizer (NF), chemical fertilizer (CF), and bio-organic fertilizer (BIO) were paired to test six combinations: NF-NF, NF-CF, NF-BIO, CF-CF, BIO-BIO, and BIO-CF. Root morphological, anatomical traits and root nutrient concentrations, and their relationships were determined. Trees receiving BIO had significantly higher lateral root numbers, activity, total lateral root length, and specific root length than trees receiving no BIO, while the effects on root tissue density were the direct opposite. Compared with CF-CF treatment, root xylem thickness, stele diameter, vessel diameter, and number of vessels all increased in response to BIO-CF treatment. Root growth was synergistically promoted in BIO-CF, with increased special root length and root nitrogen concentration, but root tissue density and the carbon:nitrogen ratio were reduced. Intriguingly, the synergistic effect resulted in greater trunk girth without sacrificing height, compared to trees receiving CF or BIO alone. Conclusions: The combination of BIO and CF improves root traits and tree growth, suggesting that using bio-organic fertilizer as a supplement to reduce the application rate of chemical fertilizer is beneficial to orchard ecosystems.

matter, alleviate soil acidification, and increase soil microbial activities, all of which improve crop yield [13]. Unlike chemical fertilizers, organic fertilizers promote the growth of crop roots by decomposing organic matter releasing humus. Humus influences root morphology, it induces proliferation of lateral roots and root hairs, and enhances root cell differentiation [14], which may be related to the presence of auxin in some humus [15]. Bio-organic fertilizer is an organic fertilizer with added functional microorganisms; these improve soil quality, promote plant growth, inhibit soil-borne diseases, and very importantly, are conducive to sustainable agricultural development [16,17].
Studies on bio-organic fertilizer have largely focused on the prevention and control of soil-borne diseases [17], relief from continuous cropping [18], the composition of soil microbial communities and their functional diversity [19,20], and aboveground biomass and yield [21]. There has been a lack of attention on the effect of bio-organic fertilizer on RSA.
Pear is the world's third largest fruit industry, and China's pear orchards account for the world's largest production area and yield [22]. The RSA of pear trees varies greatly with age, soil depth, and spatial heterogeneity of soil nutrients; the RSA of adult trees is very large and complex. Previous studies using cultivated plants and fruit trees have employed integrated excavation methods to measure RSA diameter, the number of roots, their distribution and depth, and root tissue density (RTD) [23,24]. However, this method is difficult to use when studying the RSA of adult pear trees, therefore, studies on the RSA of pear trees have largely been on small trees or saplings.
The goal of modern intensive pear production is to sustainably and predictably produce high yields.
To achieve these goals the sustainable management of soil fertility is fundamental. Long-term application of chemical fertilizer leads to a decrease in pear yield and quality [25], while the application of bio-organic fertilizer can increase pear yield and improve fruit quality (see Fig. S1, data unpublished). The application of bio-organic fertilizer in pear orchards can be toilsome and costly, and it has proven difficult to promote its use in large orchards, but it essential for maintaining soil health [13,20,21,26]. The use of bio-organic in conjunction with chemical fertilizer has proven cost effective, but there are few studies on how the RSA of pear trees respond to soil nutrient heterogeneity caused by a bioorganic/chemical fertilizer combination.

6
The main purpose of this study was to investigate the effects of a BIO-CF combination on the RSA and above ground growth of pear trees. We used split-root planting boxes to grow trees half in soil supplemented with BIO and half in soil supplemented with CF. The controls were trees grown in BIO-BIO, CF-CF, and NF-NF. We analyzed the morphology, anatomy, and chemical properties of the roots under these conditions to clarify the relationship between the root system traits, soil physicalchemical properties and plant growth.
In this study we proposed the following hypotheses: 1) Bio-organic fertilizer should increase lateral root number and length, and shorten branching distance. 2) Combined application of BIO and chemical fertilizer should have a synergistic effect on the growth of pear roots, resulting in increased trunk girth.

Effect of fertilizers on the physical and chemical properties of soil
The chemical fertilizer used in our study had little effect on soil pH and increased the soil C/N ratio by about 10%, while the bio-organic fertilizer added 0.3 -0.7 pH units and increased the soil C/N ratio by about 50%. In boxes with mixed NF-fertilizer (NF-BIO, NF-CF), the addition of BIO in the right chambers increased the SOM, NH 4 + -N, NO 3 − -N, and available K in the left (NF) chambers more than did the addition of CF in the right chambers. In the boxes containing BIO-CF, the soil available P and K, and C/N ratio were significantly reduced in the CF chambers compared to CF-CF boxes (Table S2).

Effect of fertilizers on pear tree growth
The height and girth of trees in NF-BIO boxes were about 24% and 22% greater, respectively, than trees in NF-NF boxes. Trees in NF-CF boxes were not significantly taller than trees in NF-NF boxes, but their trunk girth was about 14% greater (P< 0.05). Trees in BIO-BIO boxes were about 24% taller than trees in CF-CF boxes and their girth was greater by 14%. Trees in BIO-CF boxes were about 18% taller and 19% greater in girth than trees in the CF-CF boxes (Fig 2). These results demonstrate that the growth of the aerial parts of the pear trees were consistently greater in bio-organic fertilizer than in chemical fertilizer.

Effect of fertilizers on root morphology
7 The lateral roots of trees grown in the NF chambers of NF-BIO boxes were greater by all measures than were the lateral roots of trees grown in the NF chamber of NF-CF boxes (Fig 3). In boxes that contained the same growth media in both chambers, the 2 nd and 3 rd lateral root numbers (LRN) accounted for the largest proportion of total LRN. In NF-NF boxes they accounted for nearly 55% of the total, in CF-CF boxes the accounted for 49% and in BIO-BIO boxes they accounted for 25% ( Fig   3H). The number order of lateral roots was above 9 in BIO chambers, and below 7 in NF and CF chambers ( Fig 3G). Because of the very small number of lateral roots above 6 th order in any treatment, all lateral roots of 6 th order and above were classified as 6 th order for ease of statistical analysis (Fig 3H and I). The roots in chambers containing BIO had a significantly greater total number of lateral roots (TLRN) and greater lateral root density, and a smaller mean inter-branch distance (MID), than roots in any other media. The TLRN and lateral root density of pear trees in BIO-BIO boxes were, respectively, 2.7 and 1.8 times higher than trees in CF-CF boxes (Fig. 3A, B and F). This is due to the significantly increased proportion (up to 67 %), of 4 th , 5 th , and 6 th order lateral roots to TLRN in the BIO-BIO boxes ( Fig 3H). Compared to boxes that contained the same fertilizer in both chambers (BIO-BIO and CF-CF), in BIO-CF boxes the TLRN in BIO chambers did not change significantly, but the MID decreased by about 40%; the TLRN in the CF chambers increased by about 50%, while the MID did not change (Fig 3A and F). This is because the orders of the lateral roots (especially 2 nd to 6 th order) in the BIO chambers ( Fig 3H and S3A-E) did not change, but were more evenly distributed on their parent roots, and the length of the lateral roots above 3 rd order was increased (Fig S4B-E), while in the CF chambers the proportion of 4 th and 5 th order LRN to TLRN, LRL to TLRL were all increased ( Fig 3H and I). This data is consistent with the increase in TLRL, total lateral root surface area (TLRS), and total lateral root volume (TLRV) (Fig 3C-E) in BIO-CF boxes, indicating that the combined application of BIO and CF had a mutually beneficial effect on root growth.
The higher the root order, the smaller the lateral root diameter (LRD). The average diameter of the 2 nd lateral roots in the NF chambers of NF-BIO boxes was about 2.5 mm, and the average diameter of 8 2 nd lateral roots in all the other media was less than 2 mm (Fig 4A), which is defined as fine roots. The mean LRD in the BIO chambers of all boxes was between 0.9 mm and 1.2 mm, while the mean LRD in CF-CF or NF-NF boxes was about 0.8 mm (Fig 4F).
Specific root length (SRL) and the ratio of root surface area to root dry biomass (RSAB) of trees in NF-BIO boxes were significantly higher than of trees in NF-NF and NF-CF boxes (ANOVA F value: 31.2, pvalue <0.001) (Fig 5A, B). Root tissue density (RTD) was significantly increased in the NF chambers of NF-CF boxes, while in NF-BIO, the RTD in the NF chambers was decreased ( Fig 5C). Compared to CF-CF boxes, the SRL, RSAB, and branch intensity (BI) in the BIO-BIO boxes were increased by 67%, 94% and 30%, respectively ( Fig 5A, B and D), while the RTD was reduced by approximately 50% (Fig 5C).
In BIO-CF boxes, the SRL and RSAB in both chambers were greater, and the RTD's were smaller than in either BIO-BIO or CF-CF media ( Fig 5A, B and C).

Effects of fertilizers on root anatomy
Our results thus far indicated that a mixed application of bio-organic and chemical fertilizer had a synergistic effect on the RSA of pear trees. Therefore, to study these distinctions in root anatomy, we

Effect of fertilizers on nutrient concentration and root activity
The root carbon, and nitrogen concentration (RCC, RNC) in BIO chambers were substantially higher than in NF or CF chambers (Fig 7A and B), while the RCC/RNC ratio in NF-NF boxes was significantly higher than in any of the other boxes ( Fig 7C). Compared with NF-NF media, trees grown in BIO-BIO had significantly greater RCC and RNC, 22% and 142.3% respectively, while CF-CF grown trees had about 50% more RNC. In the NF chambers of NF-BIO boxes, the RCC and RNC were greater by 17.6% and 77% respectively, than in NF-NF boxes (Fig 7A and B).
Compared with BIO-BIO grown trees, the roots in the BIO chambers of BIO-CF grown trees had about 10% and 5% greater RCC and RNC respectively. Compared with CF-CF grown trees, the roots in the CF chambers of BIO-CF trees had about 36% greater RNC, there was no significant change in RCC (Fig 7A   and B). Trees grown in BIO had significantly greater root activity than trees not treated with BIO, and the root activity in the CF chambers of BIO-CF grown trees was about 50% higher than in CF-CF grown trees (P < 0.05) (Fig 7D). In summary, we found a close correlation between the RSA parameters and the root nutrient content of the pear trees, together these factors determined the nutrient absorption efficiency of the roots.

Redundancy analysis of RSA traits and soil factors
By RDA analysis we found that RSA parameters and root carbon and nitrogen content responded differently to soil physical-chemical factors induced by fertilization (BIO and CF) (Fig 10). RDA1 and RDA2 accounted for 46.1% and 5.9% of the total variation, respectively. Bio-organic fertilizer (NF-BIO, BIO-BIO and BIO-CF), increased soil pH and SOM promoting TLRN, TLRL, SRL, and enhanced root activities and root nutrient content, and reduced RTD and RCC/RNC. Additionally, bio-organic fertilizer improved soil NO 3 --N and NH 4 + -N levels which led to significantly increased TLRN, SRL, RA and RNC of pear roots, but had little effect on RTD and MID (Fig 8 and 10, table S1).

Discussion
In this study we found that between the chambers of split root boxes containing homogenous media (CF-CF, BIO-BIO, NF-NF), there were no significant differences in the morphology or anatomical structure of roots of pear trees (Fig 3, 6 and S5), demonstrating that the split-root boxes and systematic error were negligible in analyzing the influence of soil nutrient heterogeneity on RSA; in other words, the differences in RSA were due to the heterogeneous experimental treatments. in between chambers

BIO-CF had a significant synergistic effect on the growth of lateral roots
Plants must perceive physical and chemical changes in soil nutrient heterogeneity to optimize their root architecture system (RSA) [34], thereinto, lateral root number (LRN), lateral root density (LRD) and inter-branching distance are all parameters that reflect the high plasticity of root systems, and they are all sensitive to nutrient heterogeneity [35,36]. Lateral roots are a key factor determining the RSA of pear trees due to their taproot system. We found that bio-organic fertilizer (BIO) significantly increased the number, length and density of lateral roots of pear trees, and reduced inter-branching distance ( Fig 3A -C and F), supporting the first hypothesis proposed in this paper. Different orders of lateral roots have different functions; lateral roots less than 2 mm in diameter are the most activate part of the root system [37], playing a key role in absorbing soil nutrients and water [9, 27]. We found that while the diameter of 2 nd order and above lateral roots of trees grown in all media was 2 mm or less (Fig 4), the number and length of lateral roots in BIO-CF boxes was significantly higher than that of CF-CF boxes, indicating that the bio-organic/chemical fertilizer combination was particularly conducive to allowing pear roots to occupy a large soil space and thereby improve nutrient absorption efficiency. In addition, lateral roots grown in BIO reached about 10 orders, lateral roots grown in CFtreated were below 7 orders ( Fig 3G). Khan et al. [38] argued that the application of organic fertilizer significantly increased the number of lateral roots of oats, but did not focus on the maximum order that can be reached by lateral roots.
In order to study the mechanism of plant response to nutrient heterogeneity, the split-root is the most effective method [39,40]. Compared to the roots growing in boxes with the same media in each chamber, plants grown in boxes with a different media in each chamber can only absorb 75% of the nutrients in their own chamber and the growth of the root is asymmetrical [41]. This is consistent with the differences we saw in the growth of the pear roots treated with NF-BIO, NF-CF, and BIO-CF (Fig 3).
The heterogeneity of soil nutrients stimulates long-distance signal transduction in plants, regulating hormones such as abscisic acid (ABA), growth hormone (such as IAA), and cytokinin (CK), which affect lateral root growth, and the redistribution of photosynthetic products (for example, amino acids).
Together, these act to optimize the RSA of plants, allowing them to better exploit the soil environment [42,43]. We found that application of BIO in one chamber of split root boxes significantly increased the number and length of lateral roots in the neighboring NF chamber, while the analogous application of CF has no significant effect on the number or length of lateral roots in the neighboring NF chamber (Fig 3A and C). Interestingly, in BIO-CF boxes the TLRL, TLRS, and TLRV of pear roots were increased in both chambers over the roots of trees in BIO-BIO or CF-CF (Fig 3C-E), demonstrating an obvious synergistic effect. This BIO-CF heterogeneity may cause an alteration in the distribution of root exudates, promoting root growth toward the patch of elevated available soil nutrients [44][45][46].
Further research is however needed to determine the changes in the composition and distribution of the root exudates and their effects on the organization of rhizospheric microbes.
More than 80% of soil microbes can synthesize IAA, and tryptophan is considered the core substrate for bacterial synthesis of IAA [47,48]. High concentrations of IAA stimulate the formation and growth of lateral roots, thus increasing their surface area, which is favorable for the absorption of soil nutrients [49]. Bio-organic fertilizer contains Bacillus amyloliquefaciens SQR9 which enriches the rhizosphere and colonizes root surfaces (Fig. S6); SQR9 contains the functional gene dhaS, which encodes indole-3-pyruvate decarboxylase (IPDC) that in turn leads to production of IAA by the indole-3-pyruvic acid (IPyA) pathway [47,48], thereby stimulating the formation and growth of lateral roots.
Chemical fertilizer delivers a large amount of available nutrients over a short time frame, this high nutrient availability may result in the increased content of tryptophan in the photosynthetic products distributed to roots. In this way, beneficial microorganisms may be recruited, enriching the rhizosphere and boosting IAA production, thereby stimulating lateral root growth. We found that BIO-CF had a synergistic effect, obvious in increased trunk girth (about 19%) without a concomitant sacrifice to tree height (Fig 2 and Fig 11); this supports our second hypothesis.

BIO-CF resulted in increased SRL and reduced RTD
Root characteristics, such as RTD and SRL, reflect their role in maximizing access to and use of limited soil nutrients [50]. RTD increases as soil nutrient availability decreases [51], this tendency is associated with slower root growth and longer lifespans. Greater SRL indicates root systems have built more root length per unit dry-mass, generally these roots have higher rates nutrient and water uptake but shorter lifespans than low SRL plants [52]. In contrast, Kramer-Walter et al. [50] argued that low soil nutrients would result in increased SRL, due to root systems needing to explore more soil volume to obtain nutrients. In our study, the application of BIO in one chamber of a split root box resulted in increased SRL and decreased RTD of lateral roots in the other chamber of the split root box (Fig 5A, C and Fig 11). This may because when the pear tree roots sensed a heterogenous soil environment, the carbon input per unit of root length increased, causing a change in and redistribution of root exudates (Fig 7A), thereby prompting roots to spread over a large soil space.
Another important finding from this study is that the BIO-CF combination resulted in increased SRL and reduced the RTD on roots in each chamber ( Fig 5A, C and Fig 11).This may indicate that the lifespan of lateral roots is shortened, thereby increasing nutrient absorption efficiency [53]. Many studies suggested an intimate correlation between fine roots diameter and lifespan [54,55].
However, understanding how the relationship between fine roots diameter and lifespan of pear trees is affected by the BIO-CF heterogeneity is also a meaningful research priority.

BIO-CF resulted in increased average vessel diameter, number of vessels and their density of lateral roots
To determine how the RSA of pear trees responds to heterogeneous fertilization (BIO-CF), we used homogeneous fertilization (CF-CF and BIO-BIO) controls, and 4 th order lateral roots to analyze the relationship between anatomical structure and fertilization strategy. The root samples collected in this 13 study were mature lateral roots, (collected 120 DAB), meaning their outermost cell structure was a cork layer (Fig S5). in pear roots (Fig 6 and Fig 11). These factors improve the absorption efficiency of nutrients and water by lateral roots and are consistent with the morphology of lateral roots in BIO and BIO-CF described above.

BIO-CF resulted in increased RNC and reduced RCC/RNC
Generally, because of the rapid turnover of fine roots, the usually have a high N concentration and low C/N ratio [59]. Our results showed that in BIO chambers, RNC and root activity (RA) was higher than in NF and CF chambers (Fig 6B and D), while RCC/RNC was lower ( Fig 7C). These data indicate that BIO not only improves RA, promoting the absorption of soil available nutrients, but also may accelerate the rate of root turnover. This corresponded with our earlier observations that BIO increased SRL but reduced RTD (Fig 5A and C). The application of BIO in one chamber may also promote the absorption of soil nutrients and turnover rate of fine roots in the companion chamber, especially a CF companion chamber (Fig 7 and Fig 11). In low nutrient content conditions (NF-NF), RNC was also low (Fig 7B).

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.  Fig. 1. amyloliquefaciens SQR9 colonies per gram of fertilizer were 1 × 10 9 CFU g −1 dry weight. 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

Split-root boxes and experimental design
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. The number of second order lateral roots were counted manually, then three second order lateral roots (2 nd 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.

Sampling and measurements
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, cm 2 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 (4 th 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 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 4 th 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 (NH 4 + -N) and nitrate (NO 3 − -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.

Supplementary Information
Additional file 1: Table S1. Fertilization protocol Table S2. Properties of the soils from the split-root chambers Figure S1. Effects of different fertilizers on yield and SSC of pear.          Error bars represent 1SE of the mean (n = 9). According to LSD tests, ns indicates nonsignificant difference among fertilized chambers at P > 0.05; significant differences are indicated by different lowercase letters at P < 0.05; † means significant differences among CF-CF and BIO-BIO conditions at P < 0.05.

Figure 7
Root nutrient content and activity (A) Carbon concentration, (B) N concentration, (C) C/N ratio, (D) and root activity. Error bars represent 1SE of the mean (n = 3). † means significant differences (P < 0.05) among the NF-NF, BIO-BIO and BIO-CF boxes at P < 0.05, §means significant differences among the twelve chambers of the boxes, and * means significant differences between the left and right chambers in the same box, according to LSD (P < 0.05) tests.

Figure 8
Pearson's correlations show the relationships between root system architecture and soil properties. Bars in shades of blue represent a higher correlation coefficient (r), compared to the control, bars in shades of red represent a lower correlation coefficient (r), compared to the control. Empty grids mean no difference. * P < 0.05, ** P < 0.01. SOM, soil organic matter; TN, total soil nitrogen; AP, available phosphorus; AK, available potassium; C/N, ratio of soil organic carbon to soil total nitrogen; TLRL, total lateral root length; TLRS, total lateral root surface; TLRV, total lateral root volume; TLRN, total lateral root number; MID, mean inter-branch density; RA, root activity; SRL, specific root length; RSAB, ratio of root surface 36 area to root dry biomass; RTD, root tissue density; BI, branch intensity; MRD, mean lateral root diameter; RCC, root carbon concentration; RCN, root nitrogen concentration; RCC/RNC, ratio of root carbon concentration to root nitrogen concentration.

Figure 9
Linear regression analysis of the relationships between pear tree root traits and root nutrient content. The filled circles represent the fertilizer used: blue-NF, green-CF, and purple-BIO. TLRL, total lateral root length; TLRN, total lateral root number; SRL, specific root length; RTD, root tissue density; MRD, mean lateral root diameter; RCC, root carbon concentration; RNC, root nitrogen concentration; RCC/RNC, ratio of root carbon concentration to root nitrogen concentration. * P < 0.05, ** P < 0.01, *** P < 0.001.

Figure 11
Overview of the main RSA traits, root anatomical structure, and nutrient content in response to nutrient heterogeneity. Colored solid lines indicate primary roots and the different orders of lateral roots, line thickness indicates relative root diameter. The roots of a pear tree were placed in planting boxes such that half the roots grew in one chamber and half in a second chamber, these chambers were filled with soil and supplemented with bio-organic (right/grey square), or chemical fertilizer or unsupplemented (left/orange square). The gradually narrowing purple triangles, and their direction, represents the reduction of variables. RSA traits include global root parameters (such as total lateral root length, total lateral root number, total lateral root surface and volume), SRL and RTD. Root anatomical structures are the cork layer, xylem, phloem, and vessels. Root nutrition consisted of RCC

Supplementary Files
This is a list of supplementary files associated with this preprint. Click to download.