Inbred varieties outperformed hybrid rice varieties under dense planting with reducing nitrogen

Field experiments were conducted over two years to evaluate the effects of planting density and nitrogen input rate on grain yield and nitrogen use efficiency (NUE) of inbred and hybrid rice varieties. A significant interaction effect was observed between nitrogen input and planting density on grain yield. Higher number of panicles per square meter and spikelets per panicle largely accounted for the observed advantage in performance of inbred, relative to hybrid varieties. Compared with high nitrogen input rate, nitrogen absorption efficiency, nitrogen recovery efficiency, and partial factor productivity increased by 24.6%, 28.0%, and 33.3% in inbred varieties, and by 32.2%, 29.3%, and 35.0% in hybrids under low nitrogen input, respectively. Inbred varieties showed higher nitrogen absorption efficiency, nitrogen recovery efficiency, and partial factor productivity than hybrids, regardless of nitrogen input level. Nitrogen correlated positively with panicle number, spikelets per panicle, biomass production at flowering, and after flowering in inbred varieties but only with panicle number and biomass production at flowering in hybrids. Inbred varieties are more suitable for high planting density at reduced nitrogen input regarding higher grain yield and NUE. These findings bear important implications for achieving high yield and high efficiency in nutrient uptake and utilization in modern rice-production systems.

Yield components, biomass, and NUE. Yield component responses to plant density varied with N application rate (Tables 3 and 4). Panicle number significantly (P < 0.05) increased with N application rate and plant density for inbred and hybrid rice varieties. The number of spikelets per panicle significantly (P < 0.05) increased with N application rate but decreased with increasing planting density. The number of panicles in the treatments receiving N was 22.1% and 32.1% higher for the hybrid and the inbred varieties, respectively, relative to the N0 treatment, (Tables 3 and 4). Conversely, relative to N0, the number of spikelets per panicle in all N treatments was 27.0% and 9.9%, respectively, higher for the inbred and the hybrid varieties (Tables 3 and 4). At Jingzhou, N1×D3 was optimal for inducing greater panicle and spikelet number per panicle across treatments in both inbred and hybrid varieties (Table 3). In turn, at Hangzhou, the optimal combination treatment for promoting higher panicle and spikelets number per panicle in inbred varieties was N3×D3, while N3×D1 was the best combination treatment for the hybrid varieties under study ( Table 4).
The effects of N application rate and plant density on percent seed set were not significant (P > 0.05) for the inbred varieties but they were (P < 0.05) for the hybrid varieties. In contrast, grain filling significantly (P < 0.05) decreased with increasing N application rate in hybrid rice varieties. Grain filling in ZZY-8 and C-LYHZ decreased from 85% under N0 to 75% under N3. 1,000-grain weight did not (P > 0.05) change significantly in either rice varietal group across N application rates or planting densities. Interaction analysis showed that N application rate significantly (P < 0.05) influenced panicle number, spikelet number per panicle, percent grain filling, and 1,000-grain weight, whereas planting densities only (P < 0.05) affected significantly panicle number and spikelet number per panicle.        www.nature.com/scientificreports www.nature.com/scientificreports/ Biomass, nitrogen uptake, and NUE. Total aboveground biomass at maturity significantly (P < 0.05) increased with N application rate and planting density (Tables 5 and 6). At both sites and across N treatments, ordinary hybrids had consistently higher biomass than inbred varieties. Total aboveground biomass for hybrid varieties was 14.3% higher than that for inbred varieties (Tables 5 and 6). Total aboveground biomass at maturity under all N treatments was 10.1% and 33.7% higher in hybrids and inbred varieties compared with the N0 treatment, respectively (Tables 5 and 6). At Jingzhou, N2×D3 was the optimal combination treatment for higher biomass accumulation in both inbred and hybrid varieties. In turn, at Hangzhou, N3×D3 was optimal for elevated biomass accumulation in inbred varieties and N3×D1 was best for increased biomass accumulation in hybrid varieties.
Nitrogen uptake and NUE varied among treatments (Tables 5 and 6). N uptake and NUE under the various N application rates and planting densities were similar at both sites. Inbred varieties showed higher AE, RE, and PFP than hybrids. AE and PFP significantly (P < 0.05) increased with planting density at low N rates (N1) but significantly (P < 0.05) decreased with increasing planting density at high N rates (N2 and N3) for both types of variety. RE increased with planting density but decreased with N application rate. AE, RE, and PFP were 24.6%, 28.0%, and 33.3% higher in inbred varieties and 32.2%, 29.3%, and 35.0% higher in hybrid varieties under the N1 treatment relative to N3 (Tables 5 and 6). At Jingzhou, N1×D3 was optimal for higher AE, RE, and PFP for both inbred and hybrid varieties. In turn, at Hangzhou, N1×D3 was optimal for higher AE, RE, and PFP in hybrid varieties, while N1×D2 was optimal for higher AE, RE, and PFP in inbred varieties.
Correlation analyses of grain yield, yield components, and biomass at various nitrogen application rates and planting densities. Correlation matrices among the various grain yield components and biomass parameters for inbred and hybrid varieties are shown in Figs. 2 and 3. N application rate significantly (P < 0.01) and positively correlated with GY, P, SP, DMF, and DMAF for the inbred varieties and with GY, P, and DMF for the hybrid varieties. Additionally, there was a significant (P < 0.01) positive correlation between D and DMAF for both the inbred and hybrid varieties. The significant (P < 0.01) positive correlations among GY, P, SP, DMF, and DMAF were identical for the inbred and hybrid varieties. However, GF was significantly (P < 0.05) and negatively correlated with GY for both inbred and hybrid varieties.

Discussion
Several studies have confirmed that super hybrid rice varieties had higher yields than ordinary hybrids or inbred varieties 20,23,24 . However, it is not conclusively known whether the same holds under conditions of high planting density and reduced nitrogen application rate. The present study compared yield and NUE for inbred and hybrid rice varieties cultivated under different N rates and planting densities. Neither super nor ordinary hybrid varieties surpassed inbred varieties in terms of grain yield or NUE.
Neither the super hybrid rice variety (Y-LY900) nor the ordinary hybrid rice varieties (ZZY-8, C-LYHZ and QLYSM) showed any significant advantage over the inbred varieties (HHZ and YNSM) with regard to grain yield. There were no significant differences in grain yield among varieties or across N treatments, except relative to N0 (Tables 1 and 2). A two-year field experiment conducted by Hou et al. 18 revealed that the average grain yield of rice hybrid Liangyou 3905 was ca 9.2 ton ha −1 even under optimal 165 kg N ha −1 and 24-27 × 10 4 hills ha -1 planting density. Here, the average grain yield for inbred varieties under N1 (135 kg N ha −1 ) were 9.8 ton ha −1 at Jiangzhou and 8.1 ton ha −1 under N1 (120 kg N ha −1 ) at Hangzhou. Thus, inbred varieties achieved equal or higher grain yield than super/ordinary hybrid varieties and are relatively less dependent on exogenous nitrogen application. Huang et al. 15 proposed that high planting density at reduced nitrogen application rate increases grain yield and NUE in hybrid rice varieties even under low light-intensity stress. However, our previous studies showed that super/ordinary hybrid varieties are more sensitive to shade stress than inbred rice varieties. Shade stress at flowering caused substantially higher yield losses by super/ordinary hybrid rice varieties than it did for inbred rice varieties 25,26 . Inbred rice varieties may be better suited for high planting density at reduced nitrogen application rate than super/ordinary hybrid rice varieties.
Inbred varieties can attain equal or higher grain yield than hybrid varieties under high planting density in combination with reduced nitrogen application rate, as the former showed superior sources and sinks. High planting density and low nitrogen application rate markedly increased panicle number and spikelets per panicle in inbred varieties, compared with hybrid varieties. Correlation analyses showed that N was significantly (P < 0.01) and positively correlated with panicle number, spikelets per panicle, biomass production at flowering, and biomass production after flowering in inbred varieties. In contrast, N was significantly (P < 0.01) and positively correlated only with panicle number and biomass production at flowering in hybrid rice varieties. Compared to N0, under the other N treatments evaluated here, the number of panicles increased by 22.1% and by 32.1% in the hybrid and in the inbred varieties, respectively (Tables 3 and 4). Similarly, the number of spikelets per panicle increased by 27.0% in the inbred varieties but only by 9.9% in the hybrid varieties (Tables 3 and 4). The relatively higher number of panicles and number of spikelets per panicle in inbred varieties were attributed to an increase in number of tillers at higher planting density 27 . Thus, a higher planting density compensated for www.nature.com/scientificreports www.nature.com/scientificreports/ the negative effect of a reduced N application rate for inbred rice but not for super hybrid rice, in which case, high yield was attributed to an increase in the number of spikelets per panicle and to a greater biomass production. These factors did not apply to ordinary hybrids or inbred cultivars [20][21][22] . In contrast, these advantages were not observed at high planting density combined with reduced nitrogen application rate. Hybrid and super hybrid rice cultivars often achieve high yields under optimum growing conditions, especially at high N input. Thus, hybrid and super hybrid rice may perform better than inbred rice only at high N application rate 20,28-30 . Here, hybrids did not show any advantage over inbred varieties with respect to number of panicles per square meter, number of spikelets per panicle, or biomass production at low N input rate combined with high planting density. Hybrids had higher grain weight than inbred varieties but this discrepancy did not compensate for the detrimental treatment effects of reduced N rate.
Hybrid rice is well adapted to high N fertilizer conditions and requires large amounts of N fertilizer to produce high yields. Consequently, farmers tend to apply substantial quantities of N fertilizer aiming to ensure high grain yields. However, heavy N fertilizer application may result in low NUE because of ammonia volatilization, denitrification, surface runoff, and leaching into the soil floodwater system 31,32 . Numerous improvements in N fertilizer management practice have been developed to increase NUE in rice production. High planting density at low N input rates is widely regarded as a sustainable strategy to improve NUE. Nevertheless, few studies have focused on the relative differences in NUE between inbred and hybrid varieties sown at high density and reduced nitrogen application rate.
The response of NUE to high planting density at low nitrogen input rate observed in the experiments reported herein were consistent with previously reported results 18,19,27 . Compared with high N application rate, AE, RE and PFP increased by 24.6%, 28.0% and 33.3% in inbred varieties, and by 32.2%, 29.3% and 35.0% in hybrid varieties, respectively, under low N application rate (Table 5 and Table 6). Therefore, this type of management practice effectively improved NUE in both hybrids and inbred cultivars. Moreover, inbred varieties showed higher NAE, NRE, and PFP than hybrid rice varieties across nitrogen treatments. To the best of our knowledge, this study is the first to compare NUE between inbred and hybrid rice varieties planted at high density and low N input rate. Enhanced NUE in inbred varieties was attributed to their comparatively lower N requirements for growth and yield formation than those of hybrid varieties. NUE was negatively correlated with N application rate 33 . Excessive N fertilizer application resulted in high soil residual nitrate levels 34 . Increased soil residual-nitrate may increase the risk of nitrate leaching and low NUE. High planting density at reduced nitrogen application rate enabled inbred rice varieties to achieve both high grain yield and high NUE.

conclusions
Our study demonstrated that a higher number of panicles and of spikelets per square meter largely explained the comparatively higher yield of inbred rice varieties cultivated under high planting density combined with reduced nitrogen input rate. Furthermore, inbred varieties showed higher nitrogen absorption efficiency, nitrogen recovery rate, and partial factor productivity than hybrids under all nitrogen treatments. Increasing planting density may compensate for the negative effects of a reduced nitrogen application rate in inbred varieties. Thus, high planting density combined with reduced nitrogen application rate is better suited for rice production if inbred, rather than hybrid varieties, is used.

Methods
Site description. Field experiments were conducted in the experimental farm at Yangtze University, Jingzhou, in 2017 and in Hangzhou, Zhejiang Province in 2018. Before transplanting and fertilizing, five soil cores were collected diagonally from the 0-20 cm soil layer in the rice paddy at the two sites, and basic soil properties were analyzed after Lu 35 . The soil at the Jingzhou site was calcareous alluvial with pH 6.8, 18.5 g kg −1 organic matter, 110.5 mg kg −1 alkali-hydrolysable N, 25.0 mg kg −1 available P, and 105.5 mg kg −1 available K. The soil at the Hangzhou site was a sandy loam with pH 7.0, 7.1 g kg −1 organic matter, 237 mg kg −1 alkali-hydrolyzable N, 17.1 mg kg −1 available P, and 139 mg kg −1 available K.
Urea at 50%, 20%, 20%, and 10% was applied at transplanting, tillering, panicle initiation (PI), and heading, respectively. There were two split potassium applications in the form of KCl. The rate was 40 kg K 2 O ha-1 and 50% was applied as a basal dressing and 50% was applied as broadcast at PI. Phosphorus and zinc were broadcast as basal fertilizer in the forms of calcium superphosphate at a rate of 30 kg P 2 O 5 ha −1 and zinc sulfate at a rate of 5 kg ZnSO 4 ha −1 . Crop management followed standard cultural practices. Insects were intensively controlled with pesticides to avoid biomass and yield losses.  www.nature.com/scientificreports www.nature.com/scientificreports/ Experimental design. Treatments were arranged in a split-split plot design with N treatment as the main plot, planting density as the subplot, and varieties as sub-subplot. Three replications were included each year. Each plot was 30 m 2 . Hybrid varieties Zhongzheyou8 (ZZY-8) and C-liangyouhuazhan (C-LYHZ), and inbred varieties Huanghuazhan (HHZ) and Yangdao 6 (YD-6) were grown at Hangzhou. Hybrid varieties Y-liangyou900 (Y-LY900) and Quanliangyouhuazhan (QLYSM), and inbred varieties Huanghuazhan (HHZ) and Yuenongsimiao (YNSM) were grown at Jingzhou. These varieties are extensively planted in southern China. Varietal specifications are listed in Table 7.
Nitrogen application rates and planting densities for inbred and hybrid varieties at Jingzhou and Hangzhou are listed in Table 8 Crop management. Urea at 50%, 20%, 20%, and 10% was applied at transplanting, tillering, panicle initiation (PI), and grain filling, respectively. There were two split potassium applications using KCl at a rate of 40 kg K 2 O ha −1 ; 50% was applied as a basal dressing and 50% was applied as broadcast at PI. Phosphorus and zinc were broadcast as basal fertilizer as calcium superphosphate at a rate of 30 kg P 2 O 5 ha −1 , and zinc sulfate at a rate of 5 kg ZnSO 4 ha −1 . Crop management followed standard cultural practices. Insects were intensively controlled with pesticides to avoid biomass or yield losses.
Sampling and measurements. At tillering, plant samples were separated into straw and leaves. Then, at flowering and maturity, plant samples were separated into straw, leaves, and panicles for dry weight determination after oven-drying to constant weight at 70 °C. At maturity, twelve hills were diagonally sampled over a 5-m 2 harvest area on each plot. Three border lines were excluded to avoid border effects. Plants were hand-threshed after counting the panicles. Filled and unfilled spikelets were separated by submersion in tap water. Three 30 g of filled grain and 3 g of unfilled spikelet subsamples were removed to count the spikelets. Filled and unfilled spikelets were identified and separated after oven-drying to constant weight at 70 °C. Spikelets per panicle and grain filling percentage (100 × filled spikelet number / total spikelet number) were calculated. Grain yield was determined from a 5-m 2 area in the middle of each subplot and adjusted to a moisture content of 0.14 g H 2 O g −1 fresh weight.
Dried leaf, stem, and panicle samples were collected at heading and straw and filled-and unfilled spikelets collected at maturity were pulverized and their N content was measured with a Skalar SAN Plus segmented flow analyzer (Skalar Inc., Breda, The Netherlands). Nitrogen content of was determined by the Kjeldahl method and N uptake in each organ was determined by multiplying its dry weight by its N content. Total N uptake at heading was estimated as the sum of leaf, stem, and panicle N uptake, and total N uptake at maturity was estimated as the sum of straw, filled-, and unfilled spikelet N-uptake.
Nitrogen agronomic efficiency (NAE) was calculated by the following formula: NAE (kg kg -1 ) = [(grain yield with N treatments -grain yield without N) / amount of N fertilizer applied].
Nitrogen recovery efficiency (NRE) was calculated by the following formula: NRE (%) = [(sum of N content in all aboveground components under N treatment -sum of N content in all aboveground components without N) / amount of N fertilizer applied].
Partial factor productivity (PFP) of applied N was calculated by the following formula: N fertilizer (PFP, kg kg -1 ) = [grain yield / amount of N fertilizer applied].
Data analysis. Data shown are means subjected to ANOVA for significant differences subsequently separated by the least significant difference (LSD) test at 0.05 and 0.01 levels of significance. ANOVA was also performed on the N application rate, planting density, and their interactive effects. A path analysis of grain yield and yield components was also performed. The statistical software used for these analyses was SPSS v. 17.0 (IBM Corp., Armonk, NY, USA).