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Article

Reinforcing Nitrogen Nutrition Through Partial Substitution with Organic Nitrogen Enhances the Properties of Natural Rubber

by
Dongqi Jin
1,2,3,
Zhengzao Cha
2,3,
Jianhong Li
2,3,
Yanyan Huang
2,3,
Hongzhu Yang
2,3,
Hailin Liu
2,3,
Wei Luo
2,3,* and
Qinghuo Lin
2,3,*
1
School of Tropical Agriculture and Forestry, Hainan University, Haikou 570228, China
2
Rubber Research Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China
3
Danzhou Soil Environment of Rubber Plantation, Hainan Observation and Research Station, Danzhou 571737, China
*
Authors to whom correspondence should be addressed.
Forests 2024, 15(11), 1897; https://doi.org/10.3390/f15111897
Submission received: 20 September 2024 / Revised: 20 October 2024 / Accepted: 24 October 2024 / Published: 28 October 2024
(This article belongs to the Special Issue Stress Resistance of Rubber Trees: From Genetics to Ecosystem, 2nd Edition)
Browse Figures
Versions Notes

Abstract

:
The partial substitution of chemical fertilizer with organic fertilizer is a crucial practice for enhancing crop production and quality, although its impact on natural rubber has rarely been explored. In this study, a two-year field experiment was conducted to investigate the impact of different nitrogen application rates and varying proportions of organic nitrogen substitution on dry rubber yield, nitrogen nutrition, and natural rubber properties. Regarding nitrogen application, the control treatment received no nitrogen amendment, while the low-nitrogen treatment was amended with 138 g·tree−1·year−1 of nitrogen. The medium-nitrogen treatment received 276 g·tree−1·year−1 of nitrogen, and the high-nitrogen treatment received 552 g·tree−1·year−1 of nitrogen. In addition, the low-organic-nitrogen substitution treatment and medium-organic-nitrogen substitution treatment were amended with 276 g·tree−1·year−1 of nitrogen each. The results demonstrated that the 50% organic nitrogen substitution treatment resulted in the highest dry rubber yield across all sampling periods, ranging from 46.43 to 94.65 g·tree−1. Additionally, this treatment exhibited superior soil total nitrogen (1067.69 mg·kg−1), available nitrogen (84.06 mg·kg−1), and nitrogen content in roots (1.08%), leaves (3.25%), fresh rubber latex (0.27%), and raw natural rubber (0.44%) compared with other treatments. In terms of the physical properties of natural rubber, the 50% organic nitrogen substitution treatment resulted in advantages in the weight-average molecular weight (1.57 × 106 g·mol−1), number-average molecular weight (0.36 × 106 g·mol−1), plasticity retention index (97.35%), Wallace plasticity (40.25), and Mooney viscosity (81.40). For mechanical properties, natural rubber from the substitution treatment exhibited higher tensile strength (19.84 MPa), greater elongation at break (834.75%), and increased tear strength (31.07 N·mm−1). Overall, the substitution of 50% chemical nitrogen fertilizer with organic nitrogen fertilizer improved nitrogen nutrition in rubber trees by introducing organic nitrogen input, resulting in remarkable enhancements in natural rubber properties. Therefore, the incorporation of organic fertilizer as a substitution for 50% of chemical fertilizer is demonstrated as an effective strategy for improving both the yield and properties of natural rubber.

Graphical Abstract

1. Introduction

Natural rubber (NR), a biopolymer with a high molecular weight that is primarily derived from the rubber tree (Hevea brasiliensis Muell. Arg.), is technically referred to as 1,4-polyisoprene. It serves as a vital raw material in various industries, including transportation, medicine, and national defense [1]. The exceptional physical properties of NR, such as impact and abrasion resistance, high tensile strength, efficient thermal dispersion, elasticity, and ductility at low temperatures, make it a key component in the manufacturing process of a wide range of rubber and latex products [2,3,4]. It was initially believed that cis-1,4-polyisoprene, the primary component of NR, was solely responsible for its characteristic properties [5,6]. Nevertheless, previous studies have demonstrated that a variety of non-isoprene constituents, including carbohydrates, proteins, lipids, and inorganic materials, contribute to the intricate composition of natural rubber [7,8,9]. The composition of these non-rubber components varies based on genetic factors specific to the clone, soil conditions, harvesting practices, fertilization, weather, and season, among other factors [10,11].
Fertilization is a crucial agronomic practice in agricultural production [12]. Nitrogen is vital to plant growth [13], and extensive research has been conducted on nitrogen fertilizer application for various crops, including rubber trees [11,14,15]. Recent studies have demonstrated that the judicious use of nitrogen fertilizer can enhance crop yields and stabilize crop quality [16,17,18]. An investigation into the impact of fertilization on rubber tree productivity revealed that a three-year fertilization strategy led to an 8%–13% increase in latex yield compared with non-fertilized treatment. In addition, the nitrogen content in the latex of fertilized treatments exhibited an increase of 5.10%–7.50% [11]. Significantly, the nitrogen content in NR serves as an indicator of non-glutinous constituents, including amino acids, proteins, and nitrogen-containing bases [19,20]. Among these constituents, proteins play a crucial role in influence on the synthesis rate and the elasticity properties of NR [8,21,22].
Organic nitrogen has been demonstrated to enhance crop nutrition and potentially decrease the reliance on chemical fertilizers [23]. Several studies have revealed that organic fertilizers play a crucial role in supplying vital nutrients and enhancing microbial metabolic activity within the soil. Natural fertilizers can be used in combination with synthetic fertilizers over an extended period to enhance crop yield and quality [24,25,26]. For example, a blend of synthetic organic and organic fertilizers has been shown to mitigate the release of harmful gasses while boosting crop productivity [27]. Moreover, the highest crop yields have been achieved with an organic nitrogen replacement rate of 70%, highlighting its efficacy in yield enhancement [28]. Moreover, the utilization of organic fertilizers has significantly enhanced crop quality [29,30,31]. Despite this, there is a paucity of research on the substitution of organic nitrogen in rubber tree cultivation.
It was hypothesized that substituting a portion of chemical nitrogen fertilizer using organic nitrogen fertilizer could exert a positive impact on the growth of rubber trees, as well as on the rubber latex yield and its quality. Therefore, this study aims to propose a novel science-based strategy for rubber tree fertilization that uses cow manure as an organic nitrogen fertilizer to partially substitute for chemical fertilizers, as well as to enhance nitrogen nutrition in the soil–rubber tree system, thereby improving dry rubber yield and the properties of natural rubber. The objectives of this research were (1) to determine fertilization strategies that would enhance the NR yield and quality while reducing the reliance on chemical nitrogen fertilizers; (2) to assess the response of nitrogen levels in soil, rubber tree roots, leaves, and NR to different fertilization approaches; and (3) to examine the potential correlations between nitrogen levels and NR properties across different fertilization methods. This study provides practical guidance for the effective utilization of organic nitrogen substitution in the cultivation of rubber trees, as well as for the enhancement of NR properties through nitrogen supplementation.

2. Materials and Methods

2.1. Experimental Site

The experiments in this study were conducted at an experimental farm in Danzhou, Hainan, China (109.50° E, 19.49° N; elevation: 114 m above sea level) (Figure 1). The soil type at the experimental site was identified as a Udic Ferralsol derived from granite, characterized by a pH of 5.24. It contained 1.03 g·kg−1 total nitrogen, with available nitrogen levels at 54.79 mg·kg−1, available phosphorus levels at 11.87 mg·kg−1, available potassium levels at 64.38 mg·kg−1, and organic matter content of 12.04 mg·kg−1 in the plow layer (0–20 cm) at the beginning of the experiment. The experimental area has a hot and humid climate, with an average annual maximum temperature of 30.0 °C, a minimum average annual temperature of 22.30 °C, and an annual precipitation totaling 1891 mm (Figure 2).

2.2. Experimental Design

The experimental plot was established in 2012 within a rubber plantation, utilizing clones of the Reyan 917 rubber tree. Before commencing the experiment in April 2022, routine weeding activities were carried out on-site. The trees were strategically spaced at intervals of 3 m between individuals and 7 m between rows, leading to an average density of 476 trees·ha−1.
The experiment was conducted using a split-plot design with randomized complete blocks, incorporating six distinct fertilizer treatments in the main plot (Table 1). Each treatment had three replications, for a total of 18 plots consisting of 20 trees per plot (arranged in four rows of five trees each). The fertilizers used were Tomishima macro granular urea (a chemical nitrogen fertilizer, CN, with 46% N) for nitrogen, superphosphate (16% P2O5) for phosphate, and potassium chloride (60% K2O) for potassium. These fertilizers were applied twice a year, in April and September, in equal amounts. Organic nitrogen (ON) was sourced from pure cow manure (TN 0.92%, TP 2.70 g·kg−1, TK 1.36 g·kg−1) and applied once a year in April. All fertilizers were applied into a fertilizing hole measuring 200 cm in length, 60 cm in width, and 60 cm in depth, located 1 m away from the experimental rubber tree. The chemical fertilizer was evenly distributed within the hole, after which the litter in the rubber garden was covered. The organic nitrogen substitution treatments were applied chemical fertilizer first, after which cow manure was evenly applied and finally covered with litter from the rubber garden. The comprehensive fertilization data for the six experimental treatments are provided in Table 1.

2.3. Sample Collection and Measurements

2.3.1. Sample Collection

The rubber tree sample used in the experiment was S/2 d3 (i.e., one-half of the tree circumference tapped every 3 days). Rubber tapping was conducted at 3 a.m., and the latex was harvested at 7 a.m. Fresh latex was collected in July, September, and November, and its weight was recorded. A 120 mL sample was taken from the mixed latex for laboratory analysis to determine the dry rubber content, physiological indices, and nutrient content. During the later stages of the experiment in November 2023, 1–2 L of fresh rubber latex was collected from each plot and processed into raw rubber by adding 37 g of 5% acetic acid per liter of latex. Additionally, the soil and root samples were collected from each treatment by excavating a 20 cm × 20 cm × 20 cm block from the lateral wall of the fertilization cave, which had a depth of 60 cm, facing the side where the rubber tree was located, while 40–50 mature leaves facing north and south were sampled for nutrient content analysis. Each treatment had three replicates.

2.3.2. Measurements

The yield of dry rubber was determined by multiplying the weight of fresh latex with its corresponding dry rubber content (DRC). In order to quantify the DRC, 10 g of fresh rubber latex was placed in a Petri dish and mixed with 2 mL of a 5% acetic acid solution (Guangzhou, China). The mixture was thoroughly shaken and allowed to solidify. Subsequently, the solidified latex was washed and then dried at 70 °C until a steady weight was achieved, after which the resulting DRC was measured. The DRC was calculated as the weight after drying divided by 10 and multiplied by 100%. We took 2 mL of the freshly collected latex and added 18 mL of 2.5% trichloroacetic acid solution (Guangzhou, China). We then shook the mixture thoroughly, then transferred it into a centrifuge and spun it at 4000 rpm for 8 min. Following centrifugation, we filtered the solution to obtain the samples for testing latex sucrose, inorganic phosphorus, and thiol content. The sucrose content [32], thiol content [33], and inorganic phosphorus content [34] were then measured. The available nitrogen content and total nitrogen content of the studied soils were determined following the guidelines in the Chinese standard (NY/T 1121.24-2012) [35] method. The nitrogen content in roots, leaves, and NR was tested using the continuous flow analytical system (AutoAnalyzer 3, SEAL, Analytical GmbH, Norderstedt, Germany). The particle size distribution analysis of fresh latex was performed using a laser scattering particle size analyzer (HORIBA, LA-960, Osaka, Japan). We took approximately 1000 to 2000 mL of a fresh latex mixed sample from each plot and transferred it into a sampling bucket. We filtered the latex using a 40–60 mesh sieve to eliminate impurities, and then added 37 mL of 5% acetic acid per kilogram of fresh latex to induce solidification. We soaked the resulting gel block in clean water for washing, and subsequently cut it into small pieces. We employed a tablet press to compress the gel block into thin slices measuring approximately 2 mm thick. Next, we rinsed the slices with clean water, and then placed them in a drying oven set to 70 °C for air-drying. Once the slices achieved a light-yellow and transparent appearance, we removed the raw film, packaged it, and recorded the sample. This procedure is intended for evaluating the processing and mechanical properties of natural rubber. The NR molecular weight was measured utilizing a Waters 1515 GPC apparatus, which was equipped with a differential refractive index detector. The Wallace plasticity (P0) was determined utilizing P14, Wallace, ID, USA according to the Chinese standard (GB/T 3510-2006) [36] method, while the plasticity retention index (PRI) was tested utilizing P14 (including the aging box), Wallace, ID, USA, following the Chinese standard (GB/T 3517-2022) [37] method. Mooney viscosity (VR) was measured utilizing MV-3000ASU, Gotech, Qingdao, China using the Chinese standard (GB/T 1232.1-2016) [38] method, and elongation at break, stress modulus, and tensile strength were measured utilizing Al-7000-SU1, Gotech, Qingdao, China using the Chinese standard (GB/T 528-2009) [39] method. Tear strength was measured utilizing Al-7000-SU1, Gotech, Qingdao, China using the Chinese standard (GB/T 529-2008) [40] method.

2.4. Statistical Analysis

Results were obtained from triplicate measurements and are presented as the mean ± standard error. Data analysis was performed using a one-way ANOVA test, followed by Duncan’s multiple range tests in SPSS 26 (IBM Corporation, New York, NY, USA) to determine significant statistical differences between mean values of indicators at a significance level of p < 0.05 [30]. The experimental figures were generated using Origin 2021 (Origin Lab, Northampton, MA, USA). Partial least squares discriminant analysis (PLS-DA) and principal component analysis (PCA) were conducted using the online tool available from MetaboAnalyst (https://www.metaboanalyst.ca/, accessed on 21 April 2024). Heatmap cluster analysis was carried out using TBtools-II software (v2.096).

3. Results

3.1. Rubber Yield and Latex Parameters

The results showed that different fertilization treatments significantly impacted the dry rubber yield (Figure 3). In 2022, the overall trend was an initial increase and then a decrease, while in 2023, there was a continuous rise in yield. Notably, the ON50% treatment consistently produced the highest yield across all periods among the six treatments, ranging from 46.43 g·tree−1 to 94.65 g·tree−1 per tapping. Specifically, in September 2022, the ON50% treatment led to a 39.04% increase in yield compared with the CK treatment, while in November 2022, the yield of this treatment increased by 31.72% and 32.23% compared with the MN and ON30% treatments, respectively. In July 2023, the yield saw a notable increase, ranging from 28.70% to 38.02% compared with the other treatments; in September 2023, the yield rose by 27.08% compared with the HN treatment, while the yield of ON50% increased by 27.20% and 32.26% relative to that of the LN and CK treatments in November 2023, respectively.
The physiological parameters of latex under different fertilization treatments varied across sampling periods (Figure 4). However, the DRC did not exhibit significant differences when comparing the fertilization treatments to the control treatment at any of the time points (Figure 4A). The application of the MN treatment in July 2022 resulted in a significant reduction (37.82%) in inorganic phosphorus content in the latex compared to the control treatment. In contrast, during September 2023, the ON50%, HN, and ON30% treatments exhibited notable increases (41.54%, 50.88%, and 66.36%) in the phosphorus content of the latex when compared to the CK treatment (Figure 4B). In July 2023, the ON50% treatment led to a substantial increase (20.95%) in sucrose content within the latex compared with the control. During November 2023, significant increases (10.31%, 17.81%, and 22.04%, respectively) were observed for thiol content in latex following the MN, ON50%, and ON30% treatments as compared to the control treatment (Figure 4C). The levels of latex thiol content showed an annual increase across all treatments during this two-year period. Notably, significant increases in thiol content were observed for the ON50% treatment in July 2022 when compared with both ON30% and MN treatments, with percentages of 13.69% and 11.97%, respectively. However, no significant difference was found among all treatments regarding latex thiol content during other periods (Figure 4D).

3.2. Nitrogen Nutrition

Different fertilization treatments had varying impacts on nitrogen nutrition (Figure 5 and Figure 6). The ON50% treatment significantly increased the total nitrogen content of the soils (1067.69 mg·kg−1) by 26.40% to 94.38% compared with the other treatments, indicating enhanced soil nitrogen nutrition. Additionally, the soil available nitrogen content (84.06 mg·kg−1) in this treatment increased by 67.43% and 46.50% compared with the CK and LN treatments, respectively. The ON50% treatment resulted in higher nitrogen contents in the roots and leaves of the rubber trees. Although no significant difference in the nitrogen content of the root (1.08%) was presented compared with the other treatments, a significant increase of 12.57% in the nitrogen content of the leaf (3.25%) was observed compared with the control treatment. Moreover, the ON50% treatment had the highest nitrogen content in fresh rubber latex and raw NR among all treatments. The nitrogen content in fresh rubber latex was 0.27%, which was 3.78%–9.34% higher than that in the other treatments, with a significant difference compared with the MN treatment. Additionally, the nitrogen content in processed raw NR reached 0.44%, a significant increase of 6.14%–12.13% compared with the other treatments. Overall, the results indicated that fresh rubber latex and raw NR treated with ON50% contained higher protein levels.

3.3. Particle Size and Molecular Weight of NR

The examination of the particle size distribution of fresh latex (Figure 7) and the molecular weight distribution of raw NR (Figure 8) under different fertilization treatments revealed a negative correlation between molecular weight and particle size (Table 2). Specifically, as the particle size decreased, the molecular weight increased. The mean particle size of samples in the ON50% treatment measured 342 nm, showing a significant reduction of 3.54%–5.45% compared with the other treatments. Moreover, the number-average molecular weight (Mn) concentration in this treatment was 0.36 × 106 (g·mol−1), the highest among all treatments, representing a substantial increase of 41.50% and 21.23% compared with the CK and LN treatments, respectively. The weight-average molecular weight (Mw) for the ON50% treatment was 1.57 × 106 (g·mol−1), surpassing that of other treatments and showing a significant increase of 13.04% compared with the control treatment. Furthermore, the molecular weight distribution (Mn/Mw) for this treatment was the lowest among all treatments, demonstrating significant decreases of 20.03% and 12.96% compared with the control and LN treatments, respectively. The molecular weight of NR exhibited an increase from low to high with elevated nitrogen application and organic nitrogen fertilizer replacement (Figure 8). Additionally, NR presented the highest distribution of high-molecular-weight compounds under the ON50% treatment.

3.4. Natural Rubber Properties

Analysis of physical properties (Figure 9A–C) revealed that only the Wallace plasticity (P0) of the control treatment did not meet the Chinese standard (36) (NY/T 459-2022 [41]). However, all other treatments showed significant improvement ranging from 12.59% to 19.26% compared with CK, with the ON50% treatment demonstrating the greatest enhancement. Additionally, all fertilization treatments surpassed the standard plasticity retention index (PRI) of NY/T 459-2022 (60%), with the ON50% treatment (97.35%) exhibiting the highest PRI among all treatments. The Mooney viscosity (VR) of all treatments exceeded the minimum requirement of the standard (73) (NY/T 459-2022), except for the MN treatment. The ON50% treatment recorded the highest VR value (81.40), this being 6.54%–14.65% higher than that in other treatments. The tensile strength (a mechanical property index of vulcanized NR) of NR in all fertilization treatments did not meet the NY/T 459-2022 standard (21 MPa) (Figure 9D–F). The ON50% treatment showed the best performance, with a slight 5.55% difference from the standard. Additionally, the elongation at break of all treatments exceeded the 800% of the Chinese standard (NY/T 733-2003 [42]), with the ON50% treatment (834.75%) showing significant increases of 3.11% and 3.36% compared with the ON30% and MN treatments, respectively. The tear strength of the ON50% treatment reached the highest value (31.07 N·mm−1) among all treatments, representing significant increases of 9.63%, 19.78%, and 27.08% compared with the ON30%, LN, and MN treatments. The toughness of vulcanized NR can be quantified by analyzing the area beneath the stress–strain curve. As the material’s elongation increased, the variations in stress among various fertilization treatments were more pronounced. In particular, the ON50% treatment exhibited certain advantages compared with the other treatments, with increases of 1.49%–5.43% at 100% elongation, 1.79%–7.98% at 300% elongation, and 3.56%–9.69% at 500% elongation (Figure 10).

3.5. Relationship Between Nitrogen Nutrition and Natural Rubber Properties

Spearman correlation analysis was conducted to investigate the relationship between nitrogen fertilization, nitrogen nutrition, and natural rubber properties (Figure 11). The findings of this study indicate a positive correlation between the application of organic nitrogen, total nitrogen content in soil, available nitrogen content, and both the technological properties of raw natural rubber as well as the mechanical properties of vulcanized rubber. Specifically, there was a significant positive correlation observed between organic nitrogen application and Mw. Furthermore, soil TN and soil AN were significantly positively correlated with the molecular weight of raw natural rubber. The nitrogen content in roots and leaves also exhibited a positive association with most natural rubber properties, particularly with roots’ nitrogen content showing a significant positive correlation with Mn. Additionally, it was found that the nitrogen content in fresh latex had positive correlations with various technological properties of raw natural rubber and mechanical properties of vulcanized natural rubber; specifically, VR (vulcanization rate), tear strength, elongation at break, and strain–stress showed notable associations. Moreover, there was a positive correlation observed between the nitrogen content in raw natural rubber and its weight-average molecular weight (Mw), P0 (Wallace plasticity), tensile strength, elongation at break along with other technological and mechanical properties. Conversely, the particle size of fresh rubber latex displayed negative correlations with most natural rubber properties.

3.6. Principle Component Analysis (PCA), Partial Least Squares Discriminant Analysis (PLS-DA), and Cluster Analysis of Nitrogen Nutrition and Rubber Properties

Various statistical methods, such as PCA, PLS-DA, and cluster analysis, were performed to assess the influence of various fertilization treatments on nitrogen nutrition, the physical properties of raw natural rubber, and the mechanical properties of vulcanized rubber. The PCA results revealed that PC1 accounted for 68.3% of the total variance, while PC2 explained 24.1% of the variance, indicating that there were particularly significant variations in the ON50% treatment relative to the other treatments (Figure 12A). The results obtained from PLS-DA revealed that component 1 and component 2 accounted for 67.6% and 24.5% of the variance, respectively, thereby highlighting the distinct differentiation between the ON50% treatment and other treatments (Figure 12B). Furthermore, cluster analysis identified associations among nitrogen nutrition variables, nitrogen nutrition and raw NR properties, as well as raw NR properties and vulcanized rubber mechanical properties (Figure 12C). For example, the analysis identified relationships between soil total nitrogen content and root nitrogen content, between leaf nitrogen content and raw NR nitrogen content, and between fresh rubber latex nitrogen content and VR, as well as between PRI and elongation at break. The clustering also revealed distinct groupings based on different fertilization treatments, with the ON50% treatment being prominent, indicating its significant influence on nitrogen nutrition and NR properties.

4. Discussion

4.1. Effects of Various Fertilization Treatments on the Yield and Property of Natural Rubber

The substitution of chemical nitrogen with organic nitrogen constitutes a pivotal strategy for improving crop yield and quality. In this research, the dry rubber yield of the ON50% treatment, where organic nitrogen replaced 50% of chemical nitrogen, consistently outperformed all other treatments, ranging from 46.43 to 94.65 g·tree−1 across various sampling periods (Figure 1). Additionally, the raw NR physical properties were enhanced in the ON50% treatment, with Mn and Mw values of 0.36 × 106 g·mol−1 and 1.57 × 106 g·mol−1, respectively, and resulted in superior mechanical properties in vulcanized NR. The tensile strength (19.84 MPa), tear strength (31.07 N·mm−1), and elongation at break (834.75%) all showed significant differences compared with the other treatments (Table 1; Figure 9 and Figure 10). The main reason for this is that organic fertilizers can provide a wide range of nutrients that are gradually released, ensuring an extended period of nutrient availability to support sustained crop growth and development [43]. Additionally, organic nitrogen sources often promote healthier root systems in crops, which are essential for efficient nutrient and water uptake [44]. Through the provision of nutrients in a manner that aligns with natural and sustainable practices, organic nitrogen sources have the potential to enhance crop nutrient utilization efficiency, thereby resulting in improved yields and quality [31].
Although limited research has been conducted on the impact of organic nitrogen substitution on natural rubber yield and quality, several previous studies have reported findings consistent with those of this study. For instance, substituting organic fertilizers for a portion of chemical fertilizers led to a 26.3% increase in dry rubber yield compared with treatments using only chemical fertilizers [45]. Moreover, reducing the usage of nitrogen, phosphorus, and potassium chemical fertilizers while increasing organic fertilizer application increased the dry rubber yield [44]. In summary, the strategy of substituting organic nitrogen for chemical nitrogen is beneficial for enhancing both crop yield and quality, while concurrently promoting environmental sustainability through reduced reliance on synthetic fertilizers and mitigating their associated ecological impacts.

4.2. Effects of Different Fertilization Treatments on Nitrogen Nutrition in the Soil–Natural Rubber System

Concerning nitrogen nutrition, a 12-year study on substituting chemical nitrogen with organic nitrogen in rubber plantations, where the total nitrogen content of the studied soil in the organic nitrogen substitution treatment increased by approximately 39.58%–63.16% compared with the no fertilization treatment, was conducted [46]. Similarly, organic nitrogen substitution led to a significant 68.91% increase in available nitrogen content in rubber plantation soil compared with chemical fertilizer treatment [45]. These research results were consistent with these findings, showing that the 50% organic nitrogen substitution treatment resulted in a substantial increase in soil total nitrogen content ranging from 26.40% to 94.38% compared with other treatments. The available nitrogen increased by 67.43% compared with the control treatment, and the nitrogen content in the roots and leaves of the ON50% treatment reached peak levels. Notably, the nitrogen content, which influences NR protein content, and the protein content in non-rubber components significantly impact the properties of NR [47,48]. Chemical additives and centrifugation are commonly employed to alter the protein content [49,50,51].
This research conducted long-term field experiments with various fertilization treatments, resulting in slight changes in the nitrogen content of NR. The ON50% treatment increased the nitrogen content in fresh latex by 3.78%–9.34% compared with other fertilization methods, resulting in a rise in the nitrogen content of raw NR of 6.14%–12.13% (Figure 6). This treatment also exhibited superior performance in NR properties. The mean particle size of fresh latex decreased to 342 nm (Figure 7), with Mn and Mw values of 0.36 × 106 g·mol−1 and 1.57 × 106 g·mol−1, respectively, reaching peak levels (Figure 7; Table 1). This aligns with previous studies where deproteinization and centrifugation methods were utilized to produce varying particle sizes and molecular weights of raw NR. In this study, the particle size of fresh latex increased from 106 nm to 628 nm, while Mn and Mw decreased from 1.56 × 106 g·mol−1 and 2.21 × 106 g·mol−1 to 0.41 × 106 g·mol−1 and 1.63 × 106 g·mol−1, respectively [51]. An increase in nitrogen content generally leads to a reduction in particle size and an increase in molecular weight.

4.3. Potential Correlations Between Nitrogen Levels and Natural Rubber Properties

Proteins are critical in the cross-linking process of NR networks by binding to the end groups of polyisoprene chains, thereby enhancing various properties of NR [52,53]. Indicators for assessing the physical properties of NR include Wallace plasticity (P0), the PRI, and Mooney viscosity (VR) [19,47]. Previous research has found a negative correlation between nitrogen content and the P0 of NR, with lower nitrogen content associated with higher P0 values [54]. Conversely, an increase in the nitrogen content of fresh rubber latex from 0.23% to 0.26% during fertilization treatment resulted in a corresponding increase in P0 from 34 to 42 [11]. This research further supports a positive relationship between P0 and the nitrogen levels in fresh latex as well as in raw NR (Figure 11). Research on cloned NR with varying protein contents indicated that a higher protein content (2.98%) led to a higher plasticity retention rate (92%) [47]. In terms of rubber PRI, fertilization treatment resulted in an increase ranging from 0.6% to 5.1% compared with non-fertilization treatment [11]. Consistent with previous studies, the ON50% treatment in this study had a nitrogen content of 0.44%, equivalent to a protein content of 2.74%, and achieved a PRI of 97.35%, the highest among all fertilization treatments. Additionally, all nitrogen application treatments except for MN resulted in increases in protein content ranging from 1.27% to 7.87% compared with CK (Figure 6 and Figure 9B). A previous report suggested a negative correlation between VR and NR nitrogen content [54]; in contrast, the results of this study demonstrated a positive correlation (Figure 11).
This research demonstrated that NR with high nitrogen content exhibits exceptional mechanical properties in vulcanized rubber such as elongation at break, tensile strength, and modulus stress. These results are consistent with previous studies [51,55,56] and are supported by similar research. The 50% organic nitrogen substitution treatment in this study resulted in significant improvements in dry rubber yield, nitrogen nutrition, and NR properties compared with other fertilization methods. Furthermore, analytical techniques such as PCA, PLS-DA, and heatmap clustering further validated the efficacy of this fertilization approach. This outcome is congruent with existing studies suggesting that substituting chemical nitrogen with organic nitrogen can enhance both crop yield and quality. Overall, this study confirms that substituting 50% of chemical nitrogen fertilizer with organic nitrogen over a two-year field experiment period can effectively enhance dry rubber yield while improving the properties of NR through increased nitrogen nutrition in the soil–rubber tree system. Future research should focus on elucidating the intricate network systems involving nitrogen transformation, microbial components, and mineral nutrition to better understand their influence on the nitrogen metabolism of rubber trees and subsequently regulate the mechanisms underlying the properties of NR.

5. Conclusions

The dry rubber yield, nitrogen nutrition, and NR properties were significantly influenced by different nitrogen levels and varying proportions of organic nitrogen substitution. Among all treatments, the medium-organic-nitrogen substitution (50%) treatment (ON50%) exhibited the highest dry rubber yield. Compared with the control treatment, the ON50% treatment showed a remarkable increase in available nitrogen by 67.43%. Additionally, compared with other fertilization methods, the ON50% treatment resulted in an increase of 3.78%–9.34% and 6.14%–12.13% in nitrogen content in fresh latex and raw natural rubber (NR), respectively, leading to improved physical properties of raw NR and superior mechanical properties in vulcanized NR. Notably, significant differences were observed for tensile strength (19.84 MPa), tear strength (31.07 N·mm−1), and elongation at break (834.75%), including Mooney viscosity, the plasticity retention index, Wallace plasticity, and the distribution of molecular weights between the ON50% treatment and other treatments. The findings from this two-year study provide valuable insights and recommendations for optimizing nitrogen fertilizer application strategies in rubber tree cultivation. Moving forward, future research will focus on exploring how this fertilization method can further enhance the properties of NR.

Author Contributions

D.J.: methodology, investigation, data curation, writing—review and editing. Z.C.: methodology, investigation, data curation, writing—original draft. J.L.: methodology, writing—review and editing. Y.H.: data curation, Writing—review and editing. H.Y.: data curation. H.L.: methodology, investigation. W.L.: supervision, conceptualization, writing—review and editing. Q.L.: funding acquisition, supervision, writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Major Science and Technology Plan Project of Hainan Province (ZDKJ2021004), the National Key R&D Program of China (2022YFD2301201), the Chinese Academy of Tropical Agricultural Sciences for Science and Technology Innovation Team of National Tropical Agricultural Science Center (CATASCXTD202303), the Central Public-interest Scientific Institution Basal Research Fund (1630022024001), the Earmarked Fund for China Agriculture Research System (CARS-33).

Data Availability Statement

The data that support the results of the study are available on reasonable request from the corresponding authors.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

CNchemical nitrogen fertilizer
ONorganic nitrogen fertilizer
NRnatural rubber
CKcontrol
LNlow nitrogen
MNmedium nitrogen
HNhigh nitrogen
ON30%30% organic nitrogen replacement
ON50%50% organic nitrogen replacement
DRCdry rubber content
Sucsucrose
Piinorganic phosphorus
Mnnumber-average molecular weight
Mwweight-average molecular weight
P0Wallace plasticity
PRIplasticity retention index
VRMooney viscosity

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Figure 1. Experimental location, fertilizer treatments, and the research protocol.
Figure 1. Experimental location, fertilizer treatments, and the research protocol.
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Figure 2. Daily temperature and rainfall. (1) First fertilization. (2) Second fertilization. (3) Third fertilization. (4) Fourth fertilization.
Figure 2. Daily temperature and rainfall. (1) First fertilization. (2) Second fertilization. (3) Third fertilization. (4) Fourth fertilization.
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Figure 3. Dry rubber yield under fertilization treatments. CK, control treatment; LN, low-nitrogen treatment; MN, medium-nitrogen treatment; HN, high-nitrogen treatment; ON30%, low-organic-nitrogen substitution (30%) treatment; ON50%, medium-organic-nitrogen substitution (50%) treatment. The error bars represent standard errors. Different lowercase letters indicate significant differences (p < 0.05) between fertilization treatments in the same period, and different uppercase letters indicate significant differences (p < 0.05) between months in the same year.
Figure 3. Dry rubber yield under fertilization treatments. CK, control treatment; LN, low-nitrogen treatment; MN, medium-nitrogen treatment; HN, high-nitrogen treatment; ON30%, low-organic-nitrogen substitution (30%) treatment; ON50%, medium-organic-nitrogen substitution (50%) treatment. The error bars represent standard errors. Different lowercase letters indicate significant differences (p < 0.05) between fertilization treatments in the same period, and different uppercase letters indicate significant differences (p < 0.05) between months in the same year.
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Figure 4. Latex parameters. DRC represents the dry rubber content (A), Pi represents the inorganic phosphorus content of latex (B), Suc represent the sucrose content of latex (C), and Thiol represents the thiol content of latex (D). CK, control treatment; LN, low-nitrogen treatment; MN, medium-nitrogen treatment; HN, high-nitrogen treatment; ON30%, low-organic-nitrogen substitution (30%) treatment; ON50%, medium-organic-nitrogen substitution (50%) treatment. Different lowercase letters indicate significant differences (p < 0.05) between months in the same year.
Figure 4. Latex parameters. DRC represents the dry rubber content (A), Pi represents the inorganic phosphorus content of latex (B), Suc represent the sucrose content of latex (C), and Thiol represents the thiol content of latex (D). CK, control treatment; LN, low-nitrogen treatment; MN, medium-nitrogen treatment; HN, high-nitrogen treatment; ON30%, low-organic-nitrogen substitution (30%) treatment; ON50%, medium-organic-nitrogen substitution (50%) treatment. Different lowercase letters indicate significant differences (p < 0.05) between months in the same year.
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Figure 5. Soil total nitrogen and available nitrogen. CK, control treatment; LN, low-nitrogen treatment; MN, medium-nitrogen treatment; HN, high-nitrogen treatment; ON30%, low-organic-nitrogen substitution (30%) treatment; ON50%, medium-organic-nitrogen substitution (50%) treatment. Different lowercase letters indicate significant differences (p < 0.05).
Figure 5. Soil total nitrogen and available nitrogen. CK, control treatment; LN, low-nitrogen treatment; MN, medium-nitrogen treatment; HN, high-nitrogen treatment; ON30%, low-organic-nitrogen substitution (30%) treatment; ON50%, medium-organic-nitrogen substitution (50%) treatment. Different lowercase letters indicate significant differences (p < 0.05).
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Figure 6. Nitrogen content of root, leaf and natural rubber. FR and RB represent the fresh rubber latex and raw natural rubber, respectively. CK, control treatment; LN, low-nitrogen treatment; MN, medium-nitrogen treatment; HN, high-nitrogen treatment; ON30%, low-organic-nitrogen substitution (30%) treatment; ON50%, medium-organic-nitrogen substitution (50%) treatment. Different lowercase letters indicate significant differences (p < 0.05).
Figure 6. Nitrogen content of root, leaf and natural rubber. FR and RB represent the fresh rubber latex and raw natural rubber, respectively. CK, control treatment; LN, low-nitrogen treatment; MN, medium-nitrogen treatment; HN, high-nitrogen treatment; ON30%, low-organic-nitrogen substitution (30%) treatment; ON50%, medium-organic-nitrogen substitution (50%) treatment. Different lowercase letters indicate significant differences (p < 0.05).
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Figure 7. Particle size distribution and average particle size of NR samples. CK, control treatment; LN, low-nitrogen treatment; MN, medium-nitrogen treatment; HN, high-nitrogen treatment; ON30%, low-organic-nitrogen substitution (30%) treatment; ON50%, medium-organic-nitrogen substitution (50%) treatment.
Figure 7. Particle size distribution and average particle size of NR samples. CK, control treatment; LN, low-nitrogen treatment; MN, medium-nitrogen treatment; HN, high-nitrogen treatment; ON30%, low-organic-nitrogen substitution (30%) treatment; ON50%, medium-organic-nitrogen substitution (50%) treatment.
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Figure 8. Molecular weight distribution of NR. CK, control treatment; LN, low-nitrogen treatment; MN, medium-nitrogen treatment; HN, high-nitrogen treatment; ON30%, low-organic-nitrogen substitution (30%) treatment; ON50%, medium-organic-nitrogen substitution (50%) treatment.
Figure 8. Molecular weight distribution of NR. CK, control treatment; LN, low-nitrogen treatment; MN, medium-nitrogen treatment; HN, high-nitrogen treatment; ON30%, low-organic-nitrogen substitution (30%) treatment; ON50%, medium-organic-nitrogen substitution (50%) treatment.
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Figure 9. Physical properties of NR and mechanical properties of vulcanized NR. Physical properties of NR, where P0 represents the Wallace plasticity (A), PRI represents the plasticity retention index (B), VR represents the Mooney viscosity (C). The mechanical properties of vulcanized NR, tensile strength (D), elongation at break (E), tear strength (F) are also shown. CK, control treatment; LN, low-nitrogen treatment; MN, medium-nitrogen treatment; HN, high-nitrogen treatment; ON30%, low-organic-nitrogen substitution (30%) treatment; ON50%, medium-organic-nitrogen substitution (50%) treatment. Different lowercase letters indicate significant differences (p < 0.05).
Figure 9. Physical properties of NR and mechanical properties of vulcanized NR. Physical properties of NR, where P0 represents the Wallace plasticity (A), PRI represents the plasticity retention index (B), VR represents the Mooney viscosity (C). The mechanical properties of vulcanized NR, tensile strength (D), elongation at break (E), tear strength (F) are also shown. CK, control treatment; LN, low-nitrogen treatment; MN, medium-nitrogen treatment; HN, high-nitrogen treatment; ON30%, low-organic-nitrogen substitution (30%) treatment; ON50%, medium-organic-nitrogen substitution (50%) treatment. Different lowercase letters indicate significant differences (p < 0.05).
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Figure 10. Curves for the stress–strain of vulcanized NR. CK, control treatment; LN, low-nitrogen treatment; MN, medium-nitrogen treatment; HN, high-nitrogen treatment; ON30%, low-organic-nitrogen substitution (30%) treatment; ON50%, medium-organic-nitrogen substitution (50%) treatment.
Figure 10. Curves for the stress–strain of vulcanized NR. CK, control treatment; LN, low-nitrogen treatment; MN, medium-nitrogen treatment; HN, high-nitrogen treatment; ON30%, low-organic-nitrogen substitution (30%) treatment; ON50%, medium-organic-nitrogen substitution (50%) treatment.
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Figure 11. Spearman correlations among nitrogen levels, nitrogen nutrition, and rubber natural properties. CN, chemical nitrogen fertilizer; ON, organic nitrogen fertilizer; TN, total nitrogen; AN, available nitrogen; FR N, nitrogen in fresh natural rubber; RB N, nitrogen in raw natural rubber. Asterisks * and ** indicate significances at p < 0.05 and p < 0.01, respectively. Blue and pink indicate positive and negative correlations, respectively.
Figure 11. Spearman correlations among nitrogen levels, nitrogen nutrition, and rubber natural properties. CN, chemical nitrogen fertilizer; ON, organic nitrogen fertilizer; TN, total nitrogen; AN, available nitrogen; FR N, nitrogen in fresh natural rubber; RB N, nitrogen in raw natural rubber. Asterisks * and ** indicate significances at p < 0.05 and p < 0.01, respectively. Blue and pink indicate positive and negative correlations, respectively.
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Figure 12. Relationships between various fertilization treatments, nitrogen nutrition, and natural rubber properties. Various fertilization treatments on nitrogen nutrition, raw NR and vulcanized NR properties of the principle component analysis (PCA) (A), partial least squares discriminant analysis (PLS-DA) (B), and cluster analysis (C), respectively. The cluster analysis illustrates the clustering patterns of nitrogen nutrition and natural rubber properties across different fertilization treatments, with red indicating high values and blue denoting low values. CK, control treatment; LN, low-nitrogen treatment; MN, medium-nitrogen treatment; HN, high-nitrogen treatment; ON30%, low-organic-nitrogen substitution (30%) treatment; ON50%, medium-organic-nitrogen substitution (50%) treatment.
Figure 12. Relationships between various fertilization treatments, nitrogen nutrition, and natural rubber properties. Various fertilization treatments on nitrogen nutrition, raw NR and vulcanized NR properties of the principle component analysis (PCA) (A), partial least squares discriminant analysis (PLS-DA) (B), and cluster analysis (C), respectively. The cluster analysis illustrates the clustering patterns of nitrogen nutrition and natural rubber properties across different fertilization treatments, with red indicating high values and blue denoting low values. CK, control treatment; LN, low-nitrogen treatment; MN, medium-nitrogen treatment; HN, high-nitrogen treatment; ON30%, low-organic-nitrogen substitution (30%) treatment; ON50%, medium-organic-nitrogen substitution (50%) treatment.
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Table 1. Fertilization in experimental treatments (g·tree−1·year−1).
Table 1. Fertilization in experimental treatments (g·tree−1·year−1).
TreatmentsOrganic Nitrogen (N)Chemical Nitrogen (N)Phosphorus (P2O5)Potassium (K2O)
CK0012090
LN013812090
MN027612090
HN055212090
ON30%8319312090
ON50%13813812090
Note: CK, control treatment; LN, low-nitrogen treatment; MN, medium-nitrogen treatment; HN, high-nitrogen treatment; ON30%, low-organic-nitrogen substitution (30%) treatment; ON50%, medium-organic-nitrogen substitution (50%) treatment.
Table 2. Particle size and molecular weight of NR samples from different treatments.
Table 2. Particle size and molecular weight of NR samples from different treatments.
TreatmentsParticle Size (nm)Mn × 106 (g·mol−1)Mw × 106 (g·mol−1)Mw/Mn
CK355 ± 1a0.26 ± 0.00c1.39 ± 0.04b5.43 ± 0.05a
LN362 ± 2a0.30 ± 0.02bc1.48 ± 0.04ab4.99 ± 0.25ab
MN358 ± 3a0.33 ± 0.03ab1.48 ± 0.06ab4.51 ± 0.32bc
HN359 ± 1a0.33 ± 0.01ab1.52 ± 0.01a4.68 ± 0.18bc
ON30%359 ± 1a0.33 ± 0.01ab1.54 ± 0.03a4.62 ± 0.12bc
ON50%342 ± 7b0.36 ± 0.01a1.57 ± 0.02a4.34 ± 0.07c
Note: The term Mn represents the number-average molecular weight, Mw denotes the weight-average molecular weight, and Mw/Mn refers to the distribution of molecular weights. CK, control treatment; LN, low-nitrogen treatment; MN, medium-nitrogen treatment; HN, high-nitrogen treatment; ON30%, low-organic-nitrogen substitution (30%) treatment; ON50%, medium-organic-nitrogen substitution (50%) treatment. Different lowercase letters indicate significant differences (p < 0.05).
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Jin, D.; Cha, Z.; Li, J.; Huang, Y.; Yang, H.; Liu, H.; Luo, W.; Lin, Q. Reinforcing Nitrogen Nutrition Through Partial Substitution with Organic Nitrogen Enhances the Properties of Natural Rubber. Forests 2024, 15, 1897. https://doi.org/10.3390/f15111897

AMA Style

Jin D, Cha Z, Li J, Huang Y, Yang H, Liu H, Luo W, Lin Q. Reinforcing Nitrogen Nutrition Through Partial Substitution with Organic Nitrogen Enhances the Properties of Natural Rubber. Forests. 2024; 15(11):1897. https://doi.org/10.3390/f15111897

Chicago/Turabian Style

Jin, Dongqi, Zhengzao Cha, Jianhong Li, Yanyan Huang, Hongzhu Yang, Hailin Liu, Wei Luo, and Qinghuo Lin. 2024. "Reinforcing Nitrogen Nutrition Through Partial Substitution with Organic Nitrogen Enhances the Properties of Natural Rubber" Forests 15, no. 11: 1897. https://doi.org/10.3390/f15111897

APA Style

Jin, D., Cha, Z., Li, J., Huang, Y., Yang, H., Liu, H., Luo, W., & Lin, Q. (2024). Reinforcing Nitrogen Nutrition Through Partial Substitution with Organic Nitrogen Enhances the Properties of Natural Rubber. Forests, 15(11), 1897. https://doi.org/10.3390/f15111897

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