Nitrogen Deep Placement Combined with Straw Mulch Cultivation Enhances Physiological Traits, Grain Yield and Nitrogen Use Efficiency in Mechanical Pot-Seedling Transplanting Rice

LiLin, Zhang Zheng, Tian Hua, Umair Ashraf, Yousef Alhaj Hamoud,Al Aasmi Alaa, Tang Xiangru, Duan Meiyang, Wang Zaiman, Pan Shenggang

Research Paper

Nitrogen Deep Placement Combined with Straw Mulch Cultivation Enhances Physiological Traits, Grain Yield and Nitrogen Use Efficiency in Mechanical Pot-Seedling Transplanting Rice

LiLin1, 2, Zhang Zheng1, 2, Tian Hua1, 2, Umair Ashraf1, 3, Yousef Alhaj Hamoud4,Al Aasmi Alaa5, Tang Xiangru1, 2, Duan Meiyang1, 2, Wang Zaiman6, Pan Shenggang1, 2

(Department of Botany, Division of Science and Technology, University of Education, Lahore 54770, Pakistan; College of Agricultural Science and Engineering, Hohai University, Nanjing 210098, China; ; )

To assess the effects of straw return coupled with deep nitrogen (N) fertilization on grain yield and N use efficiency (NUE) in mechanical pot-seedling transplanting (MPST) rice, the seedlings of two rice cultivars, i.e., Yuxiangyouzhan and Wufengyou 615 transplanted by MPST were applied with N fertilizer at 150 kg/hm2and straw return at 6 t/hm2in early seasons of 2019 and 2020. The experiment comprised of following treatments: CK (no fertilizer and no straw return), MDS (deep N fertilization and straw return), MBS (broadcasting fertilizer and straw return), MD (deep N fertilization without straw return), MB (broadcasting fertilizer without straw return). Results depicted that the MDS treatment significantly increased the rice yield by 41.69%?72.22% due to total above-ground biomass, leaf area index and photosynthesis increased by 54.70%?55.80%, 38.52%?52.17% and 17.89%?28.40%, respectively, compared to the MB treatment. In addition, the MDS treatment enhanced the total N accumulation by 37.74%?43.69%, N recovery efficiency by 141.45%?164.65%, N agronomic efficiency by 121.76%? 134.19%, nitrate reductase by 46.46%?60.86% and glutamine synthetase by 23.56%?31.02%, compared to the MB treatment. The average grain yield and NUE in both years for Yuxiangyouzhan were higher in the MDS treatment than in the MD treatment. Hence,deep N fertilization combined with straw return can be an innovative technique with improved grain yield and NUE in MPST in South China.

mechanical pot-seedling transplanting; nitrogen deep placement; nitrogen use efficiency; rice; straw return

Agriculture is facing the challenge of food security, water scarcity and environmental sustainability under the uprising problems of climate change, rapid growth population and environmental contamination, which can severely affect crop cultivation and production (Jensen et al, 2010; Oumarou et al, 2019). Rice is an important cereal crop and is the main staple food for the Asia population (Datta et al, 2017; Ashraf et al, 2018a, b).In China, the annual rice production is approximately 2 × 109t, which accounts for 28.7% of the world rice production (Chen et al, 2018; Li et al, 2019).With the development of the economy and the lack of rural labor, the manual broadcast fertilizer is no longer suitable for the development of Chinese agriculture (Knight et al, 2011; Tao et al, 2016). Therefore, it is necessary to develop rice production systems with new technologies and high resource use efficiencies maximizing crop yield (Wang L et al, 2019), thus the mechanization of deep fertilization is becoming popular in this regard.

Lignocellulosic crop residues have been the main target of present-day research (Shaghaleh et al, 2019). The straw is a by-product of crop production and its returning to field plays an important role in maintaining and sustaining soil productivity (Singh et al, 2004).Several field experiments have shown that straw incorporation significantly increases methane emissions in rice fields (Bhattacharyya et al, 2012; Zhang et al, 2015). However, Wang H H et al (2019) found that straw applied treatment significantly reduces methane and nitrous oxide emissions, and mulching of the soil surface using straw residue is a common approach to reduce straw burning and the negative effects of the burning on the environment such as air pollution (Oanh et al, 2011; Qu et al, 2012). Straw return also provides essential nutrients to the crop. Straw return treatment can save up to 25 kg/hm2of nitrogen (N) fertilizer (in pure N) but not reduce rice yield, compared with no straw return treatment (Eagle et al, 2001).Jiang et al (2018) reported that straw returning to field can replace potassium (K) fertilizer and effectively balance soil K deficiency, whereas Haberman et al (2019) found that straw returning to field can increase the content of water-soluble K, non-exchangeable K and mineral K in the soil, and play a role in nutrient return. Rasool et al (2019) found straw layer boosts the growth of other crops and increases yields under greenhouse conditions, whereas Liao et al (2018) showed that the straw return treatment cannot improve rice yield substantially. Therefore, the effects of straw return on rice yield need further investigations.

Mechanical pot-seedling transplanting (MPST) is an emerging technology for the transplantation of rice seedlings in fields by precise row and hill spacing without damage to the plants (Hu et al, 2014). Moreover, MPST can transplant the seedlings without root damage, and it would expect to reduce the transplant shock and sustain root activity, resulting in increased absorption of nutrients and ultimately a strong initial growth (Zhu C H et al, 2019). However, the traditional method of fertilization (i.e. applying fertilizer directly on the soil surface and/or in floods), is generally inefficient, with only 30% of N fertilizer being used by rice (Tao et al, 2016). Therefore, seedlings transplanted by MPST cannot obtain N resources, which limits rice growth and development. To overcome this problem, the use of chemical fertilizers has increased dramatically, but excessive use of chemical fertilizers often results in low N recovery and serious environmental pollution (Ju et al, 2009; Linquist et al, 2012), thus combination of deep fertilization with straw return in MPST is an alternative approach. Some researchers have discoveredthat deep application of N fertilizer can greatly improve N uptake by plants and increase the efficiency of N absorption while reducing environmental pollution (Gaihre et al, 2015, 2018; Mofijul Islam et al, 2018a). Furthermore, deep fertilization also promotes root growth and increases rice yield (Chen et al, 2021). Zhu C C et al (2019) observed that when fertilized at 5 cm depth, above-ground biomass, leaf area index, photosynthesis and yield of rice can be significantly increased. Deep application of N fertilizer can substantially reduce the ammonia volatilization by reducing the amount of ammonium nitrogen in the flood, thus improving the N use efficiency (NUE) (Huda et al, 2016; Mofijul Islam et al, 2016, 2018b). However, the effects of straw incorporation coupled with deep placement of N fertilizer on the NUE and grain yield of MPST are rarely investigated.

The deep N fertilization as well as the straw residue application to soil surface can improve the quality of soil and the productivity of several crops (Jin et al, 2016; Zhang Y N et al, 2017; Rasool et al, 2019), however, the impacts on the morphology, physiology and enzymatic activity of plants and shoot system adaption during rice growth under the integration of deep N fertilization and straw mulch cultivation are rarely investigated. Thus, understanding the plant response to the changes in soil environment is necessary to standardize the proper N and straw application rates to sustain rice production systems (Rodziewicz et al, 2014).Therefore, the field experimentalconfirmation on N assimilation and antioxidant enzyme activity and their relationships to biomass production of rice under the combination of deep N fertilization and straw mulch cultivation need to be investigated.

Moreover, rice productivity and soil N fertility should be improved within the use of accurate cropping management tactics, with emphasis on agronomic practices and new technologies that increase yield and fertilizer use efficiency of rice (Zhu C H et al, 2019; Li et al, 2020). However, few studies have been conducted so far to explore the effects of the combination of deep fertilization with straw return on yield, physiological activity and NUE of machine transplanted rice. The present study was conducted to provide new insights and detailed knowledge of the physiological interactions that regulate the N assimilation and the antioxidant enzyme system and, ultimately enhance rice production under straw mulch cultivation coupled with deep N fertilization by MPST.

Results

Chlorophyll content and net photosynthetic rate (Pn)

As shown in Table 1, the chlorophyll contents at all the crucial growth stages were significantly affected by different treatments. At the mid-tillering stage (MTS), the highest average chlorophyll contents for both rice cultivars in 2019 and 2020 were 5.81?5.91 mg/gunder the deep N fertilization and straw return (MDS) treatment, which was 36.22%?38.33% higher than the broadcasting fertilizer without straw return (MB) treatment, and 0.61%?6.21% higher than the deep N fertilization without straw return (MD) treatment, respectively. Moreover, the significant differences were found in chlorophyll contents among all the treatments for both cultivars in 2019. The average chlorophyll contents for both rice cultivars declined from the panicle initiation stage (PIS) to heading stage (HS), but in deep placement fertilizer treatments (MDS and MD), the chlorophyll contents declined more slowly than the broadcasting fertilizer treaments (MBS and MB). The deep fertilizer treatments kept higher chlorophyll content than the broadcasting fertilizer treatments at HS, and significant difference was found between MDS and MB for both cultivars. Moreover, the chlorophyll contents were decreased with the following trend: MDS > MD > MBS > MB > CK.

Pn at HS was significantly affected by different treatments. The average Pn for both rice cultivars in the two years were 23.69?24.19 μmol/(m2?s) in the MDS treatment, which was 17.89%?28.40% higher than the MB treatment and 12.09%?16.92% higher than the MBS treatment, respectively. The MDS treatment remarkably improved Pn, compared to the broadcasting fertilizer treatments (MBS and MB). Compared with the MB treatment, MBS led to 5.18%?9.82% increase in Pn for both rice cultivars.

Table 1. Effects of deep nitrogen fertilization coupled with straw return on total chlorophyll content and net photosynthetic rate (Pn) of rice in mechanical pot-seedling transplanting rice in 2019 and 2020.

CK, No fertilizer and no straw return; MDS, Deep N fertilization and straw return; MBS, Broadcasting fertilizer and straw return; MD, Deep N fertilization without straw return; MB, Broadcasting fertilizer without straw return;MTS, Mid-tillering stage; PIS, Panicle initiation stage; HS, Heading stage.

Within a column, means followed by the same lowercase letters are not significantly different at the 0.05 level according to the least significant different test among the five treatments in the same cultivars and years. *,< 0.05; **,0.01; ns, Not significant variance.

Determination of related enzymatic activities

Remarkable differences were found between the MD and MB treatments at MTS and PIS for both rice cultivars regarding NR activity. At MTS, the highest average NR activity for both rice cultivars in the two years was 18.48?23.19 μg/(g?h)in the MD treatment, which was 41.92%?62.96% higher than the MB treatment. At PIS and HS, the MDS treatment kept the highest NR activity among all the treatments in 2019, and the MD and MBS treatments were not differed significantly for Yuxiangyouzhan (YXYZ) in the two years, however, significant difference was found between the MBS and MB treatments for both rice cultivars (Fig. 1).

The GS activity was substantially affected by different N and straw return treatments. At MTS, the highest average GS activities for both rice cultivars in the two years were found in the MD treatment, and the MD and MDS treatments were differed significantly in 2019. Moreover, significant differences were found between the MD and MB treatments for both rice cultivars. At PIS, the MDS treatment remarkably improved the GS activity for YXYZ in the two years. However, the MD and MBS treatments did not differ significantly in 2020. At HS, the highest GS activity was observed in the MDS treatment, which was 4.37%?13.44% higher than that in the MD treatment, and deep fertilizer treatments significantly improved the GS activity compared with the MB treatment for both rice cultivars in 2019 (Fig. 2).

Grain yield and its related traits

Grain yield and its related traits were significantly affected by different N and straw return treatments. The highest average yield for both rice cultivars in the two years was 8.28?8.40 t/hm2in the MDS treatment, which was 41.69%?72.22% higher than the MB treatment and 0.42%?3.31% higher than the MD treatment. Moreover, no significant difference was found between the MD and MDS treatments in grain yield. The MDS remarkably increased the numbers of productive panicles per m2, compared to the MB treatment. However, no significant difference was found in numbers of productive panicles per m2and spikelets per panicle between the MDS and MD treatments. Grain filling rate of MDS treatment was remarkably higher than that in the MB treatment in 2019. There was no significant difference among all the treatments in 1000-grain weight for YXYZ, but for Wufengyou 615 (WFY615), 1000-grain weight under the deep fertilizer treatment was significantly higher than that under the broadcasting treatment (Table 2)

Fig. 1. Effects of deep nitrogen fertilization coupled with straw return on nitrate reductase (NR) activity in mechanical pot-seedling transplanting rice in 2019 and 2020.

CK, No fertilizer and no straw return; MDS, Deep N fertilization and straw return; MBS, Broadcasting fertilizer and straw return; MD, Deep N fertilization without straw return; MB, Broadcasting fertilizer without straw return;MTS, Mid-tillering stage; PIS, Panicle initiation stage; HS, Heading stage.Data are Mean ± SE (= 3). The same lowercase letters above bars at each stage are not significantly different at the 0.05 level according to the least significant different test.

Fig. 2. Effects of deep nitrogen fertilization coupled with straw return on glutamine synthetase (GS) activity in mechanical pot-seedling transplanting rice in 2019 and 2020.

CK, No fertilizer and no straw return; MDS, Deep N fertilization and straw return; MBS, Broadcasting fertilizer and straw return; MD, Deep N fertilization without straw return; MB, Broadcasting fertilizer without straw return;MTS, Mid-tillering stage; PIS, Panicle initiation stage; HS, Heading stage.Data are Mean ± SE (= 3). The same lowercase letters above bars at each stage are not significantly different at the 0.05 level according to the least significant different test.

Table 2. Effects of deep nitrogen fertilization coupled with straw return on grain yield and related traits in mechanical pot-seedling transplanting rice in 2019 and 2020.

CK, No fertilizer and no straw return; MDS, Deep N fertilization and straw return; MBS, Broadcasting fertilizer and straw return; MD, Deep N fertilization without straw return; MB, Broadcasting fertilizer without straw return.

Within a column, means followed by the same lowercase letters are not significantly different at the 0.05 level according to least significant different test among the five treatments in the same cultivars and years. *,< 0.05; **,0.01; ns, Not significant variance.

Moreover, the total above-ground biomasses (TAB) at all the crucial growth stages were significantly affected by different treatments. For both rice cultivars, no remarkable differences in TAB were found between the MDS and MD treatments during PIS to MS stages. The deep fertilizer treatments remarkably increased TAB, compared to the broadcasting fertilizer treatments. TAB in the MD treatment was the highest among all the treatments and was significantly higher than that in the MDS treatment in the two years for WFY615 at MTS. At the maturity stage (MS), the highest average TAB for both rice cultivars in the two years were 14.48?15.37 t/hm2in the MDS treatment, which was 54.70%?55.80% higher than the MB treatment, and 0.80%?5.02% higher than the MD treatment, respectively. The cumulative TAB growth rate of the deep fertilizer treatment was the highest during HS to MS, because these treatments can provide sufficient nutrients to transport to the grains (Fig. 3).

As shown in Fig. 4, the leaf area indexes (LAI) at all the critical growth stages were significantly affected by different treatments. At MTS, LAI in the MD treatment was the highest among all the treatments, whereas no remarkable difference was found between the MD and MDS treatments. Meanwhile, the MD treatment was significantly higher regarding LAI than the MBS treatment. At PIS and HS, the MDS treatment led to the highest LAI among all the treatments, and the MDS and MB treatments were differed significantly for YXYZ during both years. However, the deep fertilizer treatment led to marginal increase in LAI compared to the MBS treatment for WFY615.

Fig. 3. Effects of mechanized deep nitrogen fertilization coupled with straw return on total above-ground biomass (TAB) in mechanical pot-seedling transplanting rice in 2019 and 2020.

CK, No fertilizer and no straw return; MDS, Deep N fertilization and straw return; MBS, Broadcasting fertilizer and straw return; MD, Deep N fertilization without straw return; MB, Broadcasting fertilizer without straw return; MTS, Mid-tillering stage; PIS, Panicle initiation stage; HS, Heading stage; MS, Maturity stage. Data are Mean ± SE (= 3). The same lowercase letters above bars at each stage are not significantly different at the 0.05 level according to the least significant different test.

Fig. 4. Effects of mechanized deep nitrogen fertilization coupled with straw return on leaf area index in mechanical pot-seedling transplanting rice in 2019 and 2020.

CK, No fertilizer and no straw return; MDS, Deep N fertilization and straw return; MBS, Broadcasting fertilizer and straw return; MD, Deep N fertilization without straw return; MB, Broadcasting fertilizer without straw return; MTS, Mid-tillering stage; PIS, Panicle initiation stage; HS, Heading stage. Data are Mean ± SE (= 3). The same lowercase letters above bars at each stage are not significantly different at the 0.05 level according to the least significant different test.

Nitrogen use efficiency (NUE)

Total N accumulation (TNA), N agronomic efficiency (NAE), N recovery efficiency (NRE) and N partial factor productivity (NPFP) were significantly affected by the different treatments. The highest average TNA for both rice cultivars in the two years was 172.28? 180.95 kg/hm2in the MDS treatment, which was 1.89%?5.92% and 37.74%?43.69% higher than the MD and MB treatments, respectively. Compared with the broadcasting fertilizer treatments (MBS and MB), the MDS treatment significantly increased TNA. Regarding NRE and NAE, no significant difference was found between MDS and MD, but the MDS treatment significantly increased NAE and NRE, compared with the MB treatment. Mean NRE and NAE of both rice cultivars during both years in the MDS treatment ranged from 44.62% to 45.83% and from 19.87 to 24.45 kg/kg, respectively. N harvest index (NHI) under MDS was significantly higher than MB for WFY615 during both years. Deep fertilizer treatments significantly increased NPFP, compared to the broadcasting fertilizer treatments, and no significant difference was found between the MD and MDS treatments or between the MB and MBS treatments (Table 3).

Table 3. Effects of deep nitrogen fertilization coupled with straw return on nitrogen use efficiency in mechanical pot-seedling transplanting rice in 2019 and 2020.

TNA, Total nitrogen (N) accumulation; NPFP, N partial factor productivity; NHI, N harvest index; NRE, N recovery efficiency; NAE, N agronomic efficiency; CK, No fertilizer and no straw return; MDS, Deep N fertilization and straw return; MBS, Broadcasting fertilizer and straw return; MD, Deep N fertilization without straw return; MB, Broadcasting fertilizer without straw return.

Within a column, means followed by the same lowercase letters are not significantly different at the 0.05 level according to the least significant different test among the five treatments in the same cultivars and years. *,< 0.05; **,0.01; ns, Not significant variance.

Correlation analysis

Rice yield was significantly and positively correlated with TAB, LAI, chlorophyll content, Pn, NR, GS, TNA and NUE. The NR and GS activities were significantly and positively correlated with TNA, NRE and NAE. Furthermore, TAB also significantly positively related with LAI, Pn, chlorophyll content, TNA, NRE and NAE (Table 4).

Table 4. Correlation coefficients between grain yield and physiological traits for both rice cultivars in two years (means across years and cultivars).

TAB, Total above-ground biomass at the maturity stage; LAI, Leaf area index; Chl, Total chlorophyll content; Pn, Net photosynthetic rate; NR, Nitrate reductase activity; GS, Glutamine synthetase activity; TNA, Total nitrogen accumulation; NRE, Nitrogen recover efficiency; NAE, Nitrogen agronomy efficiency. *,0.05; **,0.01.

Discussion

Physiological activities

The total chlorophyll content, NR and GS activities are all key components involved in plant metabolism (Sarker et al, 2002). NR is a key enzyme of plant N metabolism, and is the first step in the conversion of NO3?to amino acids, while GS is a multifunctional enzyme at the center of N metabolism, involved in the regulation of N metabolism in plants (Masclaux et al, 2000). Photosynthesis is an important factor that contributes towards rice yields, and accounts for more than 90% of the total above-ground biomass of the crops (Zhao et al, 2015). In the present study, it was observed that the MDS treatment improved the NR and GS activities as well as the chlorophyll content in rice (Figs. 1 and 2; Table 1), and also enhanced Pn and LAI at HS (Table 1 and Fig. 4), compared to the MD and MB treatments. Deep placement of N fertilization might provide N in adequate amount for a long period of time that is favorable for rice growth, whereas the straw return not only ensures the nutrient supply at the later stage, but also improves the soil structure in paddy fields, such as increasing soil organic matter, total N and organic matter activity (Zhang B et al, 2013). Xu et al (2018) found that straw incorporation significantly increases the total N concentration in soil and N uptake and decreases soil NO3-N leaching. In addition, Yang et al (2020) reported that the complete decomposition of straw can promote the release of C and N from straw to soil and improve the soil nutrient status. Higher LAI is also helpful to delay the senescence of plants and extend the time of photosynthesis, which is beneficial to the transportation of dry matter to the panicle (Pan et al, 2017). The NR and GS activities were positively correlated with NAE and NRE (Table 4),indicating that maintaining a high level of physiological activity can promote N uptake and assimilation in rice.

Grain yield and its related traits

Deep fertilizer treatments significantly increased the grain yield, compared to the broadcasting fertilizer treatments (Table 2), owing primarily to the higher numbers of productive panicles and spikelets. Previously,Pan et al (2017) also found deep fertilization remarkably improves the spikelet number per panicle in direct- seeded rice treatment compared to broadcasting fertilizer treatment. Deep N placement generally promotes root growth by providing more N in the root system, ensuring longer N release time, promoting N uptake and increasing crop yield (Chen et al, 2021). This study also showed that the rice yield in the MDS treatment was higher than that in the MD treatment for YXYZ, and similar result was also observed in the MBS and MB treatments, which was in agreement with Xu et al (2010). At the early stage of straw return treatment, straw decomposition may produce a large number of microorganisms, which consume additional N and compete with rice for N absorption, but the solution for this problem is to increase the amount of N applied, leading to more fertilizer loss and an increase in the labor force (Li et al, 2016; Li et al, 2017). Deep fertilization significantly reduces N loss from ammonia volatilization and surface loss (Huda et al, 2016; Mofijul Islam et al, 2016, 2018b), and in this way, the competition between microorganisms and rice for N can be reduced. Yang et al (2004) reported that straw return can promote the transfer of nutrients from straw to grain at the late stages of plant growth, delay the occurrence of adverse senescence, and improve the grain filling and spikelet number per panicle of rice plants. Straw return can also better balance N supply and crop N demand, promote rice root growth and increase effective panicle, thus increasing crop yield (Zhang H et al, 2013). Recently, Hamoud et al (2019) also observed that the numbers of tillers and panicles are highly related to N availability in the root zone. Accordingly, the reasons for the grain yield increase in the MDS treatment can be summarized as follows: 1) At the early stage of rice growth, deep fertilization ensures an adequate supply of N fertilizer to avoid microorganisms competing with rice for N, which is conducive to the production of tillers and ensures the number of productive panicles at the later stage. 2) After the panicle stage, straw decomposition facilitates the transport of nutrients to grains, and thus improves the spikelet number per panicle in rice.

NUE

The MDS treatment substantially improved NUE including NAE, NRE and NPFP, compared to the broadcasting fertilizer treatments (Table 3). The reason might be that deep N fertilization promotes the uptake of total N by the plants, and reduces the loss of N in different ways (Zhang M et al, 2017; Yao et al, 2018). Deep N fertilization can better preserve the hydrolyzed NH4+in the soil and keep higher NH4+-N in the root area at the beginning of rice growth, which promotes root growth by providing more N deep in the inter- root zone, prolonging the availability of N and promoting plant uptake of N (Liu et al, 2015). Our study also found that NUE of MDS treatment was slightly higher than that of MD treatment. Straw return can improve soil fertility by increasing nutrients such as soil N pool and organic carbon (Su et al, 2014). Yao et al (2018) also reported that straw return promotes rice root growth and suppresses weeds, leading to the improvement of NUE. NHI of the MD treatment was lower than that of the MDS treatment (Table 3), mainly due to the adequate supply of N fertilizer for deep application. On one hand, deep N fertilization mitigates some problems caused by early decomposition of the straw by providing sufficient nutrients. For example, Wu et al (2011) observed that plant toxins (such as organic acids and reducing substances) released during straw decomposition directlycause rice leaves to yellow in the early season. Liu et al (2007) reported that the straw incorporation may consume additional N due to high C/N ratio, and the straw decomposition at the early stage may compete with rice for N uptake, resulting in insufficient N supply for crop growth and inhibiting the early growth of rice. On the other hand, deep placement of N fertilizer can complete N accumulation before PIS (Table 3), which is important for TNA at MS. In addition, Yang et al (2004) also observed that straw decomposition can provide nutrients and promote the transfer of photosynthetic products to grains. Thus, the deep fertilization and straw together contributed to the higher NUE of the MDS treatment.

Compared to MB, MDS significantly increased the grain yield and NUE in MPST. Moreover, MDS can also evidently improve N absorption, attributing to higher NR and GS activities, and leading to higher TNA, NRE and NAE. The MD treatment produced higher grain yield and NUE than the MBS and MB treatments, but lower than the MDS treatment. Therefore, our results suggest that deep N fertilization coupled with straw return may be an effective technique with the advantage of higher grain yield and NUE in MPST.

METHODS

Experimental site

MPST was developed by Changzhou YaMeiKe Mechanical Co., Ltd (Changzhou, Jiangsu Province, China) (Fig. S1). The open field experiments were performed in early seasons (March to July) of 2019 and 2020 at the Experimental Research Farm, South China Agricultural University, Guangdong Province, China (23.13o N, 113.81o E). The climate of the region is subtropical monsoon. The initial soil properties in the experimental field are shown in Table 5.

Experimental treatments and design

Two widely grown and high-yielding rice cultivarsYXYZ (inbred rice) and WFY615 (hybrid rice) were used, which had a growth period of 118 and 113 d, respectively. Field experiments were conducted in a randomized complete block design with three replicates with a plot area of 48 m2(6.0 m width × 8.0 m length). The seeds were mechanically sown in pot-seedling traysusing a sowing machine (LSPE-40AM, AMEC Corporation, Changzhou, China) with four seeds per hole for pot-seedling. The trays were 61.8 cm long and 31.5 cm wide with 448 holes. The 15-day-old seedlings were transplanted with MPST at a spacing of 30 cm × 14 cm. At 5 d after transplanting, the seedlings that did not meet the requirements of four seedlings per hill were replaced with the healthy ones. Two fertilization methods were adopted i.e., broadcasting fertilizer and mechanized deep placement of N fertilization. The broadcasting fertilizer treatment was broadcast N fertilizer manually on the soil surface as basal fertilizer at 2 d before transplanting, and deep N fertilization treatment was mechanized deep N fertilizer at once as basal fertilizer in 10 cm soil depth. No N fertilizer was applied during other growth periods. Urea (N, 46%), superphosphate (P2O5, 12%), potassium chloride (K2O, 60%) were applied as fertilizer. The rate of total N was 150 kg/hm2whereas all P and 50% K were broadcast as basal fertilizer at 2 d before transplanting, and 50% of K was broadcast at the panicle initiation stage. The experiment comprised of five treatments, i.e., CK, MDS, MBS, MD and MB. The straw return treatments, i.e., the rice straw was mechanically crushed (covered straw converted into dry matter weight was 6.0 t/hm2), and rotary tillage return to the field, whereas the rice straw was harvested from the late season of rice. The nutrient content of the straw included: 10.12 mg/g total N, 3.96 mg/g total P and 20.11 mg/gtotal K. For no straw return treatments, all straws were removed from the field. Moreover, 25?35 cm ridges were built between each plot and covered with plastic film to ensure separate drainage and irrigation. The rice crop was managed according to local conditions. In brief, the strategy for water management was the sequence of flooding, midseason drainage, re-flooding, moist intermittent irrigation and drainage. Some chemical reagents such as herbicide, imidacloprid, tricyclazole and carbendazim were applied to prevent and control of weeds, insects and diseases.

Table 5. Basic soil physical and chemical properties of initial soils.

Each item measured is a mean value of three replications.

Determinations of related enzyme activity, Pn and chlorophyll content

Approximately 25 uppermost rice leaves from each treatment were collected at MTS, PIS and HS, respectively, and then stored at -80 oC to determine enzyme activity. The NR and GS activities were determined according to Liu et al (2019). At HS, Pn in flag leaves was determined according to Zhu et al (2019). Ten representative flag leaves from each treatment were taken to determine Chl a and Chl b contents according to Du et al (2018), while the total chlorophyll content equals chlorophyll a plus chlorophyll b.

Measurements of grain yield, yield components, LAI and TAB

At MS, 1 m2rice plants with four replicates in each plot were taken randomly to calculate the number of productive panicles. Six rice plants were sampled from each treatment to determine yield components such as number of spikelets per panicle, grain filling rate and 1000-grain weight. Rice grains were harvested within 6 m2sampling area in each treatment and then threshed mechanically. The grain yield and its components were determined according to Pan et al (2017).

Six plants were taken from each plot at MTS, PIS, HS and MS based on the average tiller numbers per each treatment, and thenLAI and TAB were determined according to Pan et al (2017).

Determination of NUE

At MS, after TAB was measured, each part of rice plants was pulverized to powder to analyze total N accumulation. N concentrations were determined in accordance with Pan et al (2017), and then each portion of TNA was calculated. NUE was calculated as following formulae:

N uptake in grain () = N in grain × Grain dry mass;

N uptake in straw () = N in straw × Straw dry mass;

=+;

= (N?N) /;

= (?GY) /;

=/N;

=/N.

Where,andrepresent TNA of above-ground plant parts in the CK and N-fertilized plots, respectively;GYandrepresent grain yields in CK and N-fertilized plots, respectively;is 150 kg/hm2N fertilizer applied.

Data analysis

The experimental data were analyzed using the SPSS 19.0 software (SPSS Inc., Chicago, IL, USA). The differences among means of the experimental treatments were separated using the least significant difference (LSD) test at the 0.05 probability level. All images were plotted with Origin 9.0. Correlation analyses were made by using Statistix8.1 (Analytical Software, Tallahassee, FL, USA).

ACKNOWLEDGEMENTS

This study was supported by the Guangdong Basic and Applied Basic Research Foundation, China (Grant No. 2021A1515011255), Key-Area Research and Development Program of Guangdong Province, China (Grant No.2019B020221003), and National Natural Science Foundation of China (Grant No. 31471442). We thank Dr Kong Leilei for providing rice seeds. The authors also express their appreciations to all students and staff for their contributions to the development of this study.

Supplemental DATA

The following material is available in the online version of this article at http://www.sciencedirect.com/journal/rice-science; http://www.ricescience.org.

Fig. S1. Pictorial view of mechanical pot-seeding transplanting machine.

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9 November 2020;

25 February2021

Pan Shenggang (panshenggang@scau.edu.cn)

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http://dx.doi.org/10.1016/j.rsci.2021.12.008

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