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Article

Response of Elymus sibiricus (Siberian Wildryegrass) to Combined Application of Nitrogen and Phosphorus during Aging on the Qinghai–Tibetan Plateau

by
Rui Wu
,
Wenhui Liu
*,
Yongchao Zhang
,
Guoling Liang
,
Wen Li
and
Kaiqiang Liu
Key Laboratory of Superior Forage Germplasm in the Qinghai-Tibetan Plateau, Laboratory for Research and Utilization of Qinghai Tibet Plateau Germplasm Resources, Qinghai Academy of Animal Husbandry and Veterinary Sciences, Qinghai University, Xining 810016, China
*
Author to whom correspondence should be addressed.
Agronomy 2024, 14(7), 1543; https://doi.org/10.3390/agronomy14071543
Submission received: 25 June 2024 / Revised: 10 July 2024 / Accepted: 12 July 2024 / Published: 16 July 2024
(This article belongs to the Special Issue Fertility Management for Higher Crop Productivity)
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Abstract

:
Elymus sibiricus plays a crucial role in ecological protection and animal husbandry. However, after many years of growth, the biomass of E. sibiricus decreases, and the plants degrade. Moreover, there is no good solution to the problem of degradation of Elymus sibiricus; the addition of nitrogen (N) and phosphorus (P) fertilizers is the primary measure of cultivation management to improve yield, so it is crucial to find the appropriate level of fertilization. This study performed a two-factor split-plot experiment, including four levels of N (0, 45, 60, and 75 kg·hm−2) and four levels of P (0, 60, 75, and 90 kg·hm−2), to investigate the effect of N and P fertilizers on yield, yield components, and photosynthesis characteristics of E. sibiricus. The results showed that the forage yield in 2017 was higher than in 2018. The forage yield in 2017 was highest at N75P0 with a value of 29,926 kg·hm−2, and in 2018 it was highest at N45P0 and N75P0 with a value of 12,266 kg·hm−2 and 12,233 kg·hm−2, respectively, which demonstrates the large impact of year effects on the forage yield. All traits increased with the increase in N and P fertilizer application, but with excess fertilizer application, the photosynthesis was limited, leading to a slowdown in growth and a decrease in yield. In addition, under adequate N fertilization, the role of P fertilization was not significant (p > 0.05). N, P, and N × P can significantly (p < 0.05) affect the yield traits and forage yield of E. sibiricus. According to the PCA, it is clear that N fertilizer has the largest effect, and the growth capacity of degraded E. sibiricus grassland can be restored by adding 75 kg·hm−2 of nitrogen fertilizer.

1. Introduction

Siberian wildrye (Elymus sibiricus L.) is the model species of the genus Elymus. It is a magnificent perennial species of the tribe Triticeae, belonging to the Poaceae [1,2]. Owing to its good cold tolerance, drought tolerance, and adaptability, E. sibiricus can grow well in alpine areas in poor, weakly acidic, and alkaline soils, as well as in soils with a high humus content. It has become a dominant and established species in alpine grasslands on the Tibetan plateau [3]. Furthermore, it is an excellent forage grass for livestock owing to its high grass yield, rich crude protein content, nutrition, and good palatability [4]. E. sibiricus resources are abundant and widely dispersed in the high-altitude areas of western and northern China [5]. In recent years, owing to the increase in ecological protection and the development of animal husbandry on the Qinghai-Tibet plateau, E. sibiricus has been widely used in degraded natural grassland recovery, in grass planting to protect slopes, in planting to restore vegetation, and in planting of grasses for forage to raise livestock. Thus, E. sibiricus is an important species for the advancement of animal husbandry and the preservation of the natural environment in the region [6]. However, a trait that has been the greatest disadvantage of E. sibiricus is the rapid aging of plants and a lack of persistence. It has been demonstrated that the aboveground biomass and seed yield of E. sibiricus declined rapidly after the third or fourth year of growth, accelerating the senescence process, which was mainly affected by changes in photosynthesis, antioxidant enzyme activities, and endogenous hormones [7]. Senescence is a crucial and active process of plant growth and development, morphological construction, and response to the environment; it involves the gradual decay of tissues and organs and is directly or indirectly affected by internal and external factors [8]. Based on the classification of plant senescence types, woody perennials undergo abscission senescence while perennial herbs undergo aboveground senescence [9]. Most perennial plants, such as switchgrass and giant reed, tend to decline in nutritional value after starting their reproductive growth [10]. Above-ground organs gradually age and part of the nutrients from these organs are allocated to below-ground organs as nutrient reserves for plant regeneration in the following year [11]. In this process, perennial plants are continuously degraded by nutrient deficiencies, leading to a reduction in photosynthetic phosphorylation capacity and, consequently, a decrease in biomass [12]. Furthermore, as stands of perennial plants age in years, soil nutrients are consumed, which exacerbates soil nutrient scarcity [13]. Since the addition of N fertilizers to the soil will decrease nitrogen (N) export and slow down plant senescence [14], further studies are required to understand how the life span of E. sibiricus can be extended by nutrient addition.
The nutrient balance of the soil is essential for promoting plant growth and extending the utilization life of forages. The addition of nutrients, especially N and phosphorus (P), can alleviate the nutrient deficit to delay senescence and increase the time and efficiency of the effective photosynthetic action of leaves [15,16,17]. According to previous studies, N fertilizer treatment promotes the nutritional and reproductive growth of crops; adequate N fertilizer application can increase the leaf chlorophyll concentration, net photosynthetic rate, stomatal conductance, transpiration rate, and stomatal limitation value [18,19], while a lack of N fertilizer delays nutritional and reproductive growth and decreases plant yield and yield characteristics [15,20,21]. P is crucial for energy metabolism and is part of the structural elements of plants [22,23]; its effective supply is important for plant root development [24], seedling growth, flower bud differentiation, and assimilated distribution [25,26]. Therefore, the application of phosphorus fertilizer can also effectively promote the growth and development of plants. As the absorption and utilization of N and P by plants are interdependent, N and P fertilizers are frequently applied to cultivated grasses to augment the effective N and P in the soil [27]. N application has been found to increase plant phosphatase activity, thereby decreasing the effect of P limitation on plants [28]. Thus, fertilizer application facilitates above-ground and below-ground plant growth and balances soil nutrients and nutrient transfer. It improves plant yield, increases the photosynthetic characteristics, and extends the stand utilization life.
Therefore, given that nitrogen and phosphorus affect plant growth and physiology, determining the ideal concentrations of nitrogen and phosphorus can improve plant quality and yield for agronomic purposes. It is worth noting, however, that balanced fertilization can have the effect of maintaining yields, but over-application of fertilizers harms the natural environment, and the loss of excess phosphorus fertilizers to the aquatic environment resulting in eutrophication of aquatic resources is undesirable from both an economic and an environmental point of view [29]. Therefore, it is important to find a balance between fertilizer applications that have a beneficial effect on crops and do not harm the environment. Previous research on E. sibiricus has focused on improving seed yield and biomass in a given growing year through measures such as fertilization and row spacing [30,31,32], but little consideration has been given to the senescence and stability of E. sibiricus, and there has been insufficient research on how to improve the productivity of E. sibiricus during aging. We hypothesized that N and P fertilizers have important regulatory roles in promoting yield and photosynthesis, as well as in extending the service life of E. sibiricus. Therefore, in this study, we chose E. sibiricus in its 4th and 5th years of growth to elucidate the roles of N and P and any N × P interaction effects through the response of photosynthesis, yield traits, and forage yield to fertilizers, to reveal the roles of fertilizers in improving yield and extending service life, to explore factors affecting the growth of E. sibiricus, and to determine the ideal N and P fertilizer addition rates.

2. Materials and Methods

2.1. Study Site

The experiment was performed at the Qinghai–Tibet Plateau Perennial Forage Germplasm Resource Bank’s experimental field (100°59′ E, 36°54′ N, Xihai Town, Haibei Prefecture, Qinghai, China) during the growing seasons of 2017 and 2018. The station is located at 3159 m a.s.l, which has a plateau continental climate with a long cold period, characterized by an annual mean temperature of 0.9 °C, a maximum temperature of 30.5 °C, a minimum temperature of −33.8 °C, and ≥10 °C annual accumulated temperature of 634.5 °C. The average precipitation and temperature for the growing seasons studied and for the previous year have been presented in Table 1. Black calcium soil was in the experimental field with a pH of 8.21, 38.35 g·kg−1 of organic matter, 2.58 mg·kg−1 of alkaline N, 1.36 mg·kg−1 of fast-acting available P, and 21.69 mg·kg−1 of fast-acting available potassium.

2.2. Experimental Design and Field Management

A two-factor split-plot experiment was set up in this experiment, with N as the main plot and P as the split-plot. The following four different N fertilizer treatments were used: 0 kg·hm−2 (N0), 45 kg·hm−2 (N45), 60 kg·hm−2 (N60), and 75 kg·hm−2 (N75). Similarly, four different P fertilizer treatments were used: 0 kg·hm−2 (P0), 60 kg·hm−2 (P60), 75 kg·hm−2 (P75), and 90 kg·hm−2 (P90), giving a total of 16 treatments in 48 plots (Table 2). N60 is the medium application of nitrogen and P75 is the medium application of phosphorus, based on which other fertilization rates were set in this study, and N0 and P0 were used as controls.
The experimental material for this study was Siberian wildrye (E. sibiricus L.), which is a forage grass with high cold resistance and good quality. Seeds were sown by hand in May 2014 at a depth of 3 cm, and the plots were deeply tilled and leveled before sowing. Each plot was 15 m2 (3 m × 5 m) in size, with a row spacing of 30 cm and 10 rows per plot. The sowing rate was 22.49 kg·hm−2, and a 50 cm buffer plot was arranged between neighboring fertilizer plots; 69 kg·hm−2 total N and 36 kg·hm−2 P2O5 were used as the base fertilizer. The boundary rows were excluded from any sampling, and the experiment was performed in the 4th and 5th years of growth for E. sibiricus. During the trial period, no irrigation was applied, and grazing was prohibited; weeds were removed once after seedling emergence in the year of sowing and once each year during the re-greening period.

2.3. Measurement of Forage Yield and Yield Components

The forage yield (Yf) of E. sibiricus was measured during the flowering stage (11 August 2017 and 5 August 2018), and four replicates were taken from each plot. It was measured and converted after mowing plants at flush ground level in 1 m × 1 m sample sections. To measure natural plant height (PH), inflorescence length (Li), and stem diameter (SD) at the flowering stage, five plants from each plot were randomly selected. The PH of the plant was calculated from the base of the stem to the top of the spike. The Li was determined by taking the absolute length of the E. sibiricus inflorescence, and vernier calipers were used to measure the SD at the second stem node of the plant.

2.4. Measurement of Leaf Photosynthetic Characteristics

Four plants from each plot were chosen at random throughout the flowering stage (17 July 2017 and 5 August 2018) to determine the gas exchange and chlorophyll content. The LI-6400XT portable photosynthesis system (Li-COR Inc., Lincoln, NE, USA) was used to evaluate the net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr), and intercellular CO2 concentration (Ci) between 9:00 and 11:00 a.m. The LI-6400XT maintained the following parameters: light intensity of 1000 µmol·m−2·s−1; temperature of 25 °C; CO2 concentration of 400 µmol·mol−1; a flow rate of 0.5 L·min−1; and relative humidity of 30 ± 1.0%. In the same period, the SPAD-502 chlorophyll meter was used to determine the relative chlorophyll content (SPAD value) of flag leaves, with three replicates of each plot. The YMJ-A leaf area meter was used to measure leaf area (LA), with five replicates per plot.

2.5. Data Analyses

The ANOVA model was programmed into SPSS 21.0 software (SPSS Institute Inc., Chicago, IL, USA). The ANOVA model we used was SST = SSN + SSR + SSN×R + SSP + SSN×P + SSe2, which was the main model in the analysis of variance for the split-plot experiment. In this analysis, N and P were treated as fixed factors, R as a random factor with three blocks, and each measured indicator as a dependent variable. RStudio 3.6.1 was used for the correlation analysis among Yf, yield components, and photosynthetic characteristics. All histograms were plotted using Origin 2018. To evaluate the impact of N, P, N × P, and the year on various traits, a linear regression analysis was conducted. To detect data patterns in leaf photosynthetic characteristics, forage yield, and yield components, we performed principal component analysis (PCA) using Minitab 21 software to identify the major data axes of variation for each trait. PC1, PC2, and PC3 scores were then analyzed by two-way ANOVA to detect N, P, N × P, and year effects captured in the PCA [33].

3. Results

3.1. SPAD

In the case of single N fertilization or single P fertilization or co-applied fertilization, the additions significantly (p < 0.05) affected SPAD values in 2017 and 2018, respectively. In 2017, the SPAD values were highest under the combination of 60 kg·hm−2 N and 60 kg·hm−2 P (N60P60), showing a 31.3% increase compared to the no-treatment control. In 2018, the SPAD values were highest under the combination of 45 kg·hm−2 N and 60 kg·hm−2 P (N45P60), showing a 9.4% increase compared to the no-treatment control (Figure 1).

3.2. Leaf Gas Exchange

In the case of single N fertilization or single P fertilization, fertilizer additions significantly (p < 0.001) affected Pn, Gs, Ci, and Tr. At the same time, the interaction between N and P also significantly (p < 0.001) affected Pn, Gs, Ci, and Tr (Figure 2).
The Pn increased from 2.61 to 14.61 µmol·m−2·s−1 (the mean of two years) as the N fertilizer rate increased from 0 to 60 kg·hm−2. Subsequently, it decreased to 12.95 μmol·m−2·s−1 (the mean of two years) when the N rate reached 75 kg·hm−2. The trend was consistent across different levels of P fertilizer. The results also demonstrated that the Pn was significantly increased by the higher application of N and P compared to the control. The combination of 75 kg·hm−2 N and 60 kg·hm−2 P (N75P60) showed the highest Pn during these two years (Figure 2a).
With increasing levels of both N and P fertilizers, the Gs showed an initial increase followed by a decrease. The results also demonstrated that the Gs was significantly higher with increased application of N and P compared to the control. The combination of 60 kg·hm−2 N and 60 kg·hm−2 P (N60P60) showed the highest Gs during these two years (Figure 2b).
In 2017, the Ci fluctuated with increasing rates of N fertilizer. In 2018, the Ci initially increased and then decreased. When the P fertilizer rate increased from 0 to 75 kg·hm−2, the Ci increased from 242.06 to 288.45 µmol·mol−1 (the mean of two years) and subsequently decreased to 268.86 µmol·mol−1 (the mean of two years) as the rate was further increased to 90 kg·hm−2. The combination of 0 kg·hm−2 N and 60 kg·hm−2 P (N0P60) showed the highest Ci during these two years (Figure 2c).
When the N fertilizer rate was increased to 60 kg·hm−2, Tr reached its maximum, and it initially increased and then decreased with an increasing P fertilizer rate. The combination of 60 kg·hm−2 N and 75 kg·hm−2 P (N60P75) showed the highest Tr during these two years (Figure 2d).

3.3. Yf and Yield Components

In the case of single N fertilization, the fertilizer significantly (p < 0.001) affected PH, SD, LA, Li, and Yf during the two years, with the exception of Li in 2017. In the case of single P fertilization, the fertilizer significantly (p < 0.05) affected PH, SD, LA, Li, and Yf during the two years. When N and P fertilizers were co-applied, their combined application significantly (p < 0.05) affected these traits (Table 3).
The Yf fluctuated with the increase in the N fertilizer rate. When the N fertilizer rate increased to 75 kg·hm−2, the Yf reached its maximum. With the increase in the P fertilizer rate, Yf increased from 5174 to 7329 kg·hm−2 (the mean of two years) and then decreased to 3696 kg·hm−2 (the mean of two years), with a further increase in the P rate up to 90 kg·hm−2. The Yf in 2017 was higher than in 2018, and the combination of 75 kg·hm−2 N and 0 kg·hm−2 P (N75P0) showed the highest Yf during these two years (Table 3).
PH was highest at an N fertilizer rate of 75 kg·hm−2, reaching a value of 101.92 cm (the mean of two years). The PH initially increased with the P fertilizer rate and then decreased. The combination of 75 kg·hm−2 N and 0 kg·hm−2 P (N75P0) showed the highest PH during these two years (Table 3).
The SD fluctuated with increasing N fertilizer rates. When the N fertilizer rate increased to 45 kg·hm−2, SD was at its maximum, and it initially increased and then decreased with an increasing P fertilizer rate. The combination of 45 kg·hm−2 N and 90 kg·hm−2 P (N45P90) showed the highest SD during these two years (Table 3).
LA showed a continuous increase with increasing N fertilizer rate, with a maximum of 222.54 mm2 at 75 kg·hm−2. The LA increased as the P fertilizer rate increased and then subsequently decreased when the P rate further increased. The combination of 45 kg·hm−2 N and 90 kg·hm−2 P (N45P90) showed the highest SD during these two years (Table 3).
The Li fluctuated with the increase in N fertilizer rate, reaching a maximum of 75 kg·hm−2. The Li increased from 8.52 cm to 9.36 cm (the mean of two years) as the P fertilizer rate rose from 0 to 60 kg·hm−2, and then it decreased when the P reached 90 kg·hm−2. The combination of 60 kg·hm−2 N and 90 kg·hm−2 P (N60P90) showed the highest Li during these two years (Table 3).

3.4. Correlation Analysis between Yf, Photosynthetic Characteristics, and Yield Components

According to the correlation analysis, Yf was positively and significantly correlated with PH (r = 0.83), LA (r = 0.68), Pn (r = 0.88), and Gs (r = 0.56) (p < 0.01 for all). Yf showed significant relationships with Li (r = 0.27), Ci (−0.36), Tr (0.36), and SPAD (r = 0.32) (p < 0.05 for all). However, SD was not significantly correlated with Yf (p > 0.05). Furthermore, the PH was positively correlated with LA, Pn, Gs, and SPAD (p < 0.01). Significant correlations were also noted for LA with SD, Pn, Gs, and Tr (p < 0.01), for Pn with Gs and SPAD (p < 0.01), and for Gs with Tr and SPAD (p < 0.01) (Figure 3).

3.5. Patterns of Association between Fertilizers and Traits

The linear regression analyses of N, P, N × P, and year effects for each trait showed that PH, SD, LA, Pn, Gs, and Yf increased significantly with increasing N fertilizer application. PH, SD, LA, and Pn increased significantly as the P fertilizer application increased. However, PH, LA, and Pn were significantly reduced by the interaction of N and P fertilizers. PH, SD, LA, Li, SPAD, and Yf decreased significantly with increasing years, while the changes in photosynthetic characteristics were not significant (Table 4). Subsequent principal component analysis of these 10 traits revealed that the proportions of variance explained by the first three principal components were 43.8%, 20.9%, and 10.2%, respectively. These three principal components explained most of the data variation, and hence the remaining principal components were discarded. The results showed that PC1 concentrated on the traits of PH, LA, photosynthetic characteristics, and forage yield, with half of the indexes having PC values above 0.7. The primary contribution to PC2 comes from Li, Gs, Ci, and Tr, with the PC2 value of Li exceeding 0.7, indicating the significance of Li in PC2, while the PC3 had a primary contribution from SD with a PC value of 0.6 (Table 4).
A two-way ANOVA on PC1, PC2, and PC3 scores showed that the year effect was significantly captured by PC1 and PC2. The N effect was significantly captured by PC1 and PC3, with the highest principal component mean at N75, followed by N60. The P effect was mainly captured by PC1, with scores ranked P60 > P75 > P0 > P90. The interaction of N and P fertilizers was significantly captured by PC1 and PC2, with the highest principal component mean at N75P60, N60P75, and N60P60 ratios (Table 5).

4. Discussion

4.1. Nutrient Addition and Plant Senescence

Plant senescence is a highly regulated physiological process that contributes to their adaptation to the growing environment [34]. During growth, nutrient translocation occurs in perennial plants, which results in nutrient loss and continuous plant degradation [35]. The nutrient dysregulation hypothesis suggests that plant aging occurs because of the increased demand for nutrients by the reproductive organs of individual plants; however, the main nutrient-producing organs exhibit insufficient supply capacity and metabolic dysfunction, which leads to a decline in SPAD and photosynthesis [36]. And, it has been shown that plant senescence is closely related to the decline in photosynthesis, for example, in cereals, the intensity of photosynthesis declines from the earing stage [37]. Furthermore, oxidative stress occurs when plant metabolism changes during aging, characterized by the increased production of reactive oxygen species or the reduced activity of oxidative scavenging systems, leading to cellular senescence [38]. This study confirms these conclusions because it revealed that photosynthesis, SPAD value, Yf, and yield components in the 5th year of growth were significantly lower than those in the 4th year. Notably, we observed that these traits were lower in the control group compared with the fertilizer treatment group. Meanwhile, the linear regression analyses indicated that PH, SD, LA, Li, SPAD, and Yf decreased significantly with increasing years. These results corroborate the occurrence of senescence in E. sibiricus during growth, while also demonstrating that the application of fertilizer can promote plant growth and delay the aging process. Therefore, understanding how nitrogen is allocated to seeds and underground organs, two major carbon sinks, during senescence in perennial plants is crucial for revealing the mechanism of senescence.

4.2. Nitrogen and Phosphorus Fertilizer with Plant Growth Physiology

N is the main component of chlorophyll, which increases the chlorophyll content, rubisco enzyme activity, and intercellular CO2 concentration [39]. Simultaneously, N mitigates the generation of intracellular reactive oxygen species, augments the capability to scavenge these reactive oxygen species, and thus alleviates the decline of photosynthetic capacity [40]. The application of exogenous N contributes to a decrease in N output from senescing organs, thereby alleviating the effect of senescence [41]. Furthermore, N fertilization has a profound impact on the development of the underground root system, such as the root nodule number, root biomass, and root nodule dry weight [42]. This may be because N fertilizer addition balances soil nutrients and promotes balanced growth above and below the ground [43]. In the present study, reverse senescence was found in E. sibiricus, attributed to the application of fertilizer. That is, both SPAD and photosynthetic characteristics tended to increase and subsequently decline with an increase in N fertilizer application. However, these changes were not significantly reflected in growth indexes, which was consistent with the nutrient transfer law. The law states that the addition of exogenous N can significantly alleviate senescence. However, excessive N fertilizer application not only detriments the synthesis of photosynthetic pigments in leaves but also leads to an increase in leaf area, consequently increasing the shading area and diminishing the efficiency of light energy utilization in leaves [44]. This observation is consistent with the study’s conclusion that leaf area continued to increase with increasing N fertilizer application. In the investigation of leaf gas exchange parameters, Gs and Tr were observed to decrease significantly under conditions of high N and high P treatments in 2017. Previous studies have indicated that Ci is affected by factors such as light and stomatal conductance [45,46]. As a result, we hypothesized that the high N and high P proportions decreased leaf Gs and Tr, potentially leading to an accumulation of Ci. Excessive Ci cannot be consumed, thus acting as a limitation on photosynthesis. Combined with the study of forage yield, we found that its forage yield was significantly decreased under N75P75 and N75P90 treatments in 2017. This indicates that the decreased forage yield under these two treatments was attributed to photosynthesis limitations. Consequently, the combination of high N and P appears to be detrimental to the growth of E. sibiricus. A previous study [47] showed that P fertilization treatment improved nutrient availability, promoted nutritional growth, and increased plant biomass and seed production. Furthermore, P fertilization has been shown to increase plant biomass, plant height, seed yield, and protein content [48]. Conversely, P deficiency in plants affected xylem development and thus reduced plant water uptake, thereby decreasing Gs and Tr [49]. The application of sufficient N and P fertilizers has been reported to increase the SPAD and Gs of plants, thereby enhancing their photosynthesis capacity [50]. The present study revealed a tendency for photosynthesis, Yf, and yield components to increase and then decrease as P fertilizer usage increased. However, at a higher level of N fertilizer application, P fertilization slightly improved the Yf and yield components. This indicates that applying various amounts of N fertilizers may change how plants react to P fertilizers [51]. Therefore, determining a reasonable amount of N and P fertilizer application can effectively improve plant yield.
The analysis of how N and P application affected the yield components of E. sibiricus revealed that applying nutrients increased yield and yield components when compared to the absence of nutrient application. During senescence, older E. sibiricus may sacrifice more nutrient organs and transfer nutrients to reproductive organs, resulting in altered E. sibiricus biomass allocation and yield traits. Nutrient addition can balance soil nutrients, provide sufficient nutrients to plants, promote plant growth, and slow down the senescence of perennial plants [52].

4.3. PCA Analysis of Fertilizer Effects on Physiological Characteristics of Plant Growth

The PCA analysis indicated that the first three principal components explained most of the data variation. Notably, five traits (PH, LA, Pn, Gs, and Yf) demonstrated a correlation with PC1 scores exceeding 0.70. These traits, which represent half of all traits examined and primarily reflect plant size and photosynthetic capacity, suggest that these traits made a major contribution to the data patterns captured in PC1. Furthermore, this implies that PC1 played a major role among the three principal components. PC1 scores primarily identified differences in plant size and captured the effects of N on plant size and differences in plant size between years. Under a reasonable application of N fertilizer, the higher the application of N fertilizer, the higher the plant yield and the photosynthesis efficiency [53]. The younger plants grew better than the older plants and showed higher yields [54]. The PC1 scores in this study confirmed the above findings, with N75 having the highest PC1 value under the N effect. Additionally, P fertilizer also showed significant differences with PC values, with P60 having the highest PC1 value, but its effect was less pronounced when compared to the N effect and year effect. Therefore, it can be inferred that the yield, growth, and leaf characteristics of E. sibiricus were mainly regulated by N fertilizer and year under a single effect. This has been similarly studied in other plants, all of which showed that nitrogen application increased plant yield, reflecting the importance of nitrogen fertilization in plant growth [55,56].
Only Li exhibited scores higher than 0.7 in PC2, indicating that PC2 mainly captured the spike characteristics of E. sibiricus. Additionally, Gs, Ci, and Tr all scored above 0.5 in PC2, indicating that PC2 captured photosynthesis characteristics as well. According to the PC value analysis, PC2 was significantly correlated with the year effect (p < 0.01), significantly correlated with the N effect (p < 0.05), and not correlated with the P effect. Therefore, PC2 can be regarded as the response of Li and the photosynthesis characteristics of E. sibiricus to the year effect. Regression analysis further demonstrated a significant negative correlation between Li and the year (p < 0.05). Perennial plants exhibit senescence as they age, characterized by a reduction in photosynthesis and diminishing spike traits [54]. PC2 affects photosynthesis and Li through the year effect, which partly explains the senescence phenomenon of E. sibiricus, and these traits are crucial in contributing to the decline in the forage yield of E. sibiricus.
The highest score in PC3 was for SD, indicating that PC3 mainly captured the stem characteristics of E. sibiricus. Analysis of PC values showed that PC3 was significantly correlated with the N effect (p < 0.01), suggesting that PC3 can be regarded as the response of SD to the N effect. Regression analysis showed that SD increased with increasing N fertilizer application, and PC values confirmed that SD achieved its peak at the N75 level due to the N effect. Of course, the study has also demonstrated that N fertilizer application affects the SD of plants [53]. Beyond providing structural support, SD plays a crucial role in increasing plant yield by serving as a storage site for soluble solids, which are later utilized in grain formation [53].
In addition to the single effects of N, P, and year described above, we also conducted a study of the interaction effects of N and P fertilizers. The analysis of the PC scores showed that PC1 and PC2 were significantly correlated with N × P (p < 0.05). Although the difference between the N × P effect and the PC value was smaller than that of the other single effects, they had higher PCs than the single effect scores. The highest PC values were obtained for N75P60 and N60P60 under PC1, N60P75 and N60P60 under PC2, surpassing all PC values associated with N, P, and year effects. This finding shows that N fertilizer enhances the growth of E. sibiricus more than P fertilizer. The combination of N and P fertilizers played a major role in the growth physiology of E. sibiricus, confirming the conclusion that the uptake of nitrogen and phosphorus fertilizers by plants is interdependent and that the N fertilizer may have facilitated the response of the plant to the P fertilizer [57].
In summary, the principal component analysis categorized all traits into PC1 subject to the N effect and year effect, PC2 subject to the year effect, and PC3 subject to the N effect. Although the PC values of N × P effects were higher than those of single effects, N fertilization was still the main effect to improve the production performance of E. sibiricus. Therefore, N75 can be an ideal fertilization measure for the long-term utilization of E. sibiricus in alpine meadows.

4.4. Yield-Related traits with Forage Yield

In this study, correlation analysis showed that PH, LA, Gs, and Pn were significantly correlated with Yf (p < 0.01), indicating that these traits play a major role in the formation of E. sibiricus forage yield. According to previous studies, some agronomic traits play a direct role in yield formation, while others indirectly affect forage yield, such as alfalfa yield, which is directly or indirectly affected by plant height, stem traits, and leaf size [58]. Similarly, the yield of E. sibiricus is influenced by spikelet number, seed number, etc., and there is also a connection between various yield traits [59]. That is, the formation of E. sibiricus forage yield is not the response of a single factor, but a combination of multiple factors and complex regulatory processes. Therefore, PH and Pn can be used as key traits for future breeding of E. sibiricus, while also taking other traits into account to achieve a yield increase.

5. Conclusions

Application of N and P fertilizers to E. sibiricus in the 4th and 5th years clarified that both N, P, and N × P significantly affected photosynthetic characteristics, growth indexes, and forage yield. Excessive application of fertilizers led to a decrease in photosynthetic performance, which in turn affected growth and reduced yield. And each trait had an effect on yield, with Pn and PH being identified as the main influencing factors. Regression and PCA analyses showed that N and year were the main effects on production performance in degraded E. sibiricus grassland, while P fertilizer and N × P played a secondary role. Therefore, for degraded E. sibiricus grassland, the addition of N fertilizer can solve most of the problems. Based on the results, N75 can be used as the optimal fertilization rate to improve the longevity of E. sibiricus. In the future, subsequent measurements of nitrogen and phosphorus content in soil and plants should be conducted to better understand the mechanisms of plant–soil interactions. And the mechanisms of senescence in E. sibiricus should be explored at the physiological and molecular levels to explain the mechanism of senescence.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy14071543/s1, Table S1. Linear regression equation of all traits.

Author Contributions

Conceptualization, R.W. and W.L. (Wenhui Liu); methodology, Y.Z. and G.L.; formal analysis, K.L.; investigation, W.L. (Wen Li); data curation, R.W.; writing—original draft preparation, R.W.; writing—review and editing, W.L. (Wen Li), G.L. and Y.Z.; supervision, K.L.; project administration, W.L. (Wenhui Liu); funding acquisition, W.L. (Wenhui Liu). All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Qinghai innovation platform construction project (2024), the earmarked fund for National Natural Science Foundation of China (U20A2050), Open Competition Project to Select the Best Candidates of the National Forestry and Grassland Administration “Breeding of Excellent Grass Varieties” (202201), China Agriculture Research System (CARS-34).

Data Availability Statement

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare they have no conflicts of interest.

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Agronomy 14 01543 g001
Figure 1. SPAD value in leaves at different nitrogen (N) and phosphorus (P) application. Lowercase letters show significant differences between various P fertilizers at the same N level (p < 0.05).
Figure 1. SPAD value in leaves at different nitrogen (N) and phosphorus (P) application. Lowercase letters show significant differences between various P fertilizers at the same N level (p < 0.05).
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Figure 2. Photosynthetic characteristics of Elymus sibiricus at different nitrogen (N) and phosphorus (P) application. Lowercase letters show significant differences between various P and N fertilizer treatments at the same level (p < 0.05).
Figure 2. Photosynthetic characteristics of Elymus sibiricus at different nitrogen (N) and phosphorus (P) application. Lowercase letters show significant differences between various P and N fertilizer treatments at the same level (p < 0.05).
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Figure 3. Correlation analysis among forage yield (Yf), plant height (PH), stem diameter (SD), leaf area (LA), inflorescence length (Li), net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr), intercellular CO2 concentration (Ci), and chlorophyll content (SPAD) in different fertilizer treatment groups. The shape of the circles in the upper triangle area indicates positive and negative correlations; the flatter the circle, the stronger the correlation. The lower triangle area shows the correlation coefficient; the darker the color, the stronger the correlation.
Figure 3. Correlation analysis among forage yield (Yf), plant height (PH), stem diameter (SD), leaf area (LA), inflorescence length (Li), net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr), intercellular CO2 concentration (Ci), and chlorophyll content (SPAD) in different fertilizer treatment groups. The shape of the circles in the upper triangle area indicates positive and negative correlations; the flatter the circle, the stronger the correlation. The lower triangle area shows the correlation coefficient; the darker the color, the stronger the correlation.
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Table 1. Average precipitation and temperature for growing seasons from 2016 to 2018 of the experimental site.
Table 1. Average precipitation and temperature for growing seasons from 2016 to 2018 of the experimental site.
MonthPrecipitation (mm)Temperature (℃)
201620172018201620172018
April 24.525.518.54.23.13.8
May45.160.321.67.56.88.5
June94.750.951.911.710.411.9
July79.9112.5136.514.214.814.1
August57.6151.3112.215.612.213.7
Table 2. The fertilizer treatments of E. sibiricus.
Table 2. The fertilizer treatments of E. sibiricus.
TreatmentsNitrogen Fertilizer (kg·hm−2)Phosphorus Fertilizer (kg·hm−2)
N0P000
N45P0450
N60P0600
N75P0750
N0P60060
N0P75075
N0P90090
N45P604560
N45P754575
N45P904590
N60P606060
N60P756075
N60P906090
N75P607560
N75P757575
N75P907590
Table 3. Variance analysis and multiple comparisons for the forage yield and yield components of E. sibiricus under N and P application.
Table 3. Variance analysis and multiple comparisons for the forage yield and yield components of E. sibiricus under N and P application.
YearsNitrogen (N)
(kg·hm−2)		Phosphorus (P)
(kg·hm−2)		PH
(cm)		SD
(mm)		LA
(mm2)		Li
(cm)		Yf
(kg·hm−2)
20170082.60 ± 0.56 c2.04 ± 0.02 d161.57 ± 0.95 c8.48 ± 0.35 b6180 ± 100.00 c
6084.89 ± 0.56 b2.56 ± 0.01 c178.60 ± 5.20 b9.47 ± 0.56 a7195 ± 305.00 b
7592.43 ± 1.27 a2.90 ± 0.03 a190.57 ± 3.74 a9.94 ± 0.17 a9090 ± 572.08 a
9082.57 ± 0.55 c2.65 ± 0.05 b164.00 ± 2.17 c9.99 ± 0.49 a3175 ± 55.00 d
45093.67 ± 1.37 a3.10 ± 0.05 a180.43 ± 4.51 b10.26 ± 0.25 a23219 ± 417.50 a
6093.14 ± 0.57 a2.82 ± 0.04 b224.77 ± 4.24 a9.19 ± 0.50 b16198 ± 271.67 c
7591.13 ± 1.00 b2.96 ± 0.08 ab220.93 ± 1.50 a9.56 ± 0.56 ab15917 ± 692.50 c
9092.60 ± 0.50 ab3.01 ± 0.13 a225.70 ± 1.10 a9.40 ± 0.34 b18452 ± 524.17 b
60094.80 ± 0.46 a2.76 ± 0.05 a236.70 ± 4.97 a9.73 ± 0.13 ns21465 ± 458.33 b
6096.23 ± 0.57 a2.90 ± 0.20 a226.47 ± 2.81 b9.72 ± 0.49 ns24455 ± 111.67 a
7594.73 ± 0.84 a2.75 ± 0.07 a231.96 ± 4.16 ab9.47 ± 0.55 ns22085 ± 412.50 b
9087.57 ± 1.42 b2.38 ± 0.02 b208.53 ± 0.76 c10.13 ± 0.29 ns16103 ± 206.67 c
750102.83 ± 1.48 a2.56 ± 0.02 ns242.62 ± 1.02 a9.88 ± 0.47 b29926 ± 783.33 a
6099.37 ± 0.95 b2.64 ± 0.04 ns237.40 ± 1.65 b9.66 ± 0.37 b28285 ± 658.33 b
7596.83 ± 1.31 c2.62 ± 0.14 ns187.07 ± 3.76 d11.03 ± 0.88 a23873 ± 440.00 c
9093.63 ± 0.59 d2.65 ± 0.07 ns224.57 ± 1.98 c10.09 ± 0.34 ab22674 ± 365.83 d
SEM 0.810.043.890.091136.40
p-valueN<0.001<0.001<0.001ns<0.001
P<0.001<0.001<0.001<0.05<0.001
N × P<0.001<0.001<0.001<0.05<0.001
20180079.47 ± 0.67 b2.16 ± 0.08 c154.24 ± 10.54 b8.55 ± 0.55 a4166 ± 66.67 b
6085.60 ± 2.17 ab2.53 ± 0.06 a151.36 ± 3.83 b9.25 ± 0.55 a5233 ± 333.33 a
7590.40 ± 1.82 a2.55 ± 0.02 a198.63 ± 21.11 a8.89 ± 0.28 a5566 ± 333.33 a
9085.37 ± 5.57 ab2.36 ± 0.02 b174.99 ± 12.86 ab7.65 ± 0.30 b4216 ± 83.33 b
45092.85 ± 0.85 ns2.82 ± 0.03 b183.31 ± 4.44 b9.83 ± 0.55 ns12266 ± 233.33 a
6090.37 ± 2.72 ns2.57 ± 0.02 c209.08 ± 13.57 a8.83 ± 0.81 ns5916 ± 150.00 c
7591.23 ± 5.53 ns2.88 ± 0.02 a208.93 ± 0.40 a9.19 ± 0.05 ns7400 ± 466.67 b
9089.93 ± 0.55 ns2.90 ± 0.02 a205.33 ± 0.64 a8.90 ± 0.30 ns7666 ± 266.67 b
60092.67 ± 1.15 a2.54 ± 0.04 a190.36 ± 2.57 a8.88 ± 0.10 b10916 ± 83.33 c
6091.57 ± 1.40 ab2.54 ± 0.05 a190.17 ± 1.27 a7.17 ± 0.29 c12616 ± 16.67 a
7589.73 ± 1.21 b2.37 ± 0.02 b181.54 ± 1.29 b7.17 ± 0.29 c11766 ± 166.67 b
9084.33 ± 1.72 c2.36 ± 0.02 b177.83 ± 0.81 c10.53 ± 0.40 a10150 ± 650.00 d
750101.00 ± 1.18 a2.37 ± 0.08 b202.45 ± 0.48 a8.89 ± 0.25 c12233 ± 800.00 ns
6098.73 ± 0.47 b2.37 ± 0.02 b203.33 ± 0.55 a9.27 ± 0.15 b11300 ± 600.00 ns
7593.33 ± 0.55 c2.52 ± 0.02 a198.23 ± 0.85 b10.20 ± 0.17 a11150 ± 583.33 ns
9087.77 ± 0.93 d2.48 ± 0.01 a186.02 ± 0.47 c10.39 ± 0.20 a11683 ± 516.67 ns
SEM 0.790.032.660.15449.29
p-valueN<0.001<0.001<0.05<0.001<0.001
P<0.001<0.001<0.001<0.05<0.001
N × P<0.001<0.001<0.001<0.001<0.001
Natural plant height (PH), stem diameter (SD), leaf area (LA), inflorescence length (Li), and forage yield (Yf) in different N and P fertilizer treatments in the two years. The average of replications is used to express the data. Lowercase letters show significant differences between various P and N fertilizer treatments at the same level (p < 0.05). ns represents not significant (p > 0.05).
Table 4. Regression analysis and principal component analysis of raw data for all traits with fertilizers and years.
Table 4. Regression analysis and principal component analysis of raw data for all traits with fertilizers and years.
TraitsRaw Data Significance of Fertilizers and All TraitsPC Score Correlations with Raw Data
NPN × PYearPC1PC2PC3
PH(+) <0.001(+) 0.021(−) <0.001(−) 0.0470.866 0.067 −0.273
SD(+) 0.046(+) 0.039ns(−) 0.0270.535 0.347 0.635
LA(+) <0.001(+) 0.042(−) 0.042(−) 0.0020.871 0.015 0.042
Linsnsns(−) 0.0110.268 0.756 −0.144
Pn(+) 0.001(+) 0.020(−) 0.026ns0.782 −0.041 −0.451
Gs(+) 0.025nsnsns0.723 −0.580 0.069
Cinsnsnsns−0.233 0.618 0.160
Trnsnsnsns0.572 −0.620 0.402
SPADnsnsns(−) 0.0140.571 0.452 0.297
Yf(+) <0.001nsns(−) <0.0010.821 0.284 −0.196
Proportion of data variance explained (%)43.820.910.2
Forage yield (Yf), plant height (PH), stem diameter (SD), leaf area (LA), inflorescence length (Li), net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr), intercellular CO2 concentration (Ci), and chlorophyll content (SPAD). The regression model can be represented as Y = a + b N + c P + d N × P + e Year + ei, where Y is trait data; a, b, c, d, and e are constants determined by the regression analysis and ei is the statistical residual of each data observation, specific linear regression equations can be found in the Table S1. ns represents not significant.
Table 5. PC1, PC2, and PC3 scores from PCA analysis of Elymus sibiricus forage yield, photosynthetic characteristics, and yield components.
Table 5. PC1, PC2, and PC3 scores from PCA analysis of Elymus sibiricus forage yield, photosynthetic characteristics, and yield components.
PC1PC2PC3
Year170.3408−0.448−0.138
18−0.34080.4480.138
F value24.56 **15.47 **3.64 ns
N0−1.272−0.187−0.233
450.264−0.029−1.054
600.4160.660.001
750.593−0.4441.286
F value38.98 **4.29 *44.91 **
P0−0.1260.0480.399
600.3550.397−0.149
750.196−0.172−0.329
90−0.425−0.2730.079
F value6.37 **1.69ns4.72 *
N × PN0P0 −2.0730.5820.538
N0P60−1.329−0.614−0.234
N0P75−0.369−0.578−0.749
N0P90−1.317−0.138−0.488
N45P00.156−0.829−0.719
N45P600.5790.635−1.429
N45P750.242−0.119−1.238
N45P900.0780.196−0.832
N60P00.4320.0440.216
N60P601.0391.097−0.462
N60P750.6091.365−0.503
N60P90−0.4180.1360.754
N75P00.981 0.3941.562
N75P601.1310.4711.53
N75P750.303−1.3561.172
N75P90−0.045−1.2860.881
F value3.25 *2.90 *1.95 ns
The * represents significant at p < 0.05; ** represents significant at p < 0.01.
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Wu, R.; Liu, W.; Zhang, Y.; Liang, G.; Li, W.; Liu, K. Response of Elymus sibiricus (Siberian Wildryegrass) to Combined Application of Nitrogen and Phosphorus during Aging on the Qinghai–Tibetan Plateau. Agronomy 2024, 14, 1543. https://doi.org/10.3390/agronomy14071543

AMA Style

Wu R, Liu W, Zhang Y, Liang G, Li W, Liu K. Response of Elymus sibiricus (Siberian Wildryegrass) to Combined Application of Nitrogen and Phosphorus during Aging on the Qinghai–Tibetan Plateau. Agronomy. 2024; 14(7):1543. https://doi.org/10.3390/agronomy14071543

Chicago/Turabian Style

Wu, Rui, Wenhui Liu, Yongchao Zhang, Guoling Liang, Wen Li, and Kaiqiang Liu. 2024. "Response of Elymus sibiricus (Siberian Wildryegrass) to Combined Application of Nitrogen and Phosphorus during Aging on the Qinghai–Tibetan Plateau" Agronomy 14, no. 7: 1543. https://doi.org/10.3390/agronomy14071543

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