The quality of artificial grasslands in alpine regions is controlled by crop photosynthesis under different fertilization conditions

The Qinghai-Xizang Plateau is located in the central part of Asia, with an average altitude of approximately 4,000 m. It is one of the sources of the Yangtze River, the Yellow River, and the Lancang River¹. Its complex geographical features have not only shaped a unique ecological environment for the plateau grasslands but also played an important role in regulating the global climate. For a long time, grassland animal husbandry has been the basic industry in the Sanjiangyuan region and the fundamental means of survival for the herdsmen². However, with the intensification of climate change and population growth, traditional grassland animal husbandry is facing a severe challenge of unbalanced forage supply. The long winter with a grass shortage period of up to seven or eight months leads to a significant decline in the nutritional value of the forage. The available land area in the Sanjiangyuan region is small, and how to utilize the limited land area to balance ecological protection and economic development has become one of the urgent problems to be solved^3,4. Therefore, the establishment of high-quality and high-yield artificial grasslands proposed in this context becomes the key to solving this problem.

Exogenous nutrient addition is a key management measure for enhancing the productivity of artificial grasslands. The rational application of nitrogen fertilizer and phosphorus fertilizer has a significant impact on the growth, quality and yield of forage grass^5,6. Studies have shown that when using nitrogen fertilizer, the yield of alfalfa will increase by 8% to 25%, while when using phosphorus fertilizer, it will increase by 122% to 172%^7[,8. There is a large amount of phosphorus in the soil, but it usually exists in the form of insoluble mineral phosphates or is combined with organic matter, which makes it difficult for plants to directly absorb or utilize. This becomes a major limiting factor for plant growth and yield⁹. In agricultural and livestock production, phosphate fertilizers are usually used to meet the phosphorus requirements of plants. Due to the tendency of chemical phosphate fertilizers to highly combine with ions such as calcium, iron and aluminum, they will deposit in the form of inorganic phosphates in the soil, so the effective utilization efficiency of plants is lower than 25%^10,11. The increase in nitrogen deposition in the atmosphere will lead to soil acidification, which will affect soil respiration and ultimately affect the photosynthesis of plant leaves^12,13. Nitrogen and phosphorus in the leaves are powerful factors for photosynthesis. When the nitrogen intake increases and chlorophyll increases, the concentration and activity of enzymes involved in photosynthesis will increase^14,15, thereby improving the photosynthetic capacity of plant leaves^16,17.

The competition among different species and the proportion of leguminous plants in the forage have significant differences in their ability to fix nitrogen^18,19, so the plant nutrients and community yield may be affected by various leguminous forage species and their proportions in the legume-cereal mixed planting combinations. The net photosynthetic rate, water use efficiency (WUE), and leaf nitrogen content of the legume-mixed planting combinations are all higher than those of monoculture^20,21,22. Yellow clover (Medicago sativa ssp.) was added to the forage to increase the content of calcium (Ca), potassium (K), and magnesium (Mg), which increased the yield of the forage and also improved its nutritional components^23,24. However, due to geographical limitations, there are low nutrient content soil conditions in the Sanjiangyuan region of the Qinghai-Tibet Plateau. Therefore, some exogenous fertilizers must be added to provide nutritional support for the growth and development of the grass-planting legume-cereal combinations. The accumulation of plant nutrients may benefit from the photosynthesis of plants, as the duration of sunlight in the mountain environment is relatively long. However, no existing research has demonstrated that applying nitrogen and phosphorus fertilisers in high-altitude regions promotes nutrient accumulation through photosynthesis in plants. Therefore, this study investigates the relationship between plant photosynthesis and fertiliser-derived nutrient accumulation by cultivating an annual grass-legume mixed cropping system in high-altitude areas. The authors propose the following points: 1: Fertilisers improve leaf physiological and biochemical indicators via photosynthesis; 2: Fertilisers enhance plant quality by accumulating nitrogen and phosphorus nutrients; 3: Identifying the most effective fertiliser types for high-altitude regions.

Details about the test site in general

The field scientific observation and research station of the Ministry of Education (located at 33°24’30” north latitude, 97°18’00” east longitude, and at an altitude of 4270 m) is situated within the Sanjiangyuan grassland ecosystem in Chengduo County, Yushu Prefecture, Qinghai Province (Fig. 1). By collecting the climate data of this area and consulting relevant literature, it is determined that this region has a typical plateau continental climate. The winter is cold with low temperatures. The annual average maximum temperature is 24.1 degrees Celsius, the annual average minimum temperature is −26.6 degrees Celsius, and the annual average temperature is −1.4 degrees Celsius. The annual average precipitation is 406.00 millimeters, with 83.79% of the total precipitation concentrated from June to October. The number of cold weather days is approximately 260, and the average daily sunshine duration is about 2650.5 hours²⁵. The annual average precipitation is 406.00 millimeters, with 83.79% of the total precipitation concentrated from June to October. The pH value of the soil is 6.92, the organic matter content is 2.36%, the total nitrogen content is 9.50 g per kilogram, the available nitrogen content is 14.0 milligrams per kilogram, the total phosphorus content is 8.20 g per kilogram, the available phosphorus content is 7.0 milligrams per kilogram, the total potassium content is 13.50 g per kilogram, and the available potassium content is 76.5 milligrams per kilogram.

Test materials

The Grassland Institute of the Qinghai Provincial Academy of Animal Husbandry and Veterinary Medicine supplied the oat and forage pea varieties for testing in this experiment, QingTian No1 and Qing Jian No1, respectively. The nitrogen fertilizer used in this experiment was urea (containing N 46%) and the phosphate fertilizer was calcium superphosphate (containing P₂O₅ 12%).

Design of the experiment

This experiment was conducted from May 12th to September 20th, 2024. Based on the test results from the existing regional studies and the screening of the previous pre-experiment, the 70:30 mixed planting ratio of oats (Avena sativa L.) and forage peas (Pisum sativum L.) achieved the highest yield and quality²⁶. Based on the above experimental results, this experiment added four fertilization treatments (Table 1): applying nitrogen fertilizer alone (A1), applying phosphorus fertilizer alone (A2), applying a nitrogen-phosphorus compound fertilizer (A3), and the no-fertilization control (CK) of this study, with the fertilizer amount referring to the results of Feng et al. A randomized complete block design was adopted, and each fertilization treatment had six replicates. No additional fertilization or irrigation was carried out during the entire growth period.

The experimental field measured 3 m×5 m (15 m² total area). One day prior to sowing, the field was plowed and leveled. Seeds were sown in parallel rows at a depth of 3–4 cm, with 10 manually dug rows per plot spaced 30 cm apart. Fertilizers were broadcast at the sowing sites. Harvesting was completed on September 25th. Both plant species were sown simultaneously, with the seeding density of grasses and legumes in each row determined by the monoculture seeding rates of individual planting treatments.

Methods and indicators

Description of leaf morphology characteristics

On September 20, 2024, scans were conducted on the leaves of herbaceous and leguminous forage plants in each experimental plot. A total of six measurements were taken for each plot. The measurement work was carried out using the Yaixin-1241 Leaf Area Measuring Instrument, which measured the leaf area, leaf perimeter, leaf length, and leaf width data of both grass and legume plants, and the results were recorded^27,28.

Analysis of leaf spectral index characteristics

On September 22, 2024, using the CI-710 S plant spectrometer, the physiological and biochemical indicators of grass and legume plants in each treatment group were measured²⁹. Each experimental field underwent 6 repetitions of experiments. The following parameters were measured: green index (G), normalized vegetation index (NDVI), chlorophyll A (CPHLA), chlorophyll B (CPHLB), total chlorophyll (CPHLT), soil-plant analysis development index (SPAD), chlorophyll B (CPHLB), plant senescence reflection index (PSRI), and water band index (WBI), and the data of grass and legume experiments were recorded separately³⁰.

The formulae for the indicators are as follows

1. (1)

   : (NDVI = frac{{R800 – R680}}{{R800 + R680}}). The numerical range is from − 1 to 1, and r represents the reflectance value in the red light band.

2. (2)

   : (PSRI = frac{{R680 – R500}}{{R750}}) .The numerical range is from − 1 to 1, and r represents the reflectance value in the red light band.

3. (3)

   : (WBI = frac{{R900}}{{R970}}) .The numerical range is from 0.8 to 1.2, and Rr represents the reflectance value in the red light band.

4. (4)

   : (CPHLA = 12.72A663 – 2.59A645) .A663 and A645 are the absorbance at 663 nm and 645 nm.

5. (5)

   : (CPHLB = 22.88A645 – 4.67A663) ,A663 and A645 are the absorbance at 663 nm and 645 nm.

6. (6)

   :(CPHLT = CPHLA + CPHLB)

7. (7)

   : (G = frac{{R554}}{{R677}}), R is the reflection value in the red light band.

8. (8)

   : (SPAD = ( – 42.9 + (42.1*Math.pow(T931/T653,0.215))))

Feed quality inspection

On September 25, 2024, three 1-meter-long sample sections were randomly selected from each treatment area on both sides, and they were trimmed to be level with the ground. Subsequently, the grass samples of the mixed species of Gramineae and Fabaceae were placed in a drying oven and dried at 110 °C for 30 min, then dried to constant weight at 80 °C. The neutral detergent fiber (NDF) and acid detergent fiber (acidic detergent fiber, ADF) were analyzed using the Van Soest method; total phosphorus (TP) was determined using the sulfuric acid-peroxide digestion technique; crude protein (CP) was determined using the sulfuric acid-peroxide digestion method; soluble sugar (SS) was determined using the anhydroxyl method; total carbon (TC) and total nitrogen (TN) were determined using an elemental analyzer.

Statistical analysis

The data were summarized using Microsoft Excel 2016, and a one-way ANOVA was conducted using IBM SPSS 23.0. The data satisfied normality (Shapiro-Wilk, P > 0.05) and homogeneity of variance (Levene test, P = 0.12). The groups with significant differences were identified through Duncan’s multiple comparison test (P < 0.05). The relevant icons were drawn using the software named Origin 2021. The geographical map of the experimental area location was drawn using ArcMap 10.8.

Changes in leaf morphology under different fertilizer treatments

Each of the three fertilization techniques improved the physiological morphological characteristics of the oat leaf blades. The results in Fig. 2 show that the fertilization treatments (A1, A2, and A3) improved the morphological characteristics of the oat tees more generally. A3 leaf area rose by 31.81% (P < 0.05) (Fig. 2a), A1 leaf perimeter increased by 25.61% (P < 0.05) (Fig. 2b), A1 leaf length increased by 17.71% (P < 0.05) (Fig. 2c), and A1’s leaf width expanded by 62.50% (P < 0.05) (Fig. 1d) in comparison to the no-fertilization (CK) treatment. Out of the three fertilizer treatments, the oat leaf morphology in the A1 treatment exhibited a superior trend, and there was no discernible difference between the A2 and A3 treatments overall.

The improvement of the forage pea’s leaf area, girth, and length was more strongly influenced by different fertilization treatments (Fig. 3). The forage pea leaf area increased by 27.08%, 28.50%, and 34.69% (P < 0.05) in A1, A2, and A3 when compared to the no-fertilizer (CK) treatment; the forage pea leaf perimeter increased by 76.43% (P < 0.05) in A1 treatment; and the forage pea leaf length increased by 9.90%, 12.58%, and 13.40% (P < 0.05) in A1, A2, and A3 treatments. However, there was no discernible difference in the forage pea leaf width. Additionally, there were no notable variations among the three fertilizer applications.

Variations in the spectral index properties of leaves treated with various fertilizers

The different fertilizer treatments had a substantial impact on the soil-vegetation analysis development, but not on the oat spectral index. The results evaluated in Fig. 4 showed that the total chlorophyll amount in A3 was 17.07% (P < 0.05) higher than in A1 treatment and 15.14% (P < 0.05) higher than in CK treatment. In comparison to the A1 and A2 treatments, the A3 treatment improved the soil-vegetation analysis development indexes by 17.05% and 26.85% (P < 0.05), respectively, and by 26.44% (P < 0.05) when compared to the CK treatment. The water band index index increased by 4.81% (P < 0.05) in A2 compared to A3.Together, these findings showed that fertilizer treatment did not significantly affect the overall oat spectral index; however, the soil-vegetation analysis development indices varied significantly.

The results in Fig. 5 showed that there was no significant difference in the spectral index features of fodder pea leaves across the different fertilization treatments. While the other indexes were not substantially differ from one another, the CK treatment showed a superior trend in the total chlorophyll index, which was 17.97% and 20.25% greater than that of the A1 and A3 treatments (P < 0.05). A detailed analysis of the previously given data showed that fertilizer treatments had no appreciable effect on the overall spectral index of forage peas, but the CK treatment demonstrated a superior trend in total chlorophyll.

Variations in the quality of the feed with various fertilizer treatments

Fertilization procedures significantly improved the fodder’s quality. The results of the analysis showed that the three fertilization treatments significantly reduced the contents of acid detergent fiber and neutral detergent fiber compared to the CK treatments by 8.67% and 19.28% (P < 0.05) in the A1 treatment, 11.25% and 28.92% (P < 0.05) in the A2 treatment, and 7.78% and 25.25% (P < 0.05) in the A2 treatment (Fig. 6). Forage soluble sugar, crude protein, total phosphorus, and total nitrogen all showed a discernible improvement as compared to CK therapy. When the three fertilization treatments were compared, the A3 treatment had lower amounts of neutral and acid detergent fiber and higher levels of soluble sugar, crude protein, total phosphorus, total nitrogen, and total carbon.

Changes in plant quality under different fertilizer treatments. X-axis: The capital letters in the four treatment groups represent the separate application of nitrogen fertilizer (A1), separate application of phosphorus fertilizer (A2), application of nitrogen-phosphorus compound fertilizer (A3), and no fertilizer application as the control group (CK). (a) The Y-axis represents the change in neutral detergent fiber content. (b) The Y-axis represents the change in acidic detergent fiber content. (c) The Y-axis represents the change in crude protein content. (d) The Y-axis represents the change in soluble sugar content. (e) The Y-axis represents the change in total nitrogen content. (f) The Y-axis represents the change in total phosphorus content. (g) The Y-axis represents the change in total carbon content. * indicates significant differences, ns indicates no significant differences. In figures a, b, c, d, e, f, and g, “*” indicates significant differences among the treatments at the P < 0.05 level (Duncan’s test); “ns” indicates no significant differences (P > 0.05).

Analysis of correlations between fodder quality, morphological traits, and leaf spectral index characteristics

Figure 6’s results show that while fodder quality and oat forage leaf spectral features and overall leaf shape are positively connected, NDF and ADF indexes are substantially inversely connected. Furthermore, there is a strong negative correlation between NDF and ADF indicators and leaf shape, although there is a significant positive correlation with forage soluble sugars, crude proteins, total phosphorus, total nitrogen, and total carbon (Fig. 7). Forage soluble sugars, crude protein, total phosphorus, total nitrogen, and total carbon all showed a strong negative connection with the NDF and ADF indexes. Furthermore, the morphology of the unique blades as a whole and the spectral properties of forage pea forage blades were positively correlated, as was the forage quality.

Construction of structural equation model for forage quality, morphological characteristics and leaf spectral index features

Based on the spectral index characteristics of the leaves, the morphological characteristics of the leaves, and the quality indicators of the forage grass, a random forest model was constructed (Fig.8A) to identify the main driving factors affecting the change in crude protein. The results showed that variables such as GLA, LLC, TP, SPAD, CPHLT, fertilizer application amount (F), NDVI, PSRI, WBI, G, etc., in the grass family and leguminous plants were the main variables affecting the crude protein index. According to the analysis results of the structural equation model (SEM)(Fig.8B), the overall fit of the model was good: Fisher’s C=12.693, degrees of freedom df = 8, P= 0.123>0.05, indicating that there was no significant difference between the model and the data, and it was acceptable. The explanatory power (R²) of each endogenous variable showed that variables such as WBI-G (R² = 0.70), WBI-L (R² = 0.62), F (R² = 0.61), CP (R² = 0.55), and GLA (R² = 0.42) had a relatively high proportion of variance explanation, while the explanatory power of CPHLB-L (R²= 0.19), NDVI-L (R²=0.23*), CPHLT-G (R²=0.25*), and LLC (R²=0.26) was relatively low but partially reached a significant level (p<0.05,***p<0.001). F, WBI-G, CPHLT-L, TP, WBI-L had direct effects on CP and were significantly positively correlated (P<0.05), CPHLB-L indirectly affected CP through F, and LLC indirectly affected CP through WBI-L.

Effect of fertilizer treatments on morphological characteristics of leaves

Leaves are one of the important organs of forage. The overall morphology of leaves has a large surface area, which is conducive to the input of water and nutrients as well as the output of photosynthetic products³¹. The results of the leaf morphology indicators selected in this study indicate that fertilization treatment has a significant impact on the morphology of oat leaves. Among them, the nitrogen and phosphorus mixed application treatment results in larger leaf area for oat, while the single application of nitrogen fertilizer treatment has higher leaf circumference, length, and width, and there are significant differences between this treatment and the non-fertilization control treatment. These differences are mainly attributed to the specificity of the physiological functions of different elements³². Nitrogen is the core component of chlorophyll and photosynthetic proteins, mainly driving cell division and elongation, so single application of nitrogen fertilizer significantly increases the circumference, length, and width of the leaves; while phosphorus is involved in the construction of energy carriers (ATP) and the transportation of photosynthetic products, and is more focused on influencing the differentiation of mesophyll cells and the expansion of single leaf area. When nitrogen and phosphorus are mixed applied, they form a synergistic effect, providing a protein framework and ensuring energy supply, enabling oat to overcome nutrient limitation and form the largest leaf area³³. The differences between species can also be analyzed. Oats, as a grass plant, does not have nitrogen fixation ability and is highly sensitive to soil nitrogen. Exogenous nitrogen application directly stimulates the comprehensive increase of their leaf morphology; while for forage peas, as a leguminous plant, it can fix nitrogen through symbiotic rhizobia, and exogenous nitrogen fertilizer may trigger the “nitrogen repression” effect, causing it to prioritize resources allocation to oat plants for stem elongation to compete for light³⁴. This explains why fertilization has no significant effect on the width of pea leaves³⁵. In addition, fertilization promotes the rapid growth and height increase of oats, causing forage peas to be in a lower spatial position in the community and being shaded^36,37. The shading stress induces peas to increase leaf area and length to enhance light capture efficiency, which is consistent with the leaf response trend after adding phosphorus. This further amplifies the phenotypic differences between grass and leguminous plants among different fertilization treatments³⁸. Based on these findings, we conclude that the combination of nitrogen and phosphorus has the most significant impact on leaf morphology and is the best fertilization strategy for high-altitude regions.

Effect offertilization treatments on spectral index characteristics of leaves

The strength of photosynthetic capacity of leaves is a key parameter determining crop yield, and premature leaf senescence is an important factor limiting high yield. Its main characteristic is the functional loss of the photosynthetic apparatus in the leaf, leading to a decline in photosynthetic function. Reasonable fertilization can reduce the nutrient output of leaves after flowering, delay leaf senescence, and maintain a higher photosynthetic rate³⁹. The research results show that in oat plants under different fertilization treatments, the CPHLT and SPAD indices of the nitrogen-phosphorus combined application treatment are at a relatively high level, which is significantly improved compared to the single application of nitrogen fertilizer and no fertilization treatment. The WBI index of leaves under the single application of phosphorus fertilizer is relatively high, significantly superior to the nitrogen-phosphorus combined application treatment. Other leaf spectral characteristic indicators do not have significant advantages. The fertilization treatments have no significant impact on the spectral indices of forage peas except for CPHLT. This is mainly because oat is a C₃ grass plant, and its photosynthetic apparatus is highly sensitive to nutrient supply⁴⁰. The addition of exogenous nitrogen and phosphorus fertilizers can directly promote chlorophyll synthesis and delay the degradation of chloroplasts, so the total chlorophyll content of oat under the nitrogen-phosphorus combined application treatment significantly increases, the light energy utilization efficiency is enhanced, the leaf senescence process is effectively delayed, and this reflects the positive regulatory effect of fertilization on grass crops⁴¹. For forage peas as a leguminous plant, their roots can symbiotically fix nitrogen with rhizobia and have a lower dependence on soil exogenous nitrogen. When exogenous fertilizers are applied, peas may preferentially utilize the nitrogen fixed by themselves rather than the increased mineral nitrogen in the soil, resulting in their sensitivity to fertilization responses being much lower than that of oat⁴². This explains why the spectral characteristic indicators of pea leaves do not show significant improvement under fertilization treatments. Moreover, in the high-altitude environment background, oat plant height is already limited by natural conditions such as low temperature and strong wind. In the oat and pea mixed-sowing system, the nitrogen-phosphorus combined application promotes oat plants to obtain more sufficient nutrient supply, resulting in a significant increase in plant height and canopy density⁴³. Although forage peas are climbing plants, their upward growth height is limited by the height of oat plants. In the high-altitude conditions, the absolute value of oat plant height is relatively low, and the climbing space of peas is physically compressed, causing them to be forced to be in the lower part of the canopy⁴⁴. Therefore, under fertilization treatment, oat leaves are more lush, the canopy density is significantly increased, and they have a stronger shading effect on the lower peas, resulting in a significant reduction in the photosynthetic effective radiation received by pea leaves and a lower light energy acquisition ability than the no-fertilization treatment. This shading effect not only reduces the photosynthetic rate of pea leaves but may also induce a compensatory increase in the total chlorophyll content of peas, resulting in a phenomenon where the total chlorophyll content of peas under fertilization treatment is higher than that under no-fertilization treatment. This is essentially an adaptation mechanism in a low-light environment, rather than a superiority in nutrient supply⁴⁵. At the same time, the effects of fertilization treatment on the distribution of canopy water and metabolic activity also show species-specific differences. Under the single application of phosphorus fertilizer treatment, the water band index of leaves is higher, indicating that phosphorus fertilizer promotes the increase of water content in the plant canopy and the enhancement of metabolic rate. However, in the oat and pea mixed-sowing system, it may be affected by interspecific competition. Oat, with stronger nutrient absorption ability and plant height advantage, occupies a dominant position in water and nutrient acquisition, while peas, due to their inferior position in the canopy, have their leaf water content, metabolic activity, and photosynthetic efficiency inhibited. In addition, this study only assesses the accumulation and conversion efficiency of nutrients based on leaf spectral index characteristics⁴⁶. Although spectral parameters such as total chlorophyll content, water band index, and soil-vegetation analysis development index can well reflect the canopy structure, chlorophyll content, and water status, they cannot deeply determine the content of various substances in leaves and the activity of key enzymes⁴⁷. Therefore, spectral characteristics are difficult to fully reveal the internal mechanisms of the interaction between oat and pea under fertilization treatment in terms of photosynthetic products and canopy competition dynamics. Future research needs to combine physiological and biochemical measurements at the leaf scale to systematically clarify the underlying mechanisms of the species-specific differences in the fertilization effect in the mixed-sowing system.

Effect offertilization treatments on forage quality

The main purpose of adding exogenous fertilizers is to achieve higher target yields, corresponding forage quality, and to enhance soil fertility⁴⁸. The results of this study indicate that there are significant differences in the regulatory effects of different fertilization treatments on forage fiber components and nutritional quality. All three fertilization treatments significantly reduced the neutral detergent fiber and acid detergent fiber contents of forage, with the single application of phosphate fertilizer having the most prominent effect, reducing by 11.25% and 28.92% respectively. This is mainly because phosphorus directly participates in the regulation of plant cell wall metabolism, and phosphorus is an important component of ATP and nucleic acids, which can promote energy metabolism and protein synthesis in plants, accelerate cell division and elongation⁴⁹. Plants tend to allocate more photosynthetic products to protoplasm synthesis, resulting in a decrease in the proportion of fiber components in dry matter. Moreover, the application of phosphate fertilizer can promote root development and nutrient absorption efficiency, enhance the overall metabolic activity of the plant, accelerate the transport and transformation of carbohydrates, and increase the accumulation of soluble sugars and other non-structural carbohydrates, thereby diluting the relative content of fiber components⁵⁰. At the same time, the fertilization treatments significantly increased the contents of soluble sugars, crude protein, total phosphorus, and total nitrogen, which reflects the comprehensive promoting effect of exogenous nutrient addition on the productivity of grassland ecosystems. Among them, the nitrogen-phosphorus combined application treatment had a more significant effect compared to the no-fertilization treatment, because grass plants lack biological nitrogen fixation ability, and their growth and development are highly dependent on soil mineral nitrogen. The addition of exogenous nitrogen directly alleviates nitrogen limitation and promotes the tillering, leaf expansion, and photosynthetic carbon assimilation of grass plants⁵¹. Moreover, the addition of nitrogen activates the nitrogen fixation activity of leguminous plants through the “nitrogen stimulation effect”, and the leguminous plant root nodule nitrogen fixation system gradually plays a supplementary role in the early growth stage, forming a dual supply pattern of exogenous nitrogen and biological nitrogen fixation⁵². Based on the above analysis, nitrogen-phosphorus combined application can simultaneously meet the large demand for nitrogen by grass plants and the sensitive dependence of leguminous plants on phosphorus, avoiding the possible nutrient imbalance caused by single fertilization. Moreover, the mixed planting system itself has the nitrogen-fixing function of leguminous plants and the exogenous nitrogen-phosphorus supply forms a synergistic effect. The addition of nitrogen not only directly promotes the growth of grass plants but also indirectly improves the utilization efficiency of phosphorus through optimizing the canopy structure and extending the photosynthetic functional period, forming a positive feedback loop⁵³. The formation of the differences in fertilization effects in this study is essentially the result of the spatial heterogeneity distribution of phosphorus, the differentiation of nutrient acquisition strategies of grass and leguminous plants, and the dynamic competition and complementarity of resources among species in the mixed planting system. The enrichment characteristic of phosphorus in the surface soil determines its direct and efficient regulation of fiber components, while nitrogen-phosphorus combined application achieves multi-target synergy in improving forage yield, quality, and soil fertility through coupling the rapid response of grass plants to mineral nitrogen and the ecological service function of leguminous plants’ nitrogen fixation. This forms a positive feedback loop⁵⁴. This study shows that the mixed application of nitrogen and phosphorus can simultaneously meet the large demand for nitrogen by grass plants and the sensitive dependence of leguminous plants on phosphorus, avoiding the possible nutrient imbalance caused by single fertilization. Moreover, the mixed planting system itself has the nitrogen-fixing function of leguminous plants and the exogenous nitrogen-phosphorus supply forms a synergistic effect. The addition of nitrogen not only directly promotes the growth of grass plants but also indirectly improves the utilization efficiency of phosphorus through optimizing the canopy structure and extending the photosynthetic functional period, forming a positive feedback loop. This study provides theoretical basis and practical feasibility for its promotion and establishment in high-altitude regions.

The results of the different fertilization treatments conducted at high altitudes on the feed quality produced by the mixed planting of leguminous plants and forage grasses show that fertilization can improve the physiological and biochemical indicators of leaves through photosynthesis and enhance the feed quality. Compared with the untreated treatment, the effect is better. In all treatment groups, the group that applied a mixture of nitrogen and phosphorus showed better performance in leaf morphology, photosynthetic capacity, and nutritional components than the untreated group, and was able to improve plant quality by accumulating nitrogen and phosphorus nutrients. Therefore, for high-altitude regions, the most effective fertilizer type is the treatment of combined application of nitrogen and phosphorus. However, it should be noted that this conclusion only applies to the mixed planting combination of oats and forage pea and their respective ratios. Additionally, this research trial was a one-year short-term experiment and lacked long-term longitudinal studies. Long-term related research will be conducted in the future.