Coupling of reduced inorganic fertilizer with plant-based organic fertilizer as a promising fertilizer management strategy for colored rice in tropical regions

Tingcheng Zhao ,Aibin He ,Mohammad Nauman Khan,Qi Yin,Shaokun Song,Lixiao Nie

Research Center for Physiology and Ecology and Green Cultivation of Tropical Crops,School of Breeding and Multiplication (Sanya Institute of Breeding and Multiplication),Hainan University,Sanya 572000,China

Abstract Colored rice is a type of high-quality,high-added-value rice that has attracted increasing attention in recent years.The use of large amounts of inorganic nitrogen fertilizer in rice fields results in low fertilizer use efficiency and high environmental pollution.Organic fertilizer is a promising way to improve soil quality and sustain high yields.However,most studies focus on the effect of animal-based organic fertilizers.The effects of different ratios of plantbased organic fertilizer and inorganic fertilizer on the grain yield and quality of colored rice have rarely been reported.Therefore,a two-year field experiment was conducted in 2020 and 2021 to study the effects of replacing inorganic N fertilizers with plant-based organic fertilizers on the yield,nitrogen use efficiency (NUE),and anthocyanin content of two colored rice varieties in a tropical region in China.The experimental treatments included no nitrogen fertilization(T1),100% inorganic nitrogen fertilizer (T2),30% inorganic nitrogen fertilizer substitution with plant-based organic fertilizer (T3),60% inorganic nitrogen fertilizer substitution with plant-based organic fertilizer (T4),and 100% plantbased organic fertilizer (T5).The total nitrogen provided to all the treatments except T1 was the same at 120 kg ha-1.Our results showed that the T3 treatment enhanced the grain yield and anthocyanin content of colored rice by increasing nitrogen use efficiency compared with T2.On average,grain yields were increased by 9 and 8%,while the anthocyanin content increased by 16 and 10% in the two colored rice varieties under T3 across the two years,respectively,as compared with T2.Further study of the residual effect of partial substitution of inorganic fertilizers showed that the substitution of inorganic fertilizer with plant-based organic fertilizer improved the soil physiochemical properties,and thus increased the rice grain yield,in the subsequent seasons.The highest grain yield of the subsequent rice crop was observed under the T5 treatment.Our results suggested that the application of plantbased organic fertilizers can sustain the production of colored rice with high anthocyanin content in tropical regions,which is beneficial in reconciling the relationship between rice production and environmental protection.

Keywords: colored rice,organic fertilizer,soil quality,grain yield,anthocyanin

1.lntroduction

Rice is one of the world’s most important food crops,accounting for 25% of the world’s total calorie intake,and 60% of the Chinese population uses rice as their main food source (FAO 2004;Yuan 2014;Kusanoetal.2015).With the development of the economy and the improvement of people’s living standards,the planting structure of rice has also changed,and it has tended to develop rice with high value-added and high economic benefits (Chenetal.2021).The demand for rice with unique qualities and nutritional value is gradually increasing worldwide.Colored rice is a kind of high-quality,high-added-value rice that has attracted increasing attention in recent years.The main substances of phenolic compounds in colored rice are anthocyanins,which are the major active antioxidant components (Iqbaletal.2005;Zhangetal.2006;Yawadioetal.2007).The effects of anthocyanins include improving iron-deficiency anemia,enhancing the body’s antioxidant,anti-cancer,and anti-allergic capabilities,and preventing atherosclerosis and some diseases associated with abnormal glucose metabolism.Studies have provided conclusive evidence for its role in the etiology of chronic hyperglycemia disease(Yawadioetal.2007;Krishnanetal.2021).

With the continuous promotion of modern agricultural concepts,implementing high-quality and sustainable development is the inevitable direction of agricultural development (Liuetal.2020).However,farmers in China rely on high levels of inorganic nitrogen (N) to increase crop yields,making Chinese nitrogen fertilizer consumption the largest in the world (FAO 2014).The application of nitrogen fertilizer in China reached 33.6 Tg in 2013,accounting for 33% of the total nitrogen fertilizer consumption worldwide.In addition,rice yield growth has slowed significantly and even stagnated in many regions of China in the past 10-20 years,despite the high nitrogen inputs (Grassinietal.2013;Zhaoetal.2015).Increasing the nitrogen input eventually reaches a point where it stops increasing crop yield,and excessive N input results in most of the N being lost to the surrounding environment and in low nitrogen use efficiency (NUE),as well as soil acidification,greenhouse gas emissions,and groundwater nitrate pollution (Gallowayetal.2008;Juetal.2009;Chenetal.2010;Zhangetal.2017).Therefore,an effective fertilizer management method is urgently needed to improve soil fertility and the yield and quality of rice.

The application of organic fertilizers has been neglected due to the obvious effect of increasing production,while the nutrients in organic fertilizers are released slowly,and they are insufficient sources of nutrients (Gengetal.2019).To achieve sustainable agricultural development,green and organic agriculture must be pursued as the development direction of modern agriculture (Kilcher 2007).Previous studies have shown that the use of organic fertilizers is a key strategy for improving the soil structure and nutrient content to achieve sustainable crop growth and high economic benefits (Cuietal.2021;Zhangetal.2022).Long-term organic fertilizer application maintains the soil organic matter at a relatively stable level,improves soil quality,increases the soil organic matter content,and increases stress resistance in farmland (Xuetal.2021;Asgharetal.2022).Studies have shown that soil microorganisms are closely related to the contents of various nutrients,such as soil organic carbon.The presence of organic matter can provide microorganisms with the nutrients required for life activities,thereby promoting the development of microbial communities (Lazcanoetal.2013;Liuetal.2022).The release of nutrients from organic fertilizers is slow,so their effect on increasing crop yields cannot be reflected immediately.However,the long-term use of organic fertilizers can improve plant growth and crop quality (Yeetal.2020).Partial replacement of inorganic fertilizers with organic fertilizers significantly improved the NUE in rice and maintained the grain yield by establishing a more balanced source-sink relationship during the grain-filling phase (Zhangetal.2018;Panetal.2022).

Currently,most studies focus on the effects of animalbased organic fertilizers.However,the effects of different ratios of plant-based organic and inorganic fertilizer,rather than inorganic fertilizer alone,on the grain yield and anthocyanin content of colored rice have rarely been reported.To test this scenario,a two-year field experiment was conducted to study the effects of partially or fully replacing inorganic N fertilizers with plant-based organic fertilizers on the yield,NUE,and anthocyanin content of colored rice in the tropical region of China.

2.Materials and methods

2.1.Site description

This study was conducted in Nanbao Town (19°64´N,109°62´E),Lingao City,Hainan Province,China,during the rice growing seasons of 2020 and 2021.Hainan Province is located in the tropical region.The typical rice growing season in the experimental site lasts from the end of February to November,and the experiments were initiated on August 2,2020 (the first season) and February 24,2021 (the second season).

The pH,total N,available phosphorus (P),potassium(K),and total organic carbon in the top 20 cm of soil were 6.17,1.90 g kg-1,62.59 mg kg-1,38.86 mg kg-1,and 0.89%,respectively,in 2020,and the values were 6.12,1.10 g kg-1,60.58 mg kg-1,42.55 mg kg-1,and 1.14% in 2021.

2.2.Experimental design

Youxianghongdao (YXHD) and Suixiangheinuo (SXHN),the two main locally-grown colored rice varieties,were adopted in this study.YXHD is normally used to produce wine,while SXHN is a sticky rice that is mostly used for making traditional Chinese rice pudding.Both are also used for domestic cooking purposes.

The experiments were arranged in a randomized complete block design (RCBD) with four replications.The plot area was 15 m2(3 m×5 m) (Fig.1).The treatments included no N fertilization (T1),100%inorganic N fertilizer (T2,the representative of inorganic fertilization),70% inorganic N fertilizer+30% organic fertilizer (T3),40% inorganic N fertilizer+60% organic fertilizer (T4),and 100% organic fertilizer (T5).The total N rate for all treatments (except for T1) was 120 kg N ha-1over the two years.In all treatments,P and K were applied at 60 kg ha-1as P2O5and 100 kg ha-1as K2O,respectively.Nitrogen fertilizer was applied inthree splits (basal fertilizer:tillering fertilizer:grain-filling fertilizer at a ratio of 1:1:1),and the P and K fertilizers were applied once as basal fertilizers.The application amounts of organic fertilizer in T3-T5 were calculated based on the N content of the organic fertilizer,which was consistent with the inorganic fertilizer treatment (T2).To make the P and K rates consistent with T2,calcium superphosphate and potassium chloride,respectively,were added to treatments T3-T5.The total application rates of organic fertilizer in T3-T5 were 1,800,3,600,and 6,000 kg ha-1,respectively.The organic fertilizer used in this study was a plant-based organic fertilizer manufactured by China National Offshore Oil Corporation(CNOOC) Fudao (Hainan) Chemical Co.,Ltd.,and the contents of N,P2O5,and K2O were 2.02,1.9,and 1.69%,respectively.Organic fertilizer was incorporated into the soil during land preparation.

Fig.1 Field trial design (photographed at the heading-early grain-filling stage).T1,no N fertilization;T2,100% inorganic N fertilizer;T3,70% inorganic N fertilizer+30% organic fertilizer;T4,40% inorganic N fertilizer+60% organic fertilizer;T5,100%organic fertilizer.YXHD and SXHN represent the varieties of Youxianghongdao and Suixiangheinuo,respectively.

Pregerminated seeds were sown in nurseries on August 2,2020 and February 24,2021.The transplanting dates were August 23,2020 and March 21,2021.All plots were plowed and puddled before transplanting.Seedlings were transplanted into the paddy soil with a hill spacing of 20 cm×20 cm,with three seedlings per hill.A shallow water layer (3-5 cm) was maintained during the whole rice growth period.Careful management was carried out to fully control diseases and pests,including hanging bird protection nets and laying down anti-rat mulch film at maturity to avoid crop yield losses.

After harvesting the colored rice of SXHN and YXHD,the ridges of the previous plots were maintained to study the residual effect of the partial substitution of inorganic fertilizer.All plots were uniformly planted with YXHD after the plot was re-levelled to a hill spacing of 20 cm×20 cm,with three seedlings per hill.Only inorganic fertilizers were applied in all the plots.The rates were the same at 60 kg N ha-1,60 kg P2O5ha-1,and 100 kg K2O ha-1for all treatments (of N:P:K) in all plots,and only the N fertilizer was applied as basal fertilizer,tillering fertilizer,and grain-filling fertilizer in a ratio of 1:1:1.Phosphorus and potassium fertilizers were used once as basal fertilizer.The sources of N,P,and K were urea (46.4% N),calcium superphosphate (12.0% P2O5),and potassium chloride(60.0% K2O),respectively.

2.3.Data recorded

Soil physical and chemical propertiesSoil samples were collected after rice flowering.A five-point sampling method was adopted to collect the 1-20 cm layer of topsoil.Soil samples were refrigerated and transported back to the laboratory in an ice box after contaminants,such as leaves,roots,and stones,were removed.Then,each soil sample was divided into two parts,and one part was used to determine the physical and chemical properties of the soil after air drying and screening,such as soil organic matter.The other part was stored in a 4°C refrigerator to determine the other physical and chemical properties of the soil.The measurement methods for the basic soil physical and chemical indices were as follows:

Soil water content and electrical conductivity: A soil multiparameter tester (SYS-TRD,China) was used for field measurements.

pH: Deionized water with the CO2removed was used,the water and soil were mixed at a 1:1 ratio,and the mixture was stirred thoroughly.The fully stirred water and soil mixture was centrifuged.After centrifugation,the pH of the upper layer solution was measured by a pH meter(Leici,Shanghai,China).

Total organic carbon (TOC): In an oil bath at 180°C,samples were boiled for 5 min,the soil organic matter was oxidized with potassium dichromate-sulfuric acid solution,and then the excess potassium dichromate was determined by titration with standard 0.2 mol L-1ferrous sulfate.The amount of potassium dichromate consumed was used to calculate the soil organic matter content (Strickland and Sollin 1987).The reactions during oxidation and titration are as follows:

Total nitrogen (TN): Total nitrogen was analyzed using the Kjeldahl method (Bremner and Tabatabai 1972).Digesting samples with concentrated sulfuric acid accelerates the decomposition of organic matter with the help of catalysts (K2SO4,CuSO4,and Se),so that the organic nitrogen is converted into ammonia in the solution.Finally,the distilled ammonia was determined by titration.

Available phosphorus (AP): AP was measured by molybdenum blue spectrophotometry (Liaoetal.2018).

Available potassium (AK): AK was detected by a flame photometer (LY/T 1234-2015 2015).

(7) Cation exchange capacity (CEC): CEC was measured by the BaCl2-MgSO4method (Hendershot and Duquette 1986).

Plant sampling and analysisSix rice plants were sampled at the middle tillering stage (MT),panicle initiation stage (PI),full heading stage (HD),and physiological maturity stage (PM) for growth analysis.The plants were washed,and the number of tillers(including main shoots and tillers) and plant height were recorded.In addition,leaf SPAD values were determined at MT,PI,HD,and PM.After removing the roots,the plants were divided into leaves,stems,and panicles (HD and PM) and dried at 70°C to a constant weight.

Nitrogen accumulation and utilizationAt MT,PI,HD,and PM,grain samples and straw were ovendried at 70°C to constant weight and ground to powder(＜0.149 mm fragments) for TN analysis.The N concentration of the samples was analyzed using the micro-Kjeldahl method.Plant N uptake (PNU) was calculated by multiplying the dry matter yield by TN.The agronomic nitrogen use efficiency (AEN),the ratio of the difference between the grain yields of plants with and without N fertilization and the N rate,or the increase in grain yield per unit of N application,was calculated.The N recovery efficiency (REN,%) describes the nitrogen use efficiency.The fertilizer-N partial factor productivity(PFPN) and the ratio of N fertilization rice yield to N rate were calculated.AEN,REN,andPFPNwere calculated as follows:

AEN=Grain yield increase/Fertilizer N input

REN(%)=100×N uptake from fertilizer N/Fertilizer N input

PFPN=Grain yield/Fertilizer N input

Grain yield and yield componentsAt the maturity stage,six hills of rice samples were taken from each plot to determine the 1,000-grain weight,effective panicle number,spikelets per panicle (filled grain,half-filled grain,and unfilled grain),filled grain rate,and harvest index.After hand-threshing,all spikelets were submerged in tap water to separate the filled grains from the others (halffilled spikelets and unfilled spikelets).Then,a 3-m2area in the center of each plot was selected as a production testing area,and the production was manually harvested and measured.The threshed grains were naturally sundried,and an artificial air separator was used to remove the impurities and empty grains.Grain yield was adjusted to a moisture content of 0.14 g H2O g-1FW.

Anthocyanin analysisAt the maturity stage,30 normal rice panicles were randomly taken from each plot,and after natural shade drying,and they were artificially threshed and screened for filled grains.Then,the grains were dehulled,and 10 g of completely unpolished rice was collected,crushed,and ground using a hybrid ball mill at a frequency of 30.0 s-1for 1 min (MM400,RETSCH,Germany).Afterward,0.1 g of the ground sample from each treatment was stored in a self-sealing bag.A total of 1 mL of extract (mixture of hydrochloric acid and ethanol) was mixed with the ground sample,and the slurry was well homogenized and transferred to an EP tube.After the EP tube was tightly covered,the sample was extracted at 75°C in a constant temperature water bath pot for 20 min and then centrifuged for 10 min under a centrifugal force of 8,000× g at room temperature.The supernatant was taken and analyzed following the instructions for determining plant anthocyanin content in the kit by Suzhou Comin Biotechnology Co.,Ltd.,China.The sampling and analysis were repeated three times.

2.4.Weather data collection

Meteorological data,including solar radiation,temperature,and precipitation,were collected from a weather station (Qi Weather Station,Insentek,China)near the experimental field.Total precipitation,average daily solar radiation,and daily mean temperature during the rice growing seasons in 2020 and 2021 are shown in Fig.2.The total rainfall in the first season was 480.6 mm,which was 11.8% lower than in 2021 (544.9 mm).The average daily solar radiation in 2020 was 10.34 MJ m-2d-1,which was 42.8% lower than in 2021 (18.07 MJ m-2d-1).The daily mean temperatures in 2020 and 2021 were 25.1 and 27.7°C,respectively.

Fig.2 Daily mean temperature (A and B),rainfall (C and D),and solar radiation (E and F) during the rice-growing seasons at Nanbao Town,Lingao City of Hainan Province,China,in 2020 (A,C and E) and 2021 (B,D and F).

2.5.Data analysis

Data were analyzed by analysis of variance using Statistix 9.0.Differences between treatments were separated at probability levels of 0.05,0.01,and 0.001 using the LSD (least significant difference) test.The data were graphically represented with Sigmaplot 12.0.

3.Results

3.1.Soil properties

Substituting different proportions of organic fertilizer for inorganic fertilizers had significant effects on the physical and chemical properties of TN,alkaline hydrolyzable nitrogen (AN),AP,AK,pH,electrical conductivity (EC),and soil organic carbon (SOC),and the trends were consistent between the two years (Table 1).Compared to T2,the soil TN,AN,AP,AK,pH,EC and SOC increased by 24,11,8,39,5,46,and 19% under T5 in 2020,respectively,and by 15,18,18,24,6,25 and 22% in 2021.The soil TN,AP,AK,SOC,cation exchange capacity (CEC),and soil bulk density (SBD) were not significantly different between T4 and T5.In addition,compared to T2,the soil physical and chemical properties under T3 and T4 were improved.Finally,the lowest soil physical and chemical property values were observed under T1 (non-N treated),and there was no interaction between treatment and year(Table 1).

3.2.SPAD value

Partial organic fertilizer substitution for inorganic fertilizers significantly affected the rice leaf SPAD values.The leaf SPAD values under T3 were higher than those of the other treatments at each growth stage (Fig.3).The SPAD values of leaves showed a gradual downward trend from MT to PM,and the same trends were observed in 2020 and 2021.The leaf SPAD values of T3 and T2 were significantly higher than those of T1,T4,and T5 at each growth stage.Furthermore,the leaf SPAD value of T3 was significantly higher than that of T2 at the heading and maturity stage;however,there were no significant differences between T2 and T3 at the middle tillering or young panicle differentiation stages,and these relationships were consistent in the 2020 and 2021 seasons.Compared with T2,the leaf SPAD values of T3 were increased by 2.5 and 1.4% at the physiological maturity stage in 2020 and 2021,respectively.In the whole growth period of rice,the declining rates of the leaf SPAD values of rice in the treatments with organic fertilizer (T3,T4,and T5) were relatively gentle compared with those in the other treatments (T1 and T2).

Fig.3 Changes in leaf SPAD values by substituting inorganic fertilizer with a plant-based organic fertilizer in 2020 (A and B)and 2021 (C and D).T1,no N fertilization;T2,100% inorganic N fertilizer;T3,70% inorganic N fertilizer+30% organic fertilizer;T4,40% inorganic N fertilizer+60% organic fertilizer;T5,100%organic fertilizer.YXHD and SXHN represent the varieties of Youxianghongdao and Suixiangheinuo,respectively.MT,PI,HD,and PM represent the middle tillering,panicle initiation,heading,and physiological maturity stages,respectively.Bars mean SE (n=5).

Fig.4 Effects of the substitution of inorganic fertilizer with plant-based organic fertilizer on plant height (A and B),tiller number (C and D),and aboveground biomass (E and F) in 2020.T1,no N fertilization;T2,100% N inorganic fertilizer;T3,70% inorganic N fertilizer+30% organic fertilizer;T4,40% inorganic N fertilizer+60% organic fertilizer;T5,100% organic fertilizer.YXHD and SXHN represent the varieties of Youxianghongdao and Suixiangheinuo,respectively.MT,PI,HD,and PM represent the middle tillering,panicle initiation,heading,and physiological maturity stages,respectively.Error bars are mean±SE (n=6).Different lowercase letters at the same stage represent significant differences at the 0.05 level.

Fig.5 Effects of the substitution of inorganic fertilizer with plant-based organic fertilizer on plant height (A and B),tiller or panicle number (C and D),and aboveground biomass (E and F) in 2021.T1,no N fertilization;T2,100% inorganic N fertilizer;T3,70% N inorganic fertilizer+30% organic fertilizer;T4,40% inorganic N fertilizer+60% organic fertilizer;T5,100% organic fertilizer.YXHD and SXHN represent the varieties of Youxianghongdao and Suixiangheinuo,respectively.MT,PI,HD,and PM represent the middle tillering,panicle initiation,heading,and physiological maturity stages,respectively.Error bars are mean±SE (n=6).Different lowercase letters at the same stage represent significant differences at 0.05,according to the least significant difference (LSD) test.

3.3.Plant height,tiller or panicle number and aboveground biomass

Partial substitution of organic fertilizer for the inorganic fertilizers significantly affected plant height,the numberof tillers or panicles,and aboveground biomass.The same trends were observed between the two varieties and the two years (Figs.4 and 5).Plant heights at MT and PI under the different treatments were in the sequence of T2=T3＞T4=T5＞T1,while at HD and PM,the sequence was T3=T2=T4＞T5＞T1,and the results were consistent in the two years (Figs.4-A and B,5-A and B).In addition,the average plant height of SNHN was significantly greater than that of YXHD,and the average plant height in 2020 was greater than in 2021.The same results were observed in both varieties.Moreover,the tiller numbers of each cultivar at MT and PI under different treatments were in the sequence of T2=T3＞T4=T5＞T1,while the panicle numbers under T3 at HD and PM were higher than those of the other treatments compared to T2.The panicle numbers of colored rice under T3 at HD and PM were increased by 2.7 and 8.5% respectively,in 2020,and by 6.7 and 7.7% in 2021 (Figs.4-C and D,5-C and D).The tiller number and panicle number in each treatment both showed downward trends from MT to PM,and the results were consistent in 2020 and 2021.Furthermore,the aboveground biomass of T3 in 2020 was significantly greater than that of T2.Compared to T2,the aboveground biomass of YXHD and SXHN under T3 increased by 3.9 and 10.4% in 2020.However,there was no significant difference between T2 and T3 in 2021.

3.4.Nitrogen accumulation and utilization

Partial organic fertilizer substitution for the inorganic fertilizers significantly impacted nitrogen accumulation,AEN,REN,andPFPNat each growth stage.The highest values of these parameters were observed in T3 across the two varieties and two years (Table 2).The nitrogen accumulation levels of T3 at MT,PI,HD,and MS were significantly higher than in the other treatments,and were consistent between the two years.Compared with T2,the nitrogen accumulation of YXHN and SXHN in T3 increased by 9 and 8% in 2020,and by 11 and 8% in 2021.Moreover,compared with T2,theAEN,REN,andPFPNof YXHN for T3 increased in 2020 by 15,18,and 5%,respectively,and SXHN increased by 45,25,and 5% in 2021;while theAEN,REN,andPFPNof YXHN at T3 increased by 35,18,and 8%,respectively,and SXHN by 28,18,and 7%.In addition,there were no interactions forAEN,REN,orPFPNamong the treatment,variety,and year,except thatRENwas significantly influenced by the two-way interaction between treatment and year,and the three-way interaction between season,treatment,and variety significantly influencedPFPN.

3.5.Grain yield and its components

Partial organic fertilizer substitution for the inorganic fertilizers significantly affected grain yield and its components.The yield gradually decreased with an increase in the proportion of organic fertilizer that was applied.The performance of the two varieties was consistent in both years (Table 3).Compared to T2,the grain yields of YXHD and SXHN under T3 were increased by 5 and 8%,respectively,in 2020,and by 13 and 7% in 2021.This was mainly due to the higher panicle number and total above-ground biomass.The effective panicle number of YXHD under T3 was significantly higher than under the other treatments in 2020;however,there were no significant differences among all treatments in 2021.The effective panicle number of SXHN was not significantly different between T2 and T3;however,the effective panicle number of T3 was significantly higher than those of T4 and T5,which was consistent in the two years.In addition,the number of spikelets per panicle was not significantly different between T2 and T3 across the two varieties and two years,except for that of SXHN in 2020.The numbers of spikelets per panicle in T2 and T3 were significantly higher than in T4 and T5.In contrast,the differences in the grain-filling rate,1,000-grain weight,and harvest index among all treatments were insignificant.

The average grain yields of YXHD (3.98 t ha-1) and SXHN (4.08 t ha-1) in 2020 were significantly lower than in 2021 (4.77 and 5.05 t ha-1) (Table 3).No interactions were observed in grain yield,panicles m-2,spikelets per panicle,grain-filling rate,and harvest index between treatment and variety,treatment and year,or treatment and variety and year,except for 1,000-grain weight and harvest index between year and treatment.

3.6.Anthocyanin analysis

With an increase in the proportion of organic fertilizer replacing inorganic fertilizer,the grain anthocyanin contents of YXHD and SXHN showed trends of increasing first and then decreasing in the two years (Fig.6).In addition,the grain anthocyanin content of YXHD was not significantly different between T2 and T3;however,the grain anthocyanin content of SXHN under T3 was significantly higher than that under T2.Moreover,the average grain anthocyanin contents of SXHN in 2020 and 2021 were 1,044.4 and 393.1 μg g-1,respectively,which were significantly higher than those of YXHD (186.7 and 144.3 μg g-1).In addition,the average rice grain anthocyanin content in 2020 was significantly higher than in 2021.

Fig.6 Effects of the substitution of inorganic fertilizer with plant-based organic fertilizer on grain anthocyanin contents of Youxianghongdao (YXHD) and Suixiangheinuo (SXHN) in 2020 (A) and 2021 (B).T1,no N fertilization;T2,100% inorganic N fertilizer;T3,70% inorganic N fertilizer+30% organic fertilizer;T4,40% inorganic N fertilizer+60% organic fertilizer;T5,100% organic fertilizer.Error bars are mean±SE (n=4).Different lowercase letters between different treatments for the same variety represent significant differences at 0.05,according to the least significant difference (LSD) test.The break points in the SXHN plots are 250-900 and 210-290 in A and B,respectively.

3.7.Grain yield of rice in the subsequent season and its components

The application of organic fertilizer significantly improved the yield of the subsequent rice crop,and it increased with an increase in the ratio of organic fertilizers to inorganic fertilizers (Table 4).Compared to T2,rice yields in the subsequent season under the T5 treatment increased by 11% (2020) and 23% (2021),respectively.The main reason for the increase in grain yield was that the aboveground biomass and effective panicle number of the subsequent rice crop under T5 were higher than those under T2.Among them,the effective paniclenumber of the subsequent rice crop under the treatment of T5 was significantly higher than that of T2 in 2020.Still,there were no significant differences between the two treatments in 2021.The number of spikelets per panicle and the seed setting rate of the subsequent rice crop under T5 were significantly higher than those under T3.Other than that comparison,the treatments had no significant differences in 1,000-grain weight or harvest index.

Table 2 Effects of the substitution of inorganic fertilizer with plant-based organic fertilizer on nitrogen accumulation in rice at different growth stages and nitrogen use efficiencies1)

4.Discussion

4.1.Organic fertilizers promoted the sustainable supply of soil nutrients and improved subsequent rice yields

Soil physio-chemical properties and nutritional status were significantly improved with the application of organic fertilizers,which significantly increased the yield of thesubsequent rice crop in the T5 treatment (Tables 1 and 4).Generally,the soil in Hainan Province is acidic,with low organic matter content and weak nutrient supply and retention capabilities,which reflects the soil conditions in most tropical areas (Zhangetal.2003).In Hainan Province,rice is usually intensively rotated with melons and vegetables,which require more nutrients,accelerating the mineralization and decomposition of soil organic matter (Luo and Luo 2001;Lietal.2007;Jiangetal.2019).This process exhausts soil organic matter and depletes most soil nutrients,reducing the soil fertility for future sustainable crop production.Organic fertilizers can have long-term effects on soil nutrient content,which reflects the amount of nutrients available to crops in the current season and contributes to improved nutrient uptake in subsequent rice seasons (Gutseretal.2005;Celaetal.2011;Pengetal.2023).These findings are consistent with our results that T5 outyielded T2,as shown in Table 4.

Table 3 Effects of the substitution of inorganic fertilizer with plant-based organic fertilizer on grain yield and its components in colored rice

4.2.Organic-inorganic fertilizer system enhanced nitrogen accumulation and utilization

Table 4 Effects of substituting inorganic fertilizer with plant-based organic fertilizer on rice grain yield and its components in the subsequent season

Coupling of 30% inorganic nitrogen with organic fertilizer(T3) significantly increased nitrogen accumulation,REN,AEN,andPFPNcompared with the application of only inorganic fertilizer (T2) (Table 2).On the one hand,this may be because inorganic fertilizers release nutrients rapidly,and organic fertilizers release nutrients more slowly and steadily,which results in more of the nitrogen in the inorganic fertilizers being absorbed and utilized by rice (Roba 2018).Applying organic fertilizers enhances the preservation soil nutrients and eliminates the downward movement of minerals,thereby reducing nitrogen leaching (Caietal.2016).In addition,partial organic fertilizer substitution for inorganic fertilizer can improve nitrogen use efficiency compared to the application of only inorganic fertilizer,which may be related to improving soil quality and increasing nitrogen retention capacity (Mehasenetal.2012),and was consistent with our results.Studies have shown that partial organic fertilizer substitution for inorganic fertilizers improved the characteristics of the soil nitrogen supply,increased the content of soil mineral nitrogen,promoted the rapid growth of microorganisms,and correspondingly improved the ability of microorganisms to maintain nitrogen nutrients (Baaruetal.2007;Maetal.2010).Moreover,some studies have reported that organic fertilizers combined with nitrogen fertilizers could balance the crop nutrient supply and demand,and improve plant nutrient assimilation and nitrogen use (Liuetal.2008;Shangetal.2014).Therefore,the 30% organic fertilizer substitution for inorganic fertilizer is a highly efficient nitrogen management model for colored rice.

4.3.Synergetic effects of organic-inorganic fertilizers on grain yield and anthocyanin content in colored rice

In the present study,30% inorganic N fertilizer substitution with organic fertilizer had significant effects on grain yield and grain anthocyanin content of colored rice;and the T3 treatment had the best performance (Table 3;Fig.6).It significantly improved root morphological traits (root length,surface area,diameter,and volume),which was consistent with the results of Iqbal (2019)and Hayatu (2022).This improvement may be related to the significantly higher nitrogen accumulation and nitrogen use efficiency with the T3 treatment than the other treatments (Table 3).Studies have documented that grain anthocyanin accumulation in colored rice was regulated by grain nitrogen content (Chenetal.2016).In addition,organic nitrogen application upregulated the expression of genes related to anthocyanin synthesis(Fongfonetal.2021).However,compared to T2,both the yield and the grain anthocyanin content of the T5 treatment showed downward trends.The application of an augmented amount of organic nutrients substituting for the nutrients in inorganic fertilizers led to a downward trend in the number of effective panicles per unit area.This may be due to the limited release of nutrients in the soil during decomposition when there is more organic fertilizer in the soil,which affects the root development and rice production (Xuetal.2008).Nitrogen from organic fertilizers often shows little effect on crop growth in the season of application due to the slow-release characteristics of organic fertilizers (Gutseretal.2005).In addition,the polysaccharides such as hemicellulose and cellulose in plant-based organic fertilizers and increased levels of aromatic compounds in organic fertilizers are known to decelerate the mineralization process (Sradnick and Feller 2020).

The grain anthocyanin of SXHN in 2020 was significantly higher than that in 2021 (Fig.6).Low temperature and short periods of sunshine are beneficial for grain anthocyanin accumulation in colored rice(Zhang 2006).Studies have shown that light conditions regulate the expression of anthocyanin synthesis genes,and weak light conditions reduce the synthesis of grain anthocyanin (Albertetal.2009).The solar radiation and daily average temperature during the grain-filling period in 2020 were significantly lower than those in 2021 (Fig.2).Therefore,the difference in grain anthocyanin of SXHN between the two years may be related to temperature and solar radiation.In addition,the average grain yields of YXHD (3.98 t ha-1) and SXHN (4.08 t ha-1) in 2020 were significantly lower than in 2021 (4.77 and 5.05 t ha-1,respectively) (Table 3).Studies have reported that low solar radiation during the rice growth stage can interfere with the re-accumulation of photosynthetic products,thereby reducing grain yield (Mauroetal.2011;Liuetal.2019).In this study,the total solar radiation increased by 66.1% in 2021 compared to 2020,and the grain-filling rate in 2021 was significantly higher than that in 2020(Fig.2;Table 3).Therefore,the differences in grain yield of the two varieties between the two years may be closely related to the difference in solar radiation.

5.Conclusion

In terms of grain yield,grain anthocyanin content,and N use efficiency of colored rice in the current season under organic fertilizer incorporation,the best performance was observed under the T3 treatment.The partial substitution of inorganic fertilizer with organic fertilizer improved the soil physio-chemical properties and thus increased the rice grain yield in the subsequent seasons.The highest grain yield of the subsequent rice crop was observed under the T5 treatment.These results suggested that the application of plant-based organic fertilizers can sustain the production of colored rice in tropical regions.However,how long the residual effect of organic fertilizer lasts,and the mechanisms underlying the residual effect of plant-based organic fertilizer,especially the microbial mechanisms,remain unknown and should be addressed in future studies.

Acknowledgements

This work is supported by the National Natural Science Foundation of China (32060430 and 31971840) and the Research Initiation Fund of Hainan University,China(KYQD (ZR) 19104).

Declaration of competing interest

The authors declare that they have no conflict of interest.