Determining the Necessity of Applying N and P Fertilizer in a Mature Subtropical Torreya Grandis Orchard

Background In managed orchards, fertilizer treatments facilitate both high productivity and environmental pollution. Because economic prot takes priority over environmental cost, increasing amounts of chemical nitrogen and phosphorus fertilizer have been used in mature subtropical Torreya grandis orchards. However, given the magnitude of global nitrogen deposition, it’s worth considering whether heavy fertilizer treatment is necessary. Methods To elucidate the balance between T. grandis nutrient demands and the fertilizer supply, we determined the C, N, and P foliar and soil concentrations ([C], [N], [P]) at ve orchards undergoing long-term varied intensity fertilizer treatments. Results After documenting the dynamic variation of available plant nutrients and the corresponding resorption eciency, we found that increasing the fertilizer supply elevated foliar [P], yet foliar [C] and [N] remained stable. Because T. grandis was already equipped with a high nutrient content, the increased foliar [P] levels decreased C:P and N:P ratios. These results demonstrate that extra fertilizer in the N-saturated environment disturbs P-limitation. Furthermore, we also found that fertilizer supply failed to improve carbon accumulation, which in addition to soil nutrient content and leaf [P], highly impacted productivity. Conclusions Thus, based on the results of this study, there are ample reasons to propose rejecting N addition in the present orchards, and we recommend organic management as a more conducive method to realize sustainable development.


Introduction
In order to facilitate a rapid increase in soil nutrients and guarantee pro table productivity, escalating amounts of chemical nitrogen (N) and phosphorus (P) rich fertilizer have been applied in T. grandis orchards, without any scienti c management guidance. N and P are key nutrients that play pivotal roles In the eld conditions, N input to an N-limited ecosystem, such as boreal forests, will improve net primary productivity (NPP) through a direct fertilizing effect on vegetation (Lebauer and Treseder 2008a). as a direct result from global change, N deposition in many regions of the world (i.e., United States, western Europe, and China) currently exceeds 10 kg N ha − 1 yr − 1 , especially in tropical and subtropical areas (Liu et al. 2013;Meunier et al. 2016),where over 80-120 kg N ha − 1 yr − 1 has been reported ). Therefore, as N deposition levels continue to accelerate, N-limitation has been subsequently alleviated (Tao and Hunter 2012), and there is a high likelihood that the ecosystem is shifting to an N-enriched status (Yu et al. 2018). N deposition has also changed the soil stoichiometry by accelerating the soil P cycle in tropical and subtropical forests. Speci cally, excessive N in temperate forests has been shown to alter the biogeochemical cycles of essential plant nutrients (Ferretti et  sites and noted that P de ciency is becoming progressively more problematic. Anthropogenic alternation of regional P and N cycling has led to large areas of southern China forests transmuting due to humaninduced P-limitation (Du et al. 2016). Thus, NPP has transformed from being N limited to P limited in many forest ecosystems (LeBauer and Treseder 2008b).
Under intensive management, long-term addition of balanced compound fertilizer (e.g., N:P:K = 15:15:15) may cause excess P in orchards, as plants generally require less P than N (Macy 1936). P is an essential element for nucleic acids and membrane lipids. Although P sensing and signaling are not fully understood, there appears to be a series of physiological processes in plants that are either stimulated or suppressed in response to P supply (Fang et al. 2009). Unlike the immobile N in the plant cell wall, most leaf P is hydrolyzable and, therefore, more easily resorbed (Ågren 2008;McGroddy et al. 2004). In fact, due to its indiscriminate uptake, greater variability of foliar P has been reported even if it's not needed at that growth stage (Ostertag 2010). Species in P-poor environments, such as subtropical evergreen trees, are equipped with a corresponding adaptation mechanism, which makes them more susceptible to toxic eutrophication under excessive P addition (Musick 1978 predominantly focused on the effect of single N or P addition on leaf nutrients or resorption; thus, the effect of intensive P addition on PRE in subtropical forests is not well understood.
Due to the high economic value of its nuts, T. grandis has become one of the most important commercial tree species in southeast China. During the past three decades, the planting area of T. grandis has steadily increased. As a result, farmers are facing escalating economic pressure, which has led to excessive fertilizer consumption, and ultimately deterioration of the soil's physical and chemical properties. Thus, it is critical to determine the optimal amount of fertilizer necessary to achieve ideal growth. A comprehensive understanding of fertilizer impact on crop quantity and soil quality is critical for improving fertilizer treatment strategies in economic considerations and maintaining a healthy soil environment. To solve the above problems, we examined the C, N, and P stoichiometry of soil as well as green and senescent leaf of a mature T. grandis, in ve orchards plots with varying fertilizer treatments. The objective of this study was to: 1) evaluate whether the continuously increasing N deposition in subtropical forests has alleviated the N-limitation, making P the limiting factor restricting plant growth; (2) determine the optimal fertilizer supply; and (3) assess whether large amounts of fertilizer, i.e., N and/or P, negatively impacts the plants. These results are expected to provide fertilization guidelines and recommendations to help farmers reduce costs and soil pollution, while ensuring optimal production.

Study site
The study was conducted at the origination locale of T.  Table 1, and fertilization was applied twice, each in April and July, at a depth of 10 cm below the soil's surface. Organic fertilizer was applied in plots F1, F2, and F4, in order to provide more organic matter.

Sample collection and measurements
Soil samples were collected at the same time when leaf samples were collected. Each soil sample was a mixture of three soil collections from a 0-20 cm depth, and each location was randomly selected along the diagonal of each plot. The soil was dried and sifted to measure the particle size distribution and other soil chemical properties. In each plot, three green leaf sample groups (30 leaves for each group) were collected from the apricus healthy shoots of random plants in the middle of the canopy. Similarly, three Data analysis To examine the relative nutrient limitation among the treatments, we calculated nutrient resorption e ciency (NuRE) using the following equation: Given the large data set collected, we also tested the relationships between log-transformed nutrient stoichiometry of green and senescent leaves, by applying a type II linear regression model (SMA, standardized major axis; Y ~ X) using the lmodel2 package in R (3.6.1). Y is the [C], [N], [P] or stoichiometry ratios in the foliar samples and X is the related variables in the senescent leaves; slope > 1 indicated dependence of Y variation on X, slope < 1 indicated an independence of Y on X, and slope = 1 indicated a synchronous change of X and Y.
Signi cant difference of each dependent variable (C, N, P stoichiometry of soil, foliar and nutrient resorption) among fertilizer treatments were tested by one-way analysis of variance (ANOVA) followed by Least Signi cant Difference (LSD) and Tamhane's T2. Distance correlation analysis was performed to assess the nutrient content correlation among the soil, green leaves, and senescent leaves. Results were considered signi cant when P < 0.05. All statistical analysis was performed using the SPSS software (version 20.0, SPSS Inc., Chicago, USA) and R (3.6.1).

Results
Effects of fertilization on soil nutrient characteristics Fertilization treatments signi cantly affected soil chemical properties. Soil nutrient stoichiometry showed a strong positive correlation with the nutrient addition gradient (Fig. 1). Concentrations were ranged from 18.17 ~ 34.17 g/kg (OC), 1.67 ~ 3.38 g/kg (TN), 1.56 ~ 4.45 mg/kg (TP), 132 ~ 299 mg/kg (HN), and 225 1081.00 mg/kg (AP) depending on the speci c treatment. Compared to the control, adding fertilizer increased the soil's TN, TP, HN, and Olsen-P by a maximum of 102%, 185%, 127%, and 380%, respectively. The addition of organic fertilizer raised the soil pH in samples F1, F2, and F4 above that of F0 and F3. Yet, by comparing samples F0, F2, and F3, it is apparent that organic fertilizer had no signi cant effect on soil organic carbon (SOC) content. Finally, as the fertilizer concentration increased, the soil N:P and C:P signi cantly decreased (Figs. 1g, h), but C:N (11.00 ± 0.87) remained stable throughout the four experimental treatments.

Effects of fertilizer on foliar nutrient characteristics
Fertilizer treatment increased foliar nutrient contents, which depended on the nutrient element and growing status (Fig. 2). Generally, green leaf uniformly exhibited a higher nutrient concentration than senesced leaf (Fig. 2)  The C:N ratio of both green (2.15 ± 0.05) and senescent (2.47 ± 0.09) leaves showed no apparent change among the treatments (Fig. 3). The signi cant increase in [P] led to big variations of N:P and C:P in green and senescent leaves, which declined with increasing fertilizer addition, excluding the control group. The highest nutrient utilization e ciency indicated by C:P and C: N were 27.92 (F1) and 2.21 (F1) in the green leaf. In addition, there was no correlation between foliar N or P with soil pH values.

Discussion
Effect of fertilizer on soil condition

Effect of fertilizer on plant C, N, and P stoichiometry
The primary goal of this research was to evaluate whether T. grandis requires fertilization or whether adding additional nutrients through fertilization can improve tree nutrition. In the present study, we found that fertilization tended to increase the green and senescent leaves [C], [N], and [P], but only the increase in [P] was statistically signi cant (Fig. 2) . Due to the different N and P utility patterns in physiological process, regardless of species and site fertilizer supply, plants are able to store a greater percentage of inorganic P than N. Thus, following fertilizer treatment, foliage accumulated more P than N (Ostertag 2010). It was also observed that some plant species growing in P-limited environments might not downregulate P uptake when a higher supply of P is available (Ostertag 2010;Shane and Lambers 2007;Standish et al. 2007). When the environment P supply shifts from P-limited to non-limited condition, the plants may undergo an excessive P uptake, even to saturated or toxic levels. Hence, foliar [P] usually displays a much higher variation after fertilizer treatment. Therefore, it is not surprised that the foliar [P] nearly doubled after a high fertilizer treatment compared to the control group (Fig. 2). Given the generally low bioavailability of P in subtropical soils, T. grandis may have developed as an e cient mechanism to take up and accumulate P in response to the strong selective pressure (Chapin III et al. 1990; Ingestad 1974; Mulligan and Sands 1988 , but increased leaf [P] led to opposite trends between C:P and N:P and soil nutrients ( Fig. 1; Table 3).
Stoichiometry homeostasis was used to analyze plant composition, ecosystem function, and nutrient limitation, especially for key elements such as C, N, and P ratios ( Furthermore, contrary to previous research , fertilizer treatment in this study also failed to increase the aboveground biomass (indicated by a stable carbon sequstration) of T. grandis. Moreover, the fertilizer signi cantly reduced the C:P (indicating the P utilization e ciency in productivity) of T. grandis in this study, suggesting that the P utilization e ciency of plants was reduced by the increased nutrient supply (Dijkstra et al. 2016). Our analysis suggested that both P and N fertilizer might be unnecessary for T. grandis.
Effect of fertilizer on plant nutrient resorption Acquisition (root uptake) and conservation (resorption from senescent tissue) are two important biological strategies for plants to maintain balanced nutrition where soil nutrition is de cient. These processes are also important in cycling nutrients between the soil and plants (Killingbeck 1986).
Choosing between alternative strategies for plants depends on the cost (time and energy) of each process and the species characteristics. Short-term experiments have demonstrated that generally, plants will reduce nutrient utilization e ciency when the availability of that nutrient increases in the plant's natural environment (Yuan and Chen 2015). While the relationship between foliar resorption and soil nutrient concentration is not consistent (Aerts 1996; Aerts and Chapin III 1999; Yuan 2015). Our results indicated a constant NRE, which further veri ed our supposition concerning N enrichment in the orchard (Fig. 4a). However, increased P migration from senescent leaf to green leaf (PRE) in response to increasing fertilizer supply con icts with the negative correlation between fertilizer treatment and nutrient resorption (Yuan and Chen 2015). This counterintuitive result from a single-resource conservation standpoint has been observed in previous studies (Boerner 1986;Sabaté et al. 1995); yet multiple-element theory much appropriately explains variability in foliar nutrient resorption (See et al. 2015). Based on a Pearson correlation analysis, we found that green leaf [P] was not related to soil [Olsen-P], but it was highly correlated with soil N content (Table 3). It should be noted that the root system can directly uptake inorganic nutrients (Olsen-P and hydrolysable nitrogen) and therefore plays a more important role in nutrient absorption than organic nutrients. This inorganic nutrient preference may help explain the absence of a common trend in studies comparing P resorption to soil P availability (Aerts 1996;Aerts and Chapin III 1999). Under the circumstances, although the soil and leaves were characterized by a higher [P] compared to other studies of evergreen trees (Tang et al. 2018b), N enrichment was responsible for the ampli ed P-limitation (Fig. 4b), which subsequently elevated leaf P uptake ( Table 4). For an evergreen species with a long leaf life span, T. grandis exhibited a more conservative P use strategy as opposed to the external root uptake strategy, regardless of soil P availability. Similar results were also reported in some non-mycorrhizal species (Laliberté et al. 2012). This mechanism allows plants to maintain a higher nutrient absorption e ciency with relatively less cost (energy and time) but lowers the utilization e ciency and productivity (Wright and Westoby 2003).

Conclusion
As N deposition and long-term fertilizer treatment intensi ed, there was a signi cant increase in the soil N and P, which improved leaf N and P concentrations. In our study area, N enrichent was indicated by stable and decoupling relationships between the soil and leaf [N]. Although T. grandis has a high level of leaf [N] and [P], N enrichment ampli ed the physiological P limitation (de ciency). Unfortunately, the present fertilizer modes did not seem to enhance productivity (C xation). Excessive fertilizer application both wastes resources and negatively impacts nutrient uptake and soil physicochemical properties. Thus, we recommended rejecting N fertilizer addition and increasing application of organic fertilizer as needed.
Although P-limitation was demonstrated in this study area, whether the P was de cient in the soil requires further exploration through a single-P element control experiment.

Availability of data and material
All data generated or analysed during this study are included in this published article.

Competing interests
The authors declare that they have no competing interests.  Figure 1 Relationship between nutrient supply and soil nutrient contents. SOC, TN, TP, HN, Olsen-P denote total soil organic C, Total N, Total P, Hydrolysable N, and Available P, respectively. Error bars refer to ±1 standard error, P<0.05, n=3  Green leaf stoichiometry under different fertilizer treatments. Error bars refer to ±1 standard error (a, b above each row indicate the differences in fertilizer treatments, P<0.05.)