Effect of Total Nitrogen Once Applied at Different Leaf Age Stages on Yield Traits of Dense Planted Dry Direct Seeding Rice

1. Introduction

With the reduction of agricultural labor force, the increasing labor costs, and the expansion of large-scale crop production in China, the direct seeding rice, which eliminates the need for seedling raising and transplanting, has gained increasing favor among rice farmers [1,2]. The direct seeding rice is widely distributed across most rice-growing regions in China, with the largest planting area found in the middle and lower reaches of the Yangtze River, estimated to exceed 3 million hectares [2]. This region is situated in the transitional zone between the subtropical and northern temperate zones. The paddy fields here have a high multiple cropping index, and are dedicated to rice-wheat, rice-oilseed, and double cropping rice cropping systems [3]. Investigations into direct seeding rice under this multi-cropping system have revealed that as the yield of the preceding crop increases, the amount of straw returned to the field also rises. However, due to the low degree of straw chopping and uneven straw spraying of mechanical harvesting, it is often challenging to improve the quality of mechanical tillage and achieve thorough tillage, which affects the uniformity and consistency of direct seeding rice [4,5]. Specifically, for the dry direct seeding rice, the uniformity and depth of seeds placement tend to be inconsistent, which can easily result in adverse conditions for many seeds during the germination stage [4,6]. At the same time, the limited time of agricultural operations during crop rotation period and the unforeseen rainy weather during the rice sowing period would exacerbate the extent of extensive soil preparation and sowing, resulting in delayed and uneven emergence of direct seeding rice, which is detrimental to early seedlings growth. Consequently, the sowing rate of direct seeding rice in production is often more than 1.5 times higher than the recommended sowing rate in experimental research, to ensure that the entire field is seeded evenly with no gaps or breaks, and to minimize the labor required for replanting and leveling seedlings [4,5,7,8]. The initial seedling quantity of this type of direct seeding rice often approaches or even exceeds the targeted effective panicle number, resulting in a final effective panicle that is predominantly composed of the main stem [4,7,9]. This type of direct seeding rice differs from the previous studies on the population construction and yield formation characteristics of high-yield cultivation methods, such as reducing the number of initial seedlings, increasing the proportion of tiller earing, and promoting strong stems and large panicles. Following traditional nitrogen application rates and multiple fertilization methods can easily cause the rice population increased rapidly in the early growth period, and decreased rapidly in the later growth period due to fierce competition among individuals and more ineffective tillers [10,11,12]. And it would weaken individual growth and intensify competition for solar, temperature, and nutrients, which would be detrimental to achieving high yields.

For dense direct seeding rice, that the initial seedling quantity would approach or even exceed the targeted effective panicle number, our research team innovated a simplified fertilization method involving reduced nitrogen application, specifically by non-nitrogen availability in the early growth period to restrict the growing and tillering of direct-seeded rice seedlings, and then supplementing nitrogen was applied in the middle growth period [4,13,14,15]. This approach significantly improved the nitrogen absorption and increased the grain yield [13,16,17,18]. However, it remained unclear how total nitrogen once applied at different leaf age stages during the mid-to-late tillering period affected the dry matter accumulation traits of dense planted dry direct seeding rice, leading to higher yield [13,14,15,16,17,18]. Therefore, we systematically compared the effects of total nitrogen once applied at different leaf age stages during the mid-to-late tillering period on the dry matter accumulation traits of dense planted dry direct seeding rice. Our objective was to provide a total nitrogen once application mode for dense planted dry direct seeding rice to facilitate the development of nitrogen reduction and high yield cultivation in a rice-wheat cropping system.

2. Results

2.1. Leaf Area Index (LAI)

The LAIs at determined growth stages varied among treatments (Table 1). At tenth-leaf and jointing stages, the LAIs showed the same trend of L6 > L7 > L8 > L9 > L10. Compared to that of L8, the LAIs of L9, L10, L7 and L6 were smaller at booting and heading stages, and significant difference was observed between L8 and L6. With the delay of total nitrogen once applied time, the LAI was increased at maturity stage in the order L10 > L9 > L8 > L7 > L6.

2.2. Mean Leaf Area Index (MLAI)

The obvious differences were observed in MLAIs among treatments (Figure 1). The MLAIs showed the same trend of L8 > L7 > L6 > L9 > L10. Compared to that of L8, the MLAIs of L9 and L10 were on average 5.3% and 7.1% significantly smaller.

2.3. Effective Leaf Area Rate (ELAR) and High Effective Leaf Area Rate (HELR) at Heading Stage

The ELARs at heading stage showed the trend of L9 > L10 > L8 > L7 > L6 (Figure 2), and the HELRs at heading stage showed the trend of L8 > L9 > L7 > L10 > L6 (Figure 3). Insignificant differences were determined in these two parameters among treatments.

2.4. Leaf Area Duration (LAD)

The delay of total nitrogen once applied time induced a decrease in the LADs before jointing stage with the trend of L6 > L7 > L8 > L9 > L10, and significant difference was between L6 and L10 (Table 2). During the jointing to heading stage, the LAD of L8 was 1.1%~7.3 higher than other treatments. The LADs of L6, L7 and L8 were significantly lower than L9 and L10 during heading to maturity stage in 2019. In addition, the LADs of L8, L9 and L10 were showed insignificant difference and higher than L7 and L6 during heading to maturity stage in 2020.

2.5. Above-Ground Dry Matter Weight (ADW) at Mian Growth Stage

The ADWs showed the same order of L6 > L7 > L8 > L9 > L10 at tenth-leaf and jointing stages among different nitrogen application stages (Table 3). At heading, milky and maturity stages, the ADWs were increased by 3.8%~16.9, 6.5%~18.0% and 2.1%~11.8% in L8 relative to that in other treatments, respectively.

2.6. Above-Ground Dry Matter Weight (ADW) in Determined Growth Period

The ADW during jointing to heading stage of L8 was 7.4%~23.5% higher than other treatments in the two experimental years (Table 4). The ADWs during heading to maturity stage led the trend of L10 > L9 > L8 > L7 > L6, and that of L8 was 5.1% ~ 18.5% higher than L6 and L7, and was 5.4% ~ 12.6% lower than L9 and L10, respectively. With the delay of total nitrogen once applied time, the ratios of ADW before jointing stage were in the order of L6 > L7 > L8 > L9 > L10, and that during heading to maturity stage were in the opposite law. The L8 was of a higher ratio of ADW during jointing to heading stage to total.

2.7. Crop Growth Rate (CGR) and Net Assimilation Rate (NAR)

The delayed total nitrogen once applicated time increased the CGR and NAR before jointing stage with the order of L6 > L7 > L8 > L9 > L10, and decreased those during heading to maturity stage with the order of L10 > L9 > L8 > L7 > L6, respectively (Table 5). The CGR and NAR of L8 during jointing to heading stage were 7.4% ~ 23.5% and 2.2% ~ 21.2% bigger than other treatments, respectively.

2.8. Grain-Leaf Ratio Traits

With the delay of total nitrogen once applicated time, the ratio of total spikelets to leaf area index at booting stage (RTSL), ratio of total grains to leaf area index at booting stage (RTGL) and ratio of total grain weight to leaf area index at booting stage (RGWL) of Nanjing 9108 showed the same order of L8 > L7 > L6 > L9 > L10 in the two experimental years (Table 6). The RTSL, RTGL and RGWL of Yangnongxiang 28 had higher values in L8 than those in other treatments. Compared with L8, the values of RTSL, RTGL and RGWL of other treatments were 4.3%~18.3%, 2.7~18.7% and 0.1%~4.5% lower, respectively. Insignificant difference was determined in RGWL among total nitrogen once applied time treatments.

2.9. Grain Yield and Harvest Index

The grain yield of L8 was 4.0% ~ 9.9% higher than that of other treatments (Figure 4). The significant difference was in grain yield between L6 and L8. The harvest indexes of rice were in the order of L6 > L7 > L8 > L9 > L10 in response to the delayed nitrogen application stage, and insignificant differences were surveyed among L6, L7 and L8.

3. Discussion

3.1. The Above-Ground Dry Matter Accumulation Trait

Rice biological yield formation was fundamentally intertwined with the processes of above-ground dry matter production and accumulation across the plant population [20]. Rice yield was determined by both biological yield and harvest index. Theoretically, increasing biological yield or economic coefficient could increase the yield, but it was reported that enhancing rice yield hinged on increasing the dry matter accumulation of the rice population, as different opinions were present in the increasing of harvest index [21,22]. It was a true fact that while ensuring biological yield, increasing the harvest index could effectively increase the rice yield, however, even if the new cultivation technology or product significantly increased the rice yield, there may be still a limit to the increase in the rice harvest index [23,24]. In addition, if the harvest index was too high, it was easy to aggravate the contradiction between source and sink and cause lodging [22,25]. As for the extreme of rice harvest index remained to be further confirmed. So, in order to further improve rice yield, it might be necessary to cultivate rice populations with more reasonable population structure and higher biological yield, so as to lay a material foundation for high yield [26,27]. In this study, the harvest index was in the order of L6 > L7 > L8 > L9 > L10, and insignificant differences were surveyed among L6, L7 and L8, while the biological yield of L8, namely the above-ground dry matter weight at maturity, was 2.1%~11.8% higher than other treatments. Thus, it indicated that higher biological yield was one of the key characteristics for high yield of L8.

The previous studies were more consistent that, under high-yield conditions, the above-ground dry matter weight from the jointing to heading stage and from heading to maturity had a positive impact on biological yield, and in contrast, the dry matter weight before the jointing stage tended to have a negative effect [20,28]. This indicated that the accumulation and distribution of dry matter during the mid-to-late growth stages are critical for enhancing the overall biological yield of crops [29]. And a quadratic parabola relationship was lead between the above-ground dry matter weight at heading stage and biological yield. As the amount and application time of nitrogen fertilizer played a significant role in promoting and controlling photosynthetic matter production, then in the rice cultivation, nitrogen fertilizer was usually applied to properly control the above-ground dry matter weight in the rice early growth period to form a proper small amount of strong plant populations at jointing stage, and to appropriately increase an efficient photosynthetic matter production in the rice middle growth period to form a high-quality population with sufficient quantity and optimized structure at heading stage, and to greatly increased the dry matter accumulation in the rice late growth period was to form a high biological yield with source-sink coordinated population [22,26,30,31,32,33]. In this study, a new approach was explored to nitrogen fertilization management for dense-planted dry direct-seeded rice, of which the initial seedling density was almost the suitable effective panicle number of this rice type under high yield direct seeding cultivation. The novel nitrogen application method involved applying the total nitrogen at a specific leaf age stage for such dry direct-seeded rice [7,13]. And the results of dry matter accumulation traits for biological yield in this study were almost consistent with previous research [11,33,34,35]. Without basal fertilizer, one-time application of total nitrogen at leaf age during tillering period had significant effects on dry matter accumulation of rice. The dry matter accumulation of L8 was at a disadvantage in the early rice growth period in compared to L6 and L7, owing to the late nitrogen application time and the relative adversity of nitrogen starvation for the plant growth, but the above-ground dry matter weights showed insignificant difference among L6, L7 and L8. Then the dry matter accumulation after jointing stage increased obviously with the plant obtaining much-needed nitrogen nutrition, and the maximum dry matter accumulation was observed in L8 at heading, milky and maturity stages. Although the L6 and L7 had more dry matter accumulation in the early rice growth stage, but the dry matter accumulation was low after jointing, because ineffective tillers were promoted by earlier nitrogen application. Nevertheless, the nitrogen was too late applicated for L9 and L10 to form strong plant population at heading stage and high biological yield with the weakness of insufficient above-ground accumulation in the early rice growth period. Thus, the one-time application of total nitrogen too early or too late was not conducive to the accumulation of dry matter and grain yield formation of dense direct seeding rice [36]. And the nitrogen application at the eight-leaf age was more suitable, which could effectively regulate the dry matter accumulation and proportion in the main growth periods, and moderately increase the dry matter accumulation in the middle and late rice growth periods on the basis of obtaining sufficient dry matter accumulation in the early rice growth period, so as to lay a material foundation for higher biological yield and grain yield formation [37].

3.2. The Above-Ground Dry Matter Production Trait

The photosynthesis was the source of above-ground dry matter production, and the LAI, ELAR, HELR, LAD, CGR, NAR, et al. were important indexes to characterize the photosynthetic matter capacity of plant population [38,39]. The researches on the traits of rice above-ground dry matter production had consistently shown that high-yielding rice generally had higher optimal leaf area, ELAR and HELR at heading stage, as well as higher LAI, CGR, NAR from jointing to maturity stages [24,37]. In this study, with the delay of nitrogen application leaf age, the L8 had a smaller LAI than L6 and L7 in the early rice growth period, bigger LAIs at booting and heading stages, and a smaller LAI than L9 and L10 at maturity stage. In addition, the HELR at heading stage and the MLAI across the whole growth duration got higher values of L8 than other treatments. These above laid the high photosynthetic efficiency population foundation for the high-efficiency dry matter production in a comprehensive view [29,37]. The LAD, CGR and NAR showed the same trend that the L8 obtained intermediate values in the early and late growth period, and higher values in the middle growth period among treatments. This indicated that the strong photosynthetic dry matter production capacity in the middle growth period was an important feature for L8 to obtain high biological yield. From the view of grain-leaf ratio, all the RTSL, RTGL and RGWL had relative higher values in L8 among treatments. It meant that the rice population of L8 could better balance the vegetative growth and reproductive growth, so that the photosynthetic matter of leaves was more used for grain formation and enrichment, thereby increasing yield [40,41,42]. However, the CGR and NAR during heading to maturity stage of L8 were lower than L9 and L10, and the LADs during heading to maturity stage were insignificant among L8, L9 and L10. On the one hand, due to the late total nitrogen application of L9 and L10 than L8, the early growth of rice was weak, and the fertilizer effect was mainly concentrated in the middle and late growth period, then the leaves stayed green for a longer time and produced more dry matter. On the other hand, this suggest that improving the plant growth in the late growth period of L8 by cultivation technology might further increase the dry matter accumulation and yield of rice.

4. Materials and Methods

4.1. Experiment Condition and Plant Material

Field experiments were conducted in 2019 and 2020 at Jiangyan experimental station, Taizhou city, Jiangsu province, China (32°61′ N, 120°12′ E, 1.2 m altitude), and the meteorological data of rice growing season was shown in Figure 5. The experimental field soil was clay loam with 31.52 and 30.68 g kg⁻¹ organic matter, 1.85 and 1.93 g kg⁻¹ total nitrogen, 29.86 and 32.27 mg kg⁻¹ available phosphorus, 148.35 and 162.13 mg kg⁻¹ available potassium in 2019 and 2020, respectively. Nanjing 9108 and Yangnongxiang 28, the Japonica conventional rice with good taste, were used in the experiment. The test field led a rice-wheat rotation system, and the wheat yields were 7.25 and 7.46 t ha^-1 in 2019 and 2020, respectively.

4.2. Experiment Design and Cultivation

The randomized block design was adopted for the experiment with total nitrogen once applied time treatments at sixth (L6), seventh (L7), eighth (L8), ninth (L9) and tenth (L10) leaf age stages. Total 180 kg ha^-1 nitrogen was once applied at the leaf growth initial stage of each treatment with 150 kg ha^-1 urea (N content was 46%) and 740 kg ha^-1 compound fertilizer (N, P₂O₅ and K₂O contents were 15%, 15% and 15%, respectively).

According to the local rice cultivation experience, rice was seeded by the multifunctional integrated dry direct seeding machine (2BFMZ-10, Yangzhou Huilong Machinery Technology Co., Ltd. Yangzhou, China) on June 14^th and June 11^th in 2019 and 2020. The row spacing was 25 cm, and the sowing rate was 195 kg ha^-1. At the third leaf age stage, the seedlings of each plot were artificial set to 380 × 10⁴ ha^-1, which was based on the suitable effective panicle number of this type rice under high yield direct seeding cultivation. The treatment was repeated 3 times with plot area of 25 m². The earth ridges covered with black plastic films were isolated between the plots to ensure that each plot was irrigated separately. The management of water, disease, pests and weeds were carried out according to the guidance of local agricultural experts.

4.3. Sampling and Measurements

The above-ground dry matter weight and leaf area index (LAI) were measured at the sixth-leaf, tenth-leaf, jointing, booting, heading, milky, Maturity stages. Sixty plants of three points in each plot were tagged for recording tiller number. Twelve plants, randomly selected from three points in the middle of each plot were sampled. The green leaf area was measured by a leaf area meter (LI-3100, LI-COR, USA) to calculate the LAI. The calculation method of mean leaf area index (MLAI) was described by Hirooka et al. (2016) [43]. All samples were oven-dried at 80°C to a constant weight to calculate the above-ground dry matter weight.

At heading stage, except green leaf area of total stems and tillers (TLA), the green leaf area of panicle-stems and panicle-tillers was measured as effective leaf area (ELA), and the top three leaf area of panicle-stems and panicle-tillers was measured as high effective leaf area (HELA). The effective leaf area rate (ELAR) and high effective leaf area rate (HELR) were calculated using the following formulas:

ELAR (%) = ELA / TLA × 100

HELR (%) = HELA / TLA × 100

Crop growth rate (CGR), leaf area duration (LAD) and net assimilation rate (NAR) were calculated using the following formulas:

CGR (g m^-2 d^-1) = (W₂−W_l) / (t₂−t₁)

LAD (m² d m^-2) = 1/2 × (L₁+L₂) × (t₂−t₁)

NAR (g m^-2 d^-1) = [ln (L₂) − ln (L₁)] / (L₂−L₁) × (W₂−W₁) / (t₂−t₁)

Where, W₁ and W₂ were the first and second measurements of above-ground dry matter (g m^-2), L₁ and L₂ were the first and second measurements of LAI (m² m^-2), and t₁ and t₂ represented the first and second days, of measurement, respectively.

The number of panicles was determined from three representative 1 m² in the middle of each plot. The number of spikelets and filled grains per panicle and filled grain weight were determined from three rows in 0.5 m in each plot. All rice plants in an area of 10 m² at the center of each plot were hand-harvested, and grain yield was calculated. The final rice yield was adjusted to 14.5%.

The grain-leaf ratio traits, namely ratio of total spikelets to LAI at booting stage (RTSL), ratio of total grains to LAI at booting stage (RTGL) and ratio of total grain weight to LAI at booting stage (RGWL), were calculated using the following formulas:

RTSL (cm^-2) = PN × SN / L / 10000

RTGL (cm^-2) = PN × GN / L / 10000

RTGL (mg cm^-2) = GY / L × 10

Where, PN was the number of panicles per hectare (×10⁴ ha^-1), SN was the number of spikelets per panicle, GN was the number of filled grains per panicle, GY was the total grain weight (t ha^-1), and L was LAI at booting stage (m² m^-2).

4.4. Statistical Analysis

Data were analyzed using IBM SPSS Statistics 22 (SPSS, Chicago, IL, USA). Means were tested by least significant difference at P < 0.05 (LSD_0.05). Graphs were prepared by Excel 2013 (Microsoft Corporation, Washington, DC, USA), and all error bars indicated standard errors of means.

5. Conclusions

For the dense planted dry direct seeding rice, of which the initial seedling density was almost the suitable effective panicle number of this rice type under high yield direct seeding cultivation, total nitrogen once applied at eight leaf age stage got the highest grain yield in this study. The higher above-ground dry matter weight after heading stage, bigger MLAI with higher LAI at heading, increased photosynthetic matter production capacity from jointing to heading period and the effective photosynthetic matter production capacity from heading to maturity period, were important traits of high grain yield formation and higher biological yield formation of L8. This study proposed a novel nitrogen fertilization method for the dense planted direct seeding rice based on the idea of total nitrogen one-time fertilization at specific leaf age of the rice middle or late tillering period. However, whether this investigated nitrogen application leaf age was suitable for different growth types of rice varieties, different types of soil, and different soil fertility levels remained to be scientifically compared. In addition, the performance of this nitrogen application mode in long-term production practice should be carried out long-term fixed-point experiments for in-depth and detailed comparative analysis.