Nitrogen-rich animal and plant wastes as fertilizer improve the soil carbon/nitrogen ratio and plant branching and thickening of young walnut trees under deficit irrigation conditions

ABSTRACT To investigate the physiological status of plant following the application of a mixture of organic residues, nitrogen-rich meat powder and alfalfa (Medicago sativa L.) powder mixed in different proportions as fertilizers were applied to the soil of a hilly area to cultivate young walnut trees under drought and wet soil conditions, respectively. The results showed that the nitrogen-rich fertilizer treatments could significantly increase the soil water content (SWC), soil organic matter (SOM), soil organic carbon (SOC), and soil organic nitrogen (SON), total nitrogen (TN), total phosphorus (TP), total potassium (TK), available nitrogen (AN), available P (AP), available K (AK), a high soil organic carbon/nitrogen ratio under soil drought conditions. Moreover, they also significantly improved the soil nutrients contents of Ca, Mg, Fe, Mn, Cu and Zn, especially Ca and Mg in dry soil. Consequently, this nutrient-rich soil greatly promoted the plant branching and thickening with a high net leaf photosynthesis rate (Pn), water use efficiency (WUE), a low transpiration (Tr) under soil drought conditions. We proposed that high soil carbon/nitrogen ratio improved by applying nitrogen-rich fertilizers might weaken top shoot growth and regulate the plant branching and thickening of young walnut trees under deficit irrigation conditions.


Introduction
It was well-known that soil drought restrict the assimilation, utilization, and cycle of the soil nutrients supplying to plants, especially the C and N (Fan et al. 2005). Soil drought also restricts the availability of N, P, and K, leading to the deficiency of plant growth rates (Schjønning et al. 2003). Moreover, soil drought influences the feedback and interactions among plants, soil, and soil microbes by reducing microbial activity and affecting the microbial community composition, disturbing plant C inputs and relatively stable soil C pools (Sommerfeldt et al. 1988;Canarini et al. 2018). Under soil drought conditions, most vegetation displays slow growth vigor, loss of biomass, low coverage, and weak physiological regulatory functions, which are attributed to the decrease in soil nutrients and carbon storage (Farooq et al. 2009;Kaushal and Wani 2016). These phenomena are particularly commonplace in hilly areas due to seasonal drought and perennial wind erosion year-round (Yuan et al. 2016;Zhu et al. 2017;. Therefore, soil improvement to promote nutrient availability and water-holding capacity under soil drought stress has become an increasingly prominent issue for enhancing plant growth and increasing biomass and economic yield. Fertilizers made from animal or plant residues, such as plant straw, dead branches and leaves, animal blood, plucks, bones, or feathers, not only contains N, P, K, Ca, Mg, S, and trace elements, but also are rich in organic matter, humic acids, amino acids, and other plant nutrients, which could be utilized by plant roots and edaphons, improving the plant growth and enhancing the regulation functions and services of regional agroecosystems (Haraldsen et al. 2011;Mekki et al. 2016). Therefore, they could be used as soil amendments to alleviate the decline of soil function in hilly areas (Chen et al. 2017;Yin et al. 2018), and reduce heavy metal uptake (Yu et al. 2023), and improve the abundance and diversity of microbes in plant root and rhizosphere (Jin et al. 2023). Many studies have indicated that adding organic fertilizer can increase SOM levels, including SOC and SON. In addition, organic fertilizers accelerate SOM degradation and mineralization, accompanying the selective accumulation of small inorganic molecules. In turn, the decomposition of organic matter affects soil physicochemical properties, enhances soil microorganism and enzyme activity, thus promoting high crop yields above the ground. In addition, organic fertilizers could strengthen the soil aggregate structure. At present, most of the studies on organic fertilizers mainly focus on their effects on reducing the amount of chemical fertilizer application, improving the formation of soil texture, especially strengthening the soil aggregate structure Rui et al. 2022), thus promoting the soil fertility and soil nutrient supply to plants for high crop biomass, economic yields, and quality.
Some studies have demonstrated that these effects of straw return increases SOC reserves (Wang et al. 2015;Zhu et al. 2015;Hu et al. 2016). Importantly, these effects are linked to the mechanism by which organic fertilizer elevates the soil osmotic potential and thus improves the water holding capacity and fertilizer preservation (Elliott et al. 2014). Due to the increasing shortage of water resources globally, developing water-saving technology and improving the utilization efficiency of limited water have become more and more urgent in agriculture production. Developing new efficient, pollution-free, drought-resistant, and water-saving organic fertilizer from animal and plant wastes will be an effective way to solve this problem.
Adding plant-derived organic fertilizer increases the leaf C/N ratio of plants by improving overall soil fertilization capacity, and maintains soil water holding capacity (Bi et al. 2009;Qiu et al. 2015). Animal residues as fertilizer could improve the nutrient accumulation and distribution in plants, resulting in changes in plant root and branch ratios because of their rich content of nitrogen components (Okoli and Nweke 2015;Thomas et al. 2016). The study of Smith illustrates that the available N released by the blood powder fertilizers is more absorbable than inorganic nitrogen fertilizers for lettuce (Smith and Hadley 1989). The plant yield using only blood powder fertilizers is sometimes less than that using a mixture of chemical fertilizers (Blatt 1991). Blood powder fertilizers may require the auxiliary and synergetic effects of other fertilizers.
Alfalfa powder fertilizers are rich in the nitrogen-containing nutrients required for plant growth, and they improve soil nutrient and dry matter accumulation in walnut plants under soil drought stress (Ma et al. 2019;Zhang et al. 2020). The meat powder and blood powder are also rich in nitrogen, which benefit soil nutrients and plant growth. However, the coupling effects of nitrogenrich animal wastes combined with nitrogen-rich plant wastes as fertilizers on the soil nutrient composition and plant growth have not been well investigated. We hypothesized that organic fertilizers from animal and plant residues could regulate the C/N ratio and enhance the soil fertility and water-holding capacity of soil, resulting in a synergistic improvement of plant vegetative growth under soil drought conditions. Walnut (Juglans regia L.) fruits contain many nutrients that are good for human health and harbor high economic value. The tree is sensitive to abiotic stresses (Lotfi et al. 2009;Chatrabnous et al. 2018). We aimed to investigate the coupling effect of fertilizer and water driven by animal and plant source fertilizers on soil water retention, the nutrient supply and growth of young walnut plants. Moreover, this study attempts to answer the following questions: 1) Do nitrogen-rich fertilizers alter the soil C/N ratio, thus enhancing plant vegetative growth and regulating plant branch architecture under drought conditions? 2) What is the coupling effect of nitrogen-rich fertilizers and deficit irrigation on plant growth and branch architectures? 3) How do these coupling effects under soil drought conditions mediate SOM decomposition, and regulate soil organic compound conversion and inorganic nutrient release in hilly soil? 4) How do these processes promote C assimilation and the accumulation of dry matter in whole young plants?

Experimental materials
Two-year-old walnut plants of Juglans regia cv. 'Xiangling' grown with similar growth vigor from the Beijing Forest and Fruit Research Institute (China, Beijing 40°05´N 116°18´E) were collected as the experimental material. The soil texture for cultivation was loam collected from the walnut planting area in the mountainous area of Mentougou District, Beijing. This region is located at 39°48´N and 115°25´E, with an altitude of 2300 m, and is classified as having a temperate, semihumid continental monsoon climate, with an annual average wind speed of 2.7 m s −1 and 21 strong gales. The annual mean rainfall is 500 mm and 377 mm at the lowest rainfall, and approximately 76% of the annual precipitation falls in summer. The annual mean temperature is 10.2°C. The test soil pH was approximately 6.5. The SWC was 13.2%. The SOM content was 15.6 g kg −1 , and the TN, TP, and TK, Ca, Mg, iron (Fe), manganese (Mn), copper (Cu), and zinc (Zn) contents were 68.5, 33.4, 131.7, 1570.0, 303.9, 12.1, 22.1, 1.2, and 1.5 mg kg −1 , respectively. Pulverized meat powder contained TN 0.194 g kg −1 , TP 1.548 g kg −1 , TK 2.960 g kg −1 , Ca 0.251 g kg −1 , Mg 0.431 g kg −1 , Fe 0.049 g kg −1 , Mn 0.006 g kg −1 , Cu 0.002 g kg −1 , and Zn 0.0195 g kg −1 . The alfalfa powder contained TN 0.145 g kg −1 , TP 1.303 g kg −1 , TK 1.463 g kg −1 , Ca 17.780 g kg −1 , Mg 2.235 g kg −1 , Fe 0.045 g kg −1 , Mn 0.013 g kg −1 , Cu 0.002 g kg −1 , and Zn 0.009 g kg −1 (Table 1). These materials were obtained from China Agricultural Co., Ltd. This company has a fertilizer business license in China, and its products have passed the quarantine of the China Agricultural Quarantine Inspection Department and the risk assessment of the China Environmental Protection Department. A plastic pot with a diameter of 25 cm × 30 cm height were used.

Experimental treatments
To investigate the separate and mixed effects of nitrogen-rich animal and plant waste fertilizer on soil nutrients and plant growth under wet and drought soil conditions, we established three levels (0, 10 and 40 g) of meat powder and two levels (0 and 30 g) of alfalfa powder in a total of five fertilizer treatments as follows: T1 (control, CK), 0 g of meat powder and 0 g of alfalfa powder; T2, 10 g of meat powder and 0 g of alfalfa powder; T3, 40 g of meat powder and 0 g of alfalfa powder; T4, 10 g of meat powder and 30 g of alfalfa powder; T5, 40 g of meat powder and 30 g of alfalfa powder. These components were mixed into the prepared soil on a 1.5 m × 1.5 m plastic sheet and incubated for 7 months in the greenhouse of Beijing University of Agriculture.
The following spring, we selected young walnut trees of uniform height, with a 50 cm stem height, full buds, and left intact at the main stem, and we removed all the lateral shoots. We dug the trenches with shovels approximately 30 cm from the edge of the walnut tree trunk and 50 cm deep for young walnut tree uprooting. We retained 20 cm diameter soil balls covering the walnut tree roots and removed the excess soil. The trees were immediately wrapped in plastic bag to avoid water loss and taken to the laboratory. Before transplanting young walnut trees, loose soil was gently shaken from the root, and attached soil was washed from the root surface with deionized water. Next, we covered the bottom of the pot with three layers of nylon cloth to prevent the soil from seeping through the hole in the pot bottom. Then, the prepared soil containing combined fertilizer was added to approximately 1/5 of the pot height, and the remaining soil was filled to the root collar in two separate batches after placing the young trees to maintain a stretched root system, with 8 kg soil with fertilizers per pot. Finally, we immediately watered the trees with deionized water until the soil moisture was saturated after compacting the soil. After one month of slow seedling growth, we carried out the wet treatment (watering with 100 mL per pot every day) and drought treatment (watering with 100 mL per pot every 10 days). The planting pots were wrapped with plastic film to prevent the loss of surface soil moisture through evaporation. Two irrigation holes were set 5 cm from the trunk on each side, and a plastic pipe with a diameter of 2 cm and a length of 5 cm was inserted into the soil to seal the tops of the water holes with plastic film after watering. A two-factor randomized block design was used in the present experiment, with a single plant plot and three replicates. All experiments were conducted in a greenhouse at Beijing University of Agriculture at 25-28°C and 50% relative humidity. After initiating the experiment, soil physicochemical indices, plant growth, and physiological indices were detected every 20 days to observe dynamic changes. The last test was completed 80 days later, and the data for the final test were selected for analysis.

Soil sampling
Soil samples (0-25 cm depth) were obtained with a stainless-steel soil auger (4 cm diameter) at 2 locations with 10 cm away from the young tree trunk and at a depth of 20 cm and a 90° angle from the irrigation holes. Taking 200 g of fresh soil from each location to form one composite sample per pot. The collected soil samples were sieved (<2 mm) and divided into two subsamples. One was stored at 4°C to determine SWC, and the other was stored at room temperature and air-dried to detecting SOM, SOC, SON and macro-and microelements.

Analyses of soil chemical properties
The SWC was determined after drying at 105°C for 48 h in an oven. SOM and SOC contents were determined according to the K 2 Cr 2 O 7 titration method. We assayed the TN and AN contents by the Kjeldahl method and measured the TP content via the NaOH molten -molybdenum antimony colorimetric method (Lu 2000). The contents of TK, AP, AK, Ca, Mg, Fe, Mn, Cu, and Zn in the soil were determined by inductively coupled plasma-atomic emission spectroscopy (iCAP 6000 series, Thermo Fisher Scientific, UK) (Bao 2000;Rivelli et al. 2012).

Plant growth and nutrient content analyses
The height, diameter, fresh weight, and dry matter weight, among others, of the organs and tissues in the young walnut tree under different treatments were analyzed after 80 days using a straight edge ruler, Vernier callipers, and an oven (with drying at 65°C for 48 h), respectively. The leaf area measured with an LI-3000A leaf area meter (Li-Cor, Inc. Lincoln, Nebraska, U.S.A), and the SLDW was calculated as the ratio of dry weight/per unit leaf area (Manceur et al. 2008). The RWC of leaves was determined according to the method of González (González and González-Vilar 2003). After dry leaves were ground, the N content was assayed by the Kjeldahl method, and the P content was measured via the NaOH molten -molybdenum antimony colorimetric method (Lu 2000). The mineral element contents in leaves were measured and quantified using an iCAP 6000 spectrophotometer (Thermo Scientific, UK) (Hadas et al. 2004). All the measured leaves were selected from the middle of the branches in the young walnut trees.

Determination of leaf photosynthesis parameters
Instantaneous gas exchange measurements were performed on five or six recently fully expanded healthy leaves in the middle of branches of the young trees in each treatment between 9:00 and 11:00 using an LI-6400 open gas exchange system (LI-COR, Inc., Lincoln, Nebraska U.S.A). The Pn, Tr, Gs, and Ci in leaves were evaluated. The instantaneous water use efficiency was calculated as the WUE equal to Pn/Tr ratio (Ribeiro et al. 2009).

Statistical analyses
The data from different fertilizer treatments under wet or dry soil condition were analyzed by the Student's t-tests for comparisons between wet and dry soils or by one-way ANOVA for comparisons of different fertilizer treatments. Duncan's multiple range test (DMRT) was conducted to split the data. We used Duncan's new multiple range tests following one-way ANOVA to analyze the significant differences at the P < 0.05 level using SPSS 16.0 software (SPSS Inc., Chicago, IL., U.S.A). OriginPro 8.0 (OriginLab Corporation, Northampton, U.S.A) was used to map the data. Principal component analysis (PCA) and redundancy analysis (RDA) were performed using Canoco v.5.0 software ).

Soil water content and soil organic matter content
The drought treatment significantly reduced the SWC in T3 and T5 relative to the wet treatment. However, the application of fertilizers inhibited this reduction in dry soil, especially T3 and T5, which had nitrogen-rich contents (Figure 1a). These results suggested that nitrogen-rich fertilizers enhance the water-holding capacity of soil, which is beneficial for the limited water supply to roots under soil drought conditions.
Although the SOM, SOC, and SON contents in both wet and dry soils markably increased with nitrogen-rich fertilizer, the differences among fertilizer treatments were more significant in dry soil (Figure 1b-d). Relative to wet soils, soil drought led to a higher C/N ratio in T1, T2, and T4, and there were higher C/N ratios in all fertilizer treatments than in CK (T1) (Figure 1e). These results indicated that animal and plant wastes as fertilizers could effectively improve the decomposition of SOM and then the possible release of mineral elements in dry soils via synergistic and supplementary effects of nitrogen-rich fertilizer and soil water. This phenomenon is beneficial to the growth of young tree roots and improvement of drought resistance.

Soil nutrients and related stoichiometry
The fertilizer treatments significantly increased the content of soil mineral elements in both wet and dry soils, including TN, TP, and TK (Figure 2a-c), resulting in fertilizers having a higher C/N ratio in all fertilizer treatment soils than in T1 soil in wet soil (Figure 2d). With the exceptions of T4, the fertilizers associated with the deficit irrigation treatment significantly increased the soil C/N ratio in T2, T3 and T5. The fertilizer treatments significantly increased the soil C/P ratio in soils, but these effects occurred in T3, T4, and T5 of the dry soil (Figure 2e). Fertilizer treatment T5 significantly increased the N/P ratio in both wet and dry soils, but in dry soil, the fertilizer treatments displayed a significant TN gradient effect as follows: T2<T1, T3 and T4 equal to T1, T5>T1 (Figure 2f). These results suggested that animal and plant wastes as fertilizers could effectively regulate the soil stoichiometric proportion of C, N, and P and promote the C and N cycles by coupling the limited soil water under soil drought conditions. In addition, when compared with T1 (CK), all the fertilizers significantly increased the contents of the available elements, including AN, AP, AK, Ca, Mg, Fe, Mn, Cu, and Zn, in both wet and dry soils. Furthermore, the application of fertilizers associated with the soil drought treatment also significantly increased the Ca and Mg contents and slightly increased Mn and Zn contents but reduced the Fe level (Figure 3ad). These results suggested that the coupled fertilizers and the limited soil water could effectively activate the release of macroelements and microelements to the plant root zone and shape the soil nutrient framework that is beneficial to plants under soil drought conditions. . Different letters indicate differences between treatments at P < 0.05 as determined by Duncan's new multiple range test. Asterisks indicate significant differences between wet and dry soil as determined by Student's t-tests (*, P < 0.05; **, P < 0.01).

Leaf nutrients and leaf photosynthesis of young walnut trees
The fertilizers treatment T5 in wet soil and T3, T4, and T5 in dry soil significantly increased the RWC in the young walnut leaves (Figure 4a). As a result, the leaf N and P contents in both the wet and dry soils were increased to varying degrees by all fertilizer treatments, but the K levels in dry soil were decreased in wet soil but slightly increased in dry soil. In addition, the leaf Mg content in both wet and dry soils was increased by all fertilizer treatments (Figure 4b), but that of Mn was reversed. In dry soil, however, the Ca, Mg, Fe, and Cu contents in leaves were increased to varying degrees by all fertilizer treatments, while the Zn content in leaves was increased only by T3 and T5 (compared with CK). Moreover, the N, K, Cu, and Zn contents were increased by fertilizer treatments to a greater extent in dry soil than wet soil, while less P, Ca, and Mg contents were increased by fertilizer treatments in dry soil than wet soil (Figure 4b). These results suggested that the coupling of nitrogen-rich fertilizer with limited water might regulate the contents and ratio of macro-and microelements, promoting young tree branching.
Furthermore, the fertilizer treatments significantly increased the Pn but significantly reduced the Tr, Gs and Ci in young walnut leaves, resulting in an significant WUE increase in both wet and dry soils, especially in T3 and T5 ( Figure 5). These results indicated that the coupling of nitrogen-rich fertilizers and soil-limited water promoted the assimilative capacity of the leaves, possibly due to the increase in mineral elements. . Data are shown as means ± SE (n = 3). Different letters indicate differences between treatments at P < 0.05 as determined by Duncan's new multiple range test. Asterisks indicate significant differences between wet and dry soil as determined by Student's t-tests (*, P < 0.05; **, P < 0.01).

Figure 3. Soil AN, AP, and AK contents and a heatmap of soil micromineral element contents in wet and dry soil under fertilizer treatments. a. Soil alkaline nitrogen content (AN). b. Soil available phosphorus content (AP)
. c. Soil available potassium content (AK). d. Heatmap of proportional changes in soil micromineral elements after fertilizer treatment. Data are shown as means ± SE. Different letters indicate differences between treatments at P < 0.05 as determined by Duncan's new multiple range test. Asterisks indicate significant differences between wet and dry soil as determined by Student's t-tests (*, P < 0.05; **, P < 0.01).

Dry matter accumulation and vegetative growth of young walnut trees
High leaf assimilates and WUE are the basis for vegetative growth in young plants. Compared with T1, the fertilizer treatments T3, T4, and T5 in both wet and dry soil significantly increased plant stem height (SH), average shoot length (ASL), total leaf number (TLN), total leaf area (TLA), total leaf dry weight (TLDW), average shoot diameter (ASD), total shoot dry weight (TSDW), and average shoot dry weight (ASDW), resulting in a high ratio of root and shoot (R/S) in the young trees. However, the effects of nitrogen-rich fertilizers on the above growth parameters in dry soil were more significant than those in wet soil, leading to no reduction of the high R/S ratio by drought stress (Figure 6). These results indicated that fertilizers coupled with soil drought significantly affected plant branch growth and branching architecture, with major effects of fertilizer treatments (Table 2).
Specifically, regarding the top shoots, the fertilizer treatments increased the shoot length (SL), shoot diameter (SD), shoot dry weight (SDW), leaf number (LN), leaf area (LA), leaf dry weight (LDW), and specific leaf dry weight (SLDW) in young trees in both wet and dry soil. The plant SL, LN, LDW, SLDW, SD, and SDW in T1, T3, and T5 were higher, whereas the SLDW was lower in T2 and T4 in dry soil than in wet soil. The SL, LN, LA, LDW, SLDW, and SDW in young trees under the T3, T4, and T5 treatments (relative to those of CK) were also higher in dry soil. In contrast, the SL, LN, LA, and SDW of top shoots in young trees in T2 (relative to those of CK) were lower in dry soil, with the exception of the LDW and SLDW in T2 (Table S1).
In addition, the fertilizer treatments in dry soil affected the shoot traits of the second, third, and other shoots, similar to those of the top shoots in young trees. However, when compared with the characteristics of different types of shoots among the fertilizer treatments in wet soil, the SL, LN, LA, SD, and SDW of the young trees were increased by T3, T4, and T5 (relative to CK), and the LDW and SLDW of young trees were increased by T2 (relative to CK) in dry soil. Several parameters displayed differences among the 4 types of shoots. In total, the values of SL, LN, LDW, SD, and SDW were in the Figure 5. Leaf photosynthetic parameters and water use efficiency of young walnut trees in wet and dry soil under fertilizer treatments. Data are shown as means ± SE. Pn, net photosynthetic rate; Tr, transpiration rate; Gs, stomatal conductance; Ci, intercellular CO 2 concentration; WUE, leaf water use efficiency. Figure 6. Dry matter accumulation and growth indices of young walnut trees in wet and dry soil under fertilizer treatments. SH, stem height; ASL, average shoot length; TLN, total leaf number; TLA, total leaf area; TLDW, total leaf dry weight; SLDW, specific leaf dry weight; ASD, average shoot diameter; TSDW, total shoot dry weight; ASDW, average shoot dry weight; RDW, root dry weight; R/S, root: shoot ratio. Data are shown as means ± SE. Different letters indicate differences between treatments at P < 0.05 as determined by Duncan's new multiple range test. Asterisks indicate significant differences between wet and dry soil as determined by Student's t-tests (*, P < 0.05; **, P < 0.01).
order of the third shoot > the second shoot and other shoots > the top shoot in young trees. LA decreased in the order of the third shoot > the second shoot > other shoots and the top shoot, whereas the SLDW values of other shoots and the top shoot were higher than or equal to those of the third and second shoots (Table S1). These results suggested that the coupling of nitrogen-rich fertilizer and limited soil water could effectively weaken top shoot growth and motivate second and third shoots, even other shoots, by regulating the dry matter accumulation and distribution of young walnut trees. This process led to the drought-adapted branching architecture in young plants under soil drought conditions.

Principal coordinate analysis of soil and plant variables and vegetative growth
PCA indicated that the differences in soil variables between the CK and fertilizer treatments and among fertilizer treatments were dispersed along with PCoA1, while the differences among fertilizer treatments dispersed along with PCoA2. The soil variables differed significantly between the dry and wet soils. These results suggested that soil drought led to more substantial nitrogen-rich fertilizer effects, especially T3, T4, and T5 (Figure 7a).
PCA revealed the correlations of the test variables among the fertilizer treatments in both wet and dry soils. There were three variable clustering groups: T3 and T5, T2 and T4, and T1 (CK) in wet soil, and T3, T4, and T5, T2, and T1 (CK) in dry soil. The same cluster variables displayed high positive correlations, whereas those in the T1 group displayed negative correlations with those in the other groups (except for T2) along PCA1. These results explained the positive correlations of SOM, SOC, SON, TP, TP, AN, AP, and AK with the leaf morphological traits of young trees and branching shoots, the positive correlations of both Fe and Zn contents in soil and leaf WUE with the shoot morphological traits of young trees and branching shoots, and the positive correlations of SWC, TN, the soil N/P ratio, and leaf N, K, Mg, and Mn contents with leaf photosynthesis, ASD and SLDW of young trees and branching shoots. These correlations of variables between T1 and T5 were strengthened under soil drought conditions (Figure 7b) RDA indicated that SWC, SOC, and SON were the soil indicators of fertilizer treatments and drought treatments, respectively. They played an important role in explaining the mechanism of branching architecture in which soil factors regulate the plant dry matter distribution and leaf and shoot morphogenesis among different shoots of young walnut plants. This result indicated that fertilizer treatment mediating the soil variables that regulated the vegetative plant growth and branching architecture of young plants under drought conditions (Figure 7c).

Discussion
The present study indicated that applying fertilizers, combining meat powder with alfalfa powder significantly increased the SWC, SOM, SOC, and SON in the wet and the dry soils, resulting in a high soil SOC/SON ratio. In addition, fertilizer application increased the TN, TP, and TK content in the wet and dry soil, resulting in a high soil C/N ratio and C/P ratio relative to those in the CK treatment. These changes may have been conducive to the release of soil nutrients. The levels and stoichiometric properties of soil organic nutrients, as well as the TN, TP, and TK, are an important indicator of soil fertility (Zhang et al. 2017) that exert a positive influence on the activities of soil microbial communities and enzyme systems, and the properties of soil ecological functions and services, affecting the  availability of mineral elements to plants (Gul et al. 2015;Triberti et al. 2016;Vincenza et al. 2016). Dry soil usually exhibits low TN and AN levels and high C/N ratios and low N/P ratios, leading to the reduction of nitrogen and the release of the available nutrients P, K, Ca, Mg, Mn, and Zn for osmotic regulation to diminish water loss in soil . Consistently, our results indicated that whereas the fertilizers increased the content of AN, AP, AK, and microelements, soil drought decreased the content of AN in soil but did not reduce the content of AP or AK in the soil. Additionally, fertilizers also increased the content of Ca, Mg, Mn, and Zn contents but decreased the Fe and Cu contents in soil under soil drought conditions. Moreover, nitrogen-rich fertilizers increased the nutrient supply from the soil to young walnut trees, resulting in a high accumulation of N, K, and microelements in leaves under soil drought conditions relative to those of young trees in wet soil. As a result, young trees can maintain effective photosynthesis and carbon assimilation efficiency with a high WUE, allowing them to slightly increase their growth rate, accumulate additional dry matter and enhance the assimilate distribution to storage organs and tissues (Liu et al. 2013;Luo et al. 2018). Soil and leaf nutrients are fundamental for plant growth and development, influencing photoassimilate partitioning at the whole young tree level and plant branch growth. Soil drought and fertilizer application might induce young trees to generate drought-adapted vegetative growth (Ramireddy et al. 2018). In the present study, the increase in P, Ca, Mg, Fe, and Zn content in leaves, accompanied by the increase in Pn and WUE of leaves and reduction in Tr. In addition, the SH (the height of young trees), ASL, TLN, TLA, TLDW, SLDW, TSDW, ASDW, and RDW increased, whereas the ASDW did not enhance in the drought-treated plants. Moreover, soil drought increased the top shoots of plants in the T3, T4 and T5 treatments relative to the corresponding levels in the CK group. Soil drought increased SL, TLN, TLA, ASDW, and TSDW in the second, third, and other shoots in the T3, T4, and T5 treatments relative to the corresponding levels in CK group. TLA decreased in the order of the third shoot > the second shoot > other shoots = top shoot, whereas SLDW in the other shoots and the top shoot was more significant than or equal to that in the third and second shoots. Accordingly, the growth length and dry matter accumulation were decreased in the top shoot but significantly enhanced in the second and third shoots. We hypothesize that low N levels might decrease the apical dominance of young trees shoot.
PCA of the soil variables, whole young tree variables, and branching traits of various shoots revealed the differences in soil variable correlations among fertilizer treatments in both wet and dry soils. Soil drought led to differences in the correlations between variables in the T3, T4, and T5 treatments in the assembly group and the T1 (CK) variables in the assembly group. These differences explained the following results. First, there were positive correlations of soil SOM, SOC, SON, TP, AN, AP, and AK contents with leaf morphological traits of the whole plant and branching shoots. Second, there were positive correlations of Fe and Zn contents in the soil and leaf WUE with the entire plant's shoot morphological traits and branching shoots. It was reported that foliar application of Zn and boron improves the vegetative and reproductive growth of walnut (Keshavarz et al. 2011). Third, there were positive correlations of SWC, TN, the soil N/P ratio, and the leaf N, K, Mg, and Mn contents with leaf photosynthesis and the ASD and SLDW of the whole plant and branching shoots. RDA indicated that SWC, SOC, and SON were the most critical environmental variables influencing soil factors, plant growth, dry matter distribution, and leaf and shoot morphogenesis associated with the branching architecture of the young walnut trees.
under various fertilizer and water treatments. b. plant and branching variables of young walnut trees under various fertilizer and water treatments. The black characters represent soil variables, the green characters represent growth variables in the young trees, and the red characters represent branching variables in the young trees. c. RDA of the main soil variables and branching variables of young walnut trees under various fertilizer and water treatments. The numbers after the abbreviations (1, 2, 3 and 4) represent the top branch, second branch, third branch, and other branches of the young trees., Ka: the index of tapering of new shoots on the main stem; NSD: the new shoot diameter on the main stem in young walnut trees (Table S1).
Our results indicated that the application of meat and alfalfa powder improved the soil nutrient supply, leaf nutrients distributions, Pn, WUE, dry matter accumulation, and vegetative growth and branching of young walnut trees under deficient irrigation treatments. It was reported that seasonal drought limited the carbon sequestration capacity of forest ecosystems, whereas increasing atmospheric nitrogen deposition contrarily enhanced its carbon sinks ). Thus, we established a working model in which the application of nitrogen-rich fertilizer improves the soil C/N ratio, resulting in more branching and thickening growth of the young walnut trees, and a high R/S ratio in wet and dry soils ( Figure S1). In the model, the combination of nitrogen-rich animal and plant waste as fertilizer mediates the ratio of SOC/SON, C/N, and N/P stoichiometries in soil, resulting in the accumulation of available soil inorganic nutrients. This accumulation enhances soil resistance to water loss and provides a nutrient supply to plants under soil drought stress (Sary et al. 2014). The ecological stoichiometry of C, N, and P in soil is used to measure soil nutrients availability , and is the basis for understanding ecosystem stability. A stable soil ecosystem could maintain soil C and N cycles and nutrient supplies under a limited water supply, ensuring the need for water and nutrients to plants. In the model, after applying fertilizer to dry soil, young walnut tree leaves achieved a high Pn and WUE in leaf N, P, K, and microelement contents, resulting in more branching and dry matter accumulation at the whole-plant level.

Conclusions
The use of meat and alfalfa powder as nitrogen-rich fertilizer promoted the branching and thickening of young walnut trees by improving the soil nutrient content and soil C/N ratio under drought conditions. Therefore, the combination meat and alfalfa powder can be used as organic fertilizers or soil amendment, which has promising potential applications under deficit irrigation conditions.