The underlying mechanism of variety–water–nitrogen–stubble damage interactions on yield formation in ratoon rice with low stubble height under mechanized harvesting

Jingnan Zou ,Ziqin Pang ,Zhou Li ,Chunlin Guo ,Hongmei Lin ,Zheng Li ,Hongfei Chen,Jinwen Huang,Ting Chen,Hailong Xu,Bin Qin,Puleng Letuma,4,Weiwei Lin,Wenxiong Lin,#

1 College of Life Sciences, Fujian Agriculture and Forestry University (FAFU)/Fujian Key Laboratory for Agroecological Processes and Safety Monitoring, Fuzhou 350002, China

2 College of Agriculture, FAFU/Fujian Key Laboratory for Crop Physiology and Molecular Ecology, Fuzhou 350002, China

3 Institute of Oceanography, College of Geography and Oceanography, Minjiang University, Fuzhou 350108, China

4 Crop Science Department, The National University of Lesotho, Roma 00100, Lesotho

5 The Agricultural Extension Station of Pucheng County, Nanping 353400, China

Abstract Agronomic measures are the key to promote the sustainable development of ratoon rice by reducing the damage from mechanical crushing to the residual stubble of the main crop,thereby mitigating the impact on axillary bud sprouting and yield formation in ratoon rice. This study used widely recommended conventional rice Jiafuzhan and hybrid rice Yongyou 2640 as the test materials to conduct a four-factor block design field experiment in a greenhouse of the experimental farm of Fujian Agricultural and Forestry University,China from 2018 to 2019.The treatments included fertilization and no fertilization,alternate wetting and drying irrigation and continuous water flooding irrigation,and plots with and without artificial crushing damage on the rice stubble. At the same time,a 13C stable isotope in-situ detection technology was used to fertilize the pot experiment. The results showed significant interactions among varieties,water management,nitrogen application and stubble status.Relative to the long-term water flooding treatment,the treatment with sequential application of nitrogen fertilizer coupled with moderate field drought for root-vigor and tiller promotion before and after harvesting of the main crop,significantly improved the effective tillers from low position nodes. This in turn increased the effective panicles per plant and grains per panicle by reducing the influence of artificial crushing damage on rice stubble and achieving a high yield of the regenerated rice. Furthermore,the partitioning of 13C assimilates to the residual stubble and its axillary buds were significantly improved at the mature stage of the main crop,while the translocation rate to roots and rhizosphere soil was reduced at the later growth stage of ratooning season rice. This was triggered by the metabolism of hormones and polyamines at the stem base regulated by the interaction of water and fertilizer at this time. We therefore suggest that to achieve a high yield of ratoon rice with low stubble height under mechanized harvesting,the timely application of nitrogen fertilizer is fundamental,coupled with moderate field drying for root-vigor preservation and tiller promotion before and after the mechanical harvesting of the main crop.

Keywords: mechanized harvesting,ratoon rice,rice stubble,yield attributes

1.Introduction

The ability of the rice plant to regenerate new tillers after harvest is called ratooning,and it has the potential to increase rice production per unit land area (Linet al.2015;Lin 2019). With its low production cost,ratoon rice has become an important component of the rice cropping system in central and southeastern China.In tracing the development of ratoon rice cultivation technology in China,we noted that from the 1970s to the beginning of this century,the ratoon rice with hand harvesting high-cut stubble was mainly recommended,and cases with higher yields were often recorded(Chauhanet al.1985). However,the implementation of reforms and the opening-up policy in China in the 1980s promoted a massive migration of rural laborers to the cities,leading to seasonal labor shortages in agriculture.As a result,this conventional ratoon rice practice is no longer suitable for the socioeconomic situation in China.Mechanized harvesting has become the inevitable option for the sustainable development of ratoon rice (Peng 2014;Lin 2019;Wanget al.2019;Huanget al.2021;Zhenget al.2022). However,mechanized harvesting seriously damages the residual stubble of the main crop,resulting in yield losses of the ratoon crop of 60–70%(Yuet al.2013;Zhenget al.2022). Meanwhile,it often causes irregular growth and heading in the ratoon crop,leading to a reduced head rice rate,and consequently diminished grain quality of the ratoon crop (Huanget al.2021;Zhenget al.2022). In dealing with these issues,based on the characteristics of light and temperature resource endowments in the different production areas of the southeastern China,the mechanized harvesting technology of ratoon rice with a low stubble height(MHTRR) was established to support the high and stable yield formation. The MHTRR mainly consists of three key sequential steps: (1) The proper nitrogen fertilizer rate is shifted from the early growing stage of the main crop to 7–10 d before its harvesting for root-vigor preservation under the premise of constant total nitrogen fertilization amounts;(2) this is followed by the timely application of nitrogen fertilizer for promoting tiller and panicle regeneration after the main crop is harvested;(3) this is coupled with moderate field drought (dried to 33% soil water content) and mechanically cut low stubble (15–25 cm) to prevent crushing and sinking damage to the rice. Consequently,the yield of the ratoon crop in large areas has been significantly improved,and the highest annual yield of the main crop and its ratoon crop can exceed 18 t ha–1,with the yield of ratoon crop reaching more than 9 t ha–1,which is a historic breakthrough under the mechanized harvesting of ratoon rice (Yuanet al.1996;Lin 2019;Huanget al.2021). However,this technological extension is facing the problem of high yield instability in practice,which still needs to be resolved (Lin 2019;Wanget al.2019;Huanget al.2020;Zhenget al.2022).

Previous studies proposed that many factors could restrict the high and stable yield formation of the ratoon crop,such as the transplanting date,the height of the residual stubble from main crop,water and fertilizer management of the main crop and ratoon crop,and others(Donget al.2017;Chen and Wang 2018). Chauhanet al.(1985) analyzed the status of research worldwide and suggested that the grain yield of the ratoon crop varied between 0.1–8.7 t ha–1,with an average of 1.54 t ha–1. Xie(2010) made an on-farm inquiry on different varieties of ratoon rice and fertilizer management,and suggested that the yield of the ratoon crop ranged from 2.79 and 8.74 t ha–1in Fujian Province,southeastern China. Bahar and De Datta (1977) documented that the yield of the ratoon crop was around 0–2.6 t ha–1under different transplanting densities and residual stubble heights coupled with different water and fertilizer treatments,suggesting that a large variation in yield performance exists;therefore,the high-yielding potential still needs to be further exploited.Our previous studies also suggested that there are two key fertilization times that can improve the grain yield of the ratoon crop (Yanget al.2009;Linet al.2015). One is bud and root vigor preserving fertilizer that is applied at about 10 d before the harvest of the main crop,and the other is tiller-promoting fertilizer that is applied 3–7 d after the harvest of the main crop. Studies have also found that the effect of the dual-fertilization approach of bud and root vigor preserving fertilizer and tiller-promoting fertilizer on the yield of ratoon crop also depends on the nitrogen nutrition status of the main crop at the maturity stage,as well as the residual stubble height and its quality after harvesting (Wanget al.2019). We also found that the ratoon crop of low-cut stubble is mainly regenerated from the first and second nodes at the base. Applying a large amount of fertilizer to promote buds before the harvest of the main crop inevitably accelerates the emergence and elongation of axillary buds,which can easily lead to cutting damage in the stubble that is left after the harvest of the main crop. Furthermore,inappropriate application of bud-promoting and root vigor preserving fertilizer in the later stage of the main crop would also lead to significantly increased greenhouse gases,especially N2O emission,reduced nitrogen utilization efficiency,and effects on rice quality (Liet al.2020;Linet al.2022a). Therefore,it is not necessary to apply fertilizer for bud-promotion,especially under a low-cut stubble regime (Linet al.2015,2019,2022b;Lin 2019).

Aside from rational fertilization,water management also has a significant influence on the yield formation in the regeneration season. We found that there was an interaction between fertilization and irrigation mode on yield formation of the ratoon crop,especially at the later growth stage of the main crop. If the field soil was continuously flooded at the later stage of the main crop,it could result in crushing and sinking damage to the residual rice stubble by mechanical harvesting,in turn reducing the yield of the regenerated rice (Chenet al.2023). Linet al.(2015) proposed a water management pattern of planting ditch irrigation and two field soil drying operations in the main crop,which could reduce the mechanical crushing damage to the residual stubble during the harvest,and maintain higher root vitality and axillary bud sprouting activity in the later growth stage of the main crop,thereby significantly increasing the yield of the main crop and the ratoon crop (Linet al.2015).Moreover,moderate field drought at this time was also conducive to grain-filling,which was responsible for a greater abscisic acid (ABA) content in the ratoon crop under the field drought condition (Linet al.2015). Chenet al.(2023) also studied the effects of water and fertilizer management measures on the ratoon crop,and found that the application of tiller-promoting fertilizer linked with the irrigation pattern in the intermittent dry and wet mode could increase the availability of nutrients in the rhizosphere soil,promote the formation of new roots,the sprouting and elongation of axillary buds,and ultimately significantly improve the number of effective tillers and grain yield in the regenerating season rice (Chenet al.2014,2016). However,the results of those studies were mainly obtained under single factor conditions.The interactions among different levels of multiple factors and the mode of action on yield attributes of the regrown rice and its action mechanism still remain unknown,which prevents the scaling-up of the MHTRR.Accordingly,the aims of the present study were twofold:(1) The interactions of multiple factors,including variety,fertilization mode,water management and rice stubble standing status,were assessed on axillary bud sprouting and tiller regeneration,effective panicles and grain yield formation of regenerated rice from mechanically lowcut stubble;(2) on this basis,the effects of nitrogen fertilizer regulation on the accumulation and distribution of13C assimilates in the above-and below-ground parts of regenerated rice and their physiological mechanisms were analyzed. The results of this study will provide the theoretical basis for further understanding the formation of a high and stable yield,optimizing cultivation technology and promoting the sustainable development of regenerated rice from mechanically low-cut rice stubble.

2.Materials and methods

2.1.Effects of water-fertilizer coupling on the yield of ratoon rice with low stubble height

Materials and cultivation overviewThis experiment was conducted in a simple greenhouse of the experimental farm of Fujian Agriculture and Forestry University(FAFU),Fuzhou (26.08°N,119.31°E),Southeast China in 2018–2019. The greenhouse with a movable shelter was opened on sunny days and covered on rainy days to prevent rain irrigation from affecting the alternation of dry and wet conditions,and the climatic conditions are shown in Appendix A. Jiafuzhan (JFZ) conventional rice and Yongyou 2640 hybrid rice (YY2640) were selected as the test materials. In 2018,the seeds were sown and raised in a wet nursery on March 24. The seedlings were transplanted on April 24. The soil had a clay loam texture with a pH of 6.18,organic matter of 29.5 g kg-1,total N of 1.86 g kg-1,available N of 92.5 mg kg–1,total P of 0.62 g kg–1,available P of 42.8 mg kg-1,total K of 1.40 g kg–1and available K of 90.1 mg kg-1. The main crops of JFZ and YY2640 were fully headed on June 25 and July 10,and were harvested on July 26 and August 15,respectively.The stubble height of the two regenerated rice accessions was 20 cm,and the full heading dates of the JFZ and YY2640 ratoon crop accessions were on September 10 and 20,while harvesting was on October 10 and October 15,2018,respectively. Weeds were removed by hand in the main and ratoon cropping seasons. Pests and diseases were controlled by chemicals three times in the main season and only one time in the ratoon season to avoid yield losses. The same field experiment was repeated in 2019,and the heading and harvesting stages of the main crop and its ratooning rice in all treatments were controlled to happen on the same dates as in 2018.

Experimental designA four-factor split block design was adopted in the experiment,with variety as main plot,nitrogen application rate as subplot,irrigation method as split plot,and artificial crushing damage to the rice stubble as the re-split plot (CD,crushing damage zone to residual stubble;NC,non-crushing damage zone to the residual stubble). The experiment was repeated three times,the area of each block was 32 m2,of which each plot was 2 m2(the small bund between the plots was not included),and the blocks were randomly arranged.Fifty plants were planted in each plot,and the planting density in each plot under each treatment was spaced at 20 cm×20 cm with one seed per hill for JFZ and YY2640.Each plot in the block was isolated by a bund of 30 cm high and 40 cm wide,and covered with a black waterproof plastic film installed to a depth of 20 cm below the soil surface to minimize leakage between plots (Fig.1). Two nitrogen treatments,i.e.,no nitrogen fertilizer (NF) and a supplemental nitrogen fertilizer (SF) pattern were adopted,of which SF was applied as pure nitrogen at 150 kg ha–1,and the N:P:K was 1:0.5:0.8. All phosphorus fertilizer was applied on the 3rd day after the main season rice was harvested,while nitrogen fertilizer and potassium fertilizer were applied at a ratio of 1:1 at 3 and 15 d after the main crop harvesting,respectively. Other treatments were the same as those of the main crop. Water treatment was applied in two patterns of continuous flooding (CF)with about a 3 cm water layer on the field during whole growing stage,and alternative wetting and drying (AWD)of the field soil in the later growth stage of the main crop.In the AWD treatment,the field soil water was naturally dried and reduced to–15 kPa soil water potential within the 5–20 cm plough layers,and then re-irrigated. So the cycle treatment spanned the whole growing stage,during which the field soil was seriously dried to 33% soil water content or to–30–40 kPa soil water potential at 7–10 d before harvesting the main crop. The changes in soil moisture content after heading of the main season rice under the irrigation treatment and the alternate dry wet treatment are shown in Appendix B. A soil moisture sensor (Granular Matrix Senor,Irrometer Co.,Riverside,CA) was used to monitor the soil water potential every day,and the dried soil was re-watered when it reached the threshold value indicated in the design requirements.During the treatments,the roof of the simple greenhouse was covered with plastic film during rainy days,otherwise the roof was opened throughout the growth period. To explore the effects of different levels of water and fertilizer coupling on axillary bud spouting and regenerative tiller formation of axillary buds in the residual stubble,the cut heights of both conventional rice JFZ and hybrid rice YY2640 were 20 cm,and the method of artificially simulated crushing damage to rice stubble was applied to cause different degrees of damage and lodging stubble in the plots. The other field management practices of the main crop and regenerated rice were the same as that of conventional cultivation. We repeated the experiment outlined above in 2019.

Fig. 1 Schematic diagram of the field experiment design and plot arrangement. YY2640,hybrid rice cultivar;JFZ,Jiafuzhan,conventional rice cultivar. NF,no nitrogen fertilizer treatment,SF,supplying nitrogen fertilizer. CI,conventional irrigation;AWD,alternating wetting and drying irrigation. NC,the zone of non-crushing damage to residual stubble;CD,the zone of manual crushing damage to residual stubble.

2.2.Nitrogen regulation of 13C assimilate distribution in regenerated rice

Materials and cultivation overviewBased on the results mentioned above,a pot experiment was conducted in 2019 at the pilot farm of Fujian Agriculture and Forestry University to further study the regulatory effect of nitrogen fertilizer on the13C assimilate distribution in regenerated rice under the optimal treatment of AWD and normal residual stubble in standing status when cut to a low height of 20 cm as in the field test in 2018. The test varieties used were the same as in 2018. Seeds of the two rice cultivars were sown and raised in a wet nursery on March 24,the seedlings were transplanted into pots on April 24,and each pot had four hills with one seed per hill. The size of the pot was 30 cm high,and the inside diameters of the bottom and top were 30 and 23 cm,respectively. Each pot contained 12 kg of sifted soil collected from the 0–20 cm plough layers in the same experimental field. The soil had a clay loam texture with a pH of 6.18,organic matter of 29.0 g kg-1,total N of 1.88 g kg-1,available N of 94.3 mg kg–1,total P of 0.64 g kg–1,available P of 34.2 mg kg–1,total K of 1.45 g kg–1,and available K of 107.5 mg kg–1. The main crops of JFZ and YY2640 were fully headed on June 25 and July 5,and harvested on July 28 and August 12,respectively. The stubble heights of the two ratoon rice accessions were both 20 cm,and the full heading dates of the JFZ and YY2640 ratoon crop were on September 10 and 20,and harvesting was conducted on October 8 and October 12,2019,respectively. The same nitrogen treatment levels (NF and SF) were set for each variety,following the method outlined above. The total amount of fertilizer was converted to 1.32 g N/pot of pure nitrogen. The AWD irrigation mode was adopted for water management in rice ratooning as described above under the normal stubble with standing status. There were 20 pots for each treatment. Pests and diseases were controlled with chemicals three times during the whole growth duration to avoid yield losses.

The13CO2pulse labeling test method was as described by Li (2020) with a slight modification. The13CO2pulse labeling was conducted during the time course from 8:30 to 12:30 a.m.on a sunny day without rain after the full heading of the JFZ and YY2640 main and ratoon rice accessions. To avoid the influences of high temperature and fog on photosynthesis,the wall of the cylinder was sprayed with an anti-fogging agent and the cylinder was suspended in an ice bag in advance,and the cylinder column was sprayed with water every half hour. The labeling procedure was conducted as follows. Before labeling,the rice plants were allowed to photosynthesize for a certain time course in the labeling chamber,so as to consume12CO2and improve the absorption and assimilation rate of13CO2. Then the fan in the cylinder was turned on,a hose with a valve was used to close the loop connecting the suction pump CO2analyzer,and air was pumped through the inlet/injection hole to monitor the concentration of CO2in the labeling room. When the concentration had dropped to about 80 μL L–1,13CO2gas was injected with a syringe (>99%,produced by Shanghai Chemical Research Institute,China),the CO2concentration in the cylinder was controlled in the range of 350–380 mg kg–1,and the whole labeling process lasted for four hours. Finally,ordinary12CO2gas was injected into the cylinder to improve the fixation rate of13C,thus reducing the loss of13CO2. When the CO2concentration dropped to about 80 μL L–1,the labeling chamber was removed,and the whole labeling process was ended. At the same time,the ambient natural abundance of13C was determined by using the same potted plants placed 10 m away from the labeled pot as a control sample (Zanget al.2019).

Determination of yield and its componentsTwentyfive regenerated rice plants were sampled from each plot at the maturity stage of the ratoon crop. From these plants,the effective panicles generated from different nodes,grains per panicle,seed-setting percentage and 1,000-grain weight were determined.

Sample collectionLabeled and unlabeled rice plants and soil were collected at the full heading stage of the main crop and the ratoon crop. In each treatment,three pots were emptied,the rhizosphere soil was scraped off with a sharp blade,and the roots were removed. The soil samples were placed in a cool place for air drying and screened through a 100-mesh sieve,then stored at a low temperature until use. Subsequently,the rice plants were rinsed with running water and all fresh roots and above-ground plant parts were sampled. Notably,at the maturity stage of the main crop,the above-ground shoots of the main crop were divided into two parts,i.e.,the remaining stubble,which was cut based on the cut height design (20 cm),and the non-remaining stubble parts (harvested stubble parts).They were then unsheathed into stems,sheaths,leaves and panicles;among them,the flesh stems,sheaths,leaves and panicles of one plant sampled from each pot in each treatment were wrapped in tin foil,frozen in liquid nitrogen and stored in a–80°C refrigerator for the later determination of plant hormones and polyamines. At the full heading stage of the ratoon crop,the rice stubble of the ratoon crop was separated into the stems and sheaths of the main crop residual stubble and the regenerated stems,sheaths,leaves,panicles and the roots. These were packaged separately in cowhide bags,placed into an oven at 105°C for 30 min,and dried at 70°C to a constant weight for about 48 h. Finally,they were ground and screened through a 100-mesh sieve for the determination of soluble sugar and starch in the different rice stubble samples.

Test items and methodsThe samples from different parts of the above-ground screened soil and rice plants were used to detect the ratio of13C to12C,and the δ13C value of each sample was calculated after comparison with the international standard Pee Dee Belemnite (PDB)(Aitken 2020) employing an elemental analyzer/pyrolysis/isotope ratio mass spectrometer (EA/HT-IRMS,Thermo Fisher Scientific,Inc.,Bremen,Germany). The total carbon content (C%) was calculated by comparing the peak area of the sample and the working standard sample.

The net input content of13C in each part of the plant and soil was calculated by the formula:

where13Cncis13C net input content (mg/pot) of labeled samples,AT%13Clis13C abundance (%) of labeled samples,AT%13Culis13C abundance (%) of unlabeled samples,and TCis total carbon content (mg/pot) of labeled samples. The distribution rate of13C in each part of the plant and soil was calculated by the formula:

where D13Ciis the allocation rate of each part (%),T13Cncis the sum of the13C net input content in the above-mentioned roots and soil of the labeled plant samples (mg/pot).

The contents of soluble sugar and starch were determined by grinding and screening the rice stems and sheaths of the rice stubble following the method of Zou(2001). Soluble sugar was extracted by a boiling water bath,and 9.2 mol L–1HClO4was added into the remaining residue to extract the starch. The absorbance values of both were measured by the anthrone colorimetric method at the wavelength of 630 nm,and the standard linear equation of absorbance and sugar content was obtained using a sucrose standard solution. Then,the concentrations of soluble sugar and starch in the sample were converted,and the units were mg g–1DW.

High performance liquid chromatography (HPLC) was used to determine the zeatin riboside (ZR),gibberellin(GA3),abscisic acid (ABA) and indoleacetic acid (IAA) in the root and shoots (stubble and non-stubble parts) as described above. A fresh sample was ground into powder in liquid nitrogen,a 1.0 g portion was accurately weighed and added into 10 mL of pre-cooled 80% chromatographic grade methanol,mixed thoroughly,placed in a 4°C shaker for more than 16 h,and centrifuged at 4°C and 5,000 r min–1for 10 min. The supernatant was absorbed and stored in the refrigerator in the dark,and the residue was extracted three times. Then the combined extraction solution was purified according to the method described by Chen and Yong (2005). The test solution was filtered through a 0.22 μm nylon filter membrane and then injected into a Shimazu LC-20AD high performance liquid chromatography for detection.

The UV detector model was a Shimadzu SPD-20A.The determination was performed on an Inertsustain C18(4.6 mm×250 mm,ID 5 μm) with the running parameters of column temperature chamber 35°C,flow rate 0.8 mL min–1,sample size 20 μL,and detection wavelength 254 nm. The mobile phase consisted of A: acetonitrile(chromatography-pure) and B: 0.6 % acetic acid. The chromatography-grade standard reagents of ZR,GA,ABA and IAA were purchased from Sigma-Aldrich (USA).

Similarly,the contents of putrescine (Put),spermine(Spm),spermidine (Spd) and other polyamines in roots and rice stubble were analyzed by HPLC. The extraction and analysis followed the method of Duanet al.(2008) with slight modifications. The fresh sample was ground into powder in liquid nitrogen,a 1 g portion was accurately weighed and added to 3 mL 5% (v/v) precooled perchloric acid (PCA),placed in a 4°C shaker for 1 h,and centrifuged at 4°C at 15,000 r min–1for 30 min. The supernatant was used to determine the free and bound polyamines,and the precipitate was used to determine the insoluble bound polyamines. The solution was benzoylated immediately or stored in a–20°C refrigerator.

Benzoyl chloride was used as the benzoyl chloride reagent. Briefly,1 mL supernatant was fully mixed with 2 mL 2N NaOH and 20 μL benzoyl chloride stock solution in a vortex,incubated at 37°C for 30 min,and then 4 mL saturated NaCl solution was added to terminate the reaction. Then,6 mL of pre-cooled anhydrous ether was added to extract the benzoylated polyamides,and the sample was sealed and mixed gently. After centrifugation at 1,500×g for 5 min,3 mL of the ether phase was taken and freeze-dried. Finally,1 mL of chromatogram-grade methanol was added to vortex dissolve,and the solution was filtered using a 0.22 μm filter membrane,and then tested by HPLC.

The determination was performed on an ODS-C18 reverse column (4.6 mm×250 mm,ID 5 μm) with a mobile phase consisting of 64% methanol at 25°C and a flow rate of 0.5 mL min–1. The UV detector was used for detection at 254 nm.

The derivation and determination methods of the combined type and bound type polyamines were the same as those for the free polyamines after acid hydrolysis at 110°C. The final content of the combined type of polyamines was obtained by subtracting the content of free polyamines from the content of polyamines measured after acid hydrolysis. The Put,Spd and Spm chromatographic standards were obtained from Sigma-Aldrich. The contents of Put,Spd and Spm were quantified by the external standard method. The samples were analyzed with different gradient solutions after the benzoylation reaction according to the above method,and standard curves were drawn according to peak area and concentration. The polyamine content was expressed as micromoles/gram fresh weight (μmol g–1FW).

2.3.Statistical analysis

All data collation and variance analyses were conducted using Microsoft Excel 2013 and SPSS19.0 statistical software. LSD was employed for the inter-group difference test and Origin8.0 was used for mapping.DPS7.05 software was used to analyze the variances of the interactions among varieties,fertilization and water treatments,as well as stubble damage status,on yield and its components in the regenerated rice.

3.Results

3.1.Effects of different treatments on yield and its attributes in the ratoon crop with low stubble height

Analysis of yield and its components in the ratoon cropThe variance analysis showed that the yield and its components under the different treatments had no significant differences in the two-year experiments.Therefore,the mean values of the results for the two years were adopted for the statistical analysis (Table 1).The results revealed that different varieties (V),nitrogen fertilizations (N) and irrigation modes (I) had effects on the yield and its attributes,except for the crushing and non-crushing damage treatments (C),and there were interactions among the four treatment factors on yield and its components (Table 1). Regarding the effects of the individual factors,regardless of the conditions,the yield of hybrid YY2640 regenerated rice was higher than that of the conventional JFZ regenerated rice accession,which was mainly related to the factors of more effective panicles and grains per panicle in the YY2640 regenerated rice. In terms of nitrogen fertilizer treatment,the main crop factors leading to the yield gap in ratoon crop accessions were the effective panicles,grains per panicle and seed-setting percentage,among which the SF was more effective than the NF. The yield gap in ratoon crop accessions caused by different irrigation modes (AWD and CF) was related to the different seed setting percentages and effective panicles,of which the AWD treatment exhibited the better yield increase compared with CF. The single-factor effect of artificial crushing damage to rice stubble was not significant,which was mainly due to the interaction of the artificial crushing damage factor with the other three factors,implying that the consequences of crushing damage could be offset by other treatment factors and their effects. Additionally,in the two-factor interactions,there were different extents of interactions in V×N,V×I,V×C,N×I,N×C and I×C. Among them,the V×N interaction resulted in different yields between the two regenerated rice accessions,mainly due to differences in grains per panicle,seed setting percentage and effective panicles. As a whole,the grain yield performance showed the following sequence: YY2640×SF>YY2640×NF>JFZ×SF>JFZ×NF.

Table 1 Effects of water–fertilizer coupling on yield and its component factors (as their two year averages) of the ratoon crop in the non-crushing damaged and crushing damaged zones of the residual stubble of the main crop1)

The interaction between varieties and irrigation mode (V×I) also had significant effects on yield and its components,among which grains per panicle exhibited the greatest response to the V×I interaction,and the yield performance of the two regenerated rice varieties was in the order of: YY2640×AWD>YY2640×CF>JFZ×AWD>JFZ×CF.The interaction between different varieties and artificially crushing damage to the rice stubble (V×C) had no significant effect on effective panicles,but it had significant effects on the other yield attributes,especially on grains per panicle. For this interaction,the yield sequence was: YY2640×NC>YY2640×CD>JFZ×NC>JFZ×CD.Further analysis revealed that the interaction of nitrogen fertilizer treatment with irrigation mode (N×I) had the greatest effect on effective panicle,thereby impacting on the yield of the regenerated rice. The same trend was also observed in the cases of N×C and I×C,illustrating that the yield performance was in the sequence of:SF×AWD>SF×CF>NF×AWD>NF×CF. Moreover,the best yield performance of the regenerated rice accessions under the three-factor interactions were under YY2640×SF×AWD and YY2640×SF×CF,while the lowest was in the SF×CF×CD interaction.

Under the four-factor interactions,there were insignificant differences in grain yields of the ratoon crop between YY2640×SF×AWD×NC and YY2640×SF×AWD×CD,as well as between JFZ×SF×AWD×CD and JFZ×SF×AWD×NC.More interestingly,under the SF×AWD interaction,the artificial crushing damage to the residual rice stubble appeared to promote effective panicles to a certain extent. In addition,the yields of the two regenerated rice accessions in the artificial crushing damage zone of the residual stubble were slightly higher than that in the non-crushing damage zone of the residual stubble. This was attributed to reducing the impact on the residual rice stubble due to crushing damage,consequently increasing the effective panicles and grains per panicle. This enhancement in the grain yield of theregenerated rice resulted from low-cut stubble under the sequential treatments with nitrogen supplies for root-vigor preservation and tiller promotion,followed by the water treatment with AWD before and after harvesting of the main crop,in which moderate field drought was applied 7–10 d before harvesting the main crop.

Contributions of axillary bud sprouting and effective tiller formation at different nodes to yield per plant of the ratoon cropThe contributions of the effective tillers regenerated from the basal notes (total of node 1 and node 2) to yield per plant in the YY2640 and JFZ ratoon crop accessions was further determined under the fourfactor interaction. The results indicated that insignificant differences were found in the contribution rates of effective tillers regenerated from node 1 and node 2 in the stem bases to the grain yield per plant of the YY2640 ratoon crop by 86 and 85.3% under SF×AWD×CD and SF×AWD×NC,respectively. YY2640 showed much higher contribution rates of effective tillers from low position nodes under SF×AWD×CD and SF×AWD×NC than in SF×CF×NC and NF×AWD×NC,as well as in the NF×CF×NC and NF×AWD×CD conditions. The same trend was observed in the JFZ ratoon crop (Table 2),suggesting that a fertilization and water interaction significantly impacted the recovery of the crushing damage to the rice stubble,which in turn affected effective panicles and the grain yield of the ratoon crop. This finding further explains why AWD could reduce the rice yield loss caused by crushing damage to the residual rice stubble,especially coupling with the application of a moderate field soil drying as well as preserving root activity and promoting tiller fertilizer used before harvesting of the main crop. This was mainlydue to effectively promoting the tillers produced from the low position node tillers to form the higher productive panicles and grains per panicle under the water and fertilizer interaction. As a result,the same yield,or even a slightly higher yield,can be obtained in the artificial crushing damage zone as in the non-crushing damage zone (Table 2),which deepens our understanding of the above research results.

Table 2 Contribution of the tiller panicles at different nodes of different ratoon crop accessions to grain yield per plant under different water and fertilizer treatments

3.2.Nitrogen regulation of photoassimilate translocation and allocation of the ratoon crop with low stubble height

Translocation and partitioning of 13C assimilatesIn 2019,the stable isotope tracer method was used to further analyze effects of fertilization treatments. This included the effects of SF and NF for root-vigor preservation and tiller promotion on dry matter accumulation and distribution as well as its physiological mechanism in the YY2640 and JFZ ratoon crop accessions from low remaining stubble in pots during the mature stage of the main crop under the optimal experimental condition of AWD treatment and normal remaining stubble with standing status based on the result of 2018. The results revealed that the distribution of13C assimilates in the panicles of the two ratoon crop accessions was the highest,but in soil it was the lowest (Table 3). For the JFZ rice variety,the supplies of nitrogen fertilizer for root-vigor preservation and tiller promotion increased the allocation of13C assimilates to the root system,the stem and sheathof the residual stubble,the stem and sheath of harvested stubble,leaves and the regenerated buds by 33.8,164.8,13.2,70.4 and 64.8%,respectively at the ripening stage of the main crop. This reduced the relative partitioning rates of13C assimilates to the panicles of the main crop by 8.22%. The same trend was also observed in the YY2640 regenerated rice,indicating that N supplies in the same mode also increased the allocation of13C assimilates in the root system,the stem and sheath of the remaining stubble,the stem and sheath of harvested stubble,leaves and the regenerated buds by 210.5,129.1,127.9,38.9,and 314.3%,respectively. At the same time,it decreased the relative allocation rate to the panicles of the YY2640 main crop by 16.57%. These findings further confirmed that nitrogen application in this period did not impact the grain filling and yield formation of the main crop,which eliminated the concern that nitrogen application for rootvigor preservation and bud promotion before harvesting the main crop might affect the yield and quality of the main crop. Moreover,the proper application of nitrogen fertilizer at the later stage could also promote the delivery of13C assimilates to the vegetative organs at the maturity stage of the main crop,especially significant increases in the photosynthetic product allocation to root system,rice stubble and axillary buds,which in turn increased the remaining rice stubble quality,which laid the strong material foundation for rice ratooning.

Table 3 Effects of nitrogen fertilizer on the distribution of 13C assimilates in different organs and soil during the maturity stage of the main crop

We also continued to track the distributions of13C assimilates in the different organs and rhizosphere soils of regenerated rice at the full head stage (Table 4).The results revealed that the residual deposits of13C assimilates decreased to different degrees in the rhizosphere soil,root system,and remaining stubble(including stem and sheaths of the stubble) as well as in the regenerated stem and sheath of the ratoon crop(Tables 3 and 4). In particular,significant declines in the remaining stubble and regenerated sheaths at the full heading stage of the ratoon crop was noted compared with that at the maturity stage of the main crop (Tables 3 and 4). Furthermore,the partitioning dosage and its rate of13C assimilates in the rhizosphere soil and sheaths of remaining stubble in the two rice varieties was reduced slightly at the full heading stage of the ratoon crop.However,it was much more significantly reduced in the root systems and the stems of the remaining stubble at this stage under the SF×AWD treatment,suggesting that in the early grain-filling stage of the ratoon crop,the partitioning of assimilates to the below-ground root system was relatively lower,but more obviously reduced after nitrogen application before and after harvesting in the main crop relative to the NF×AWD treatment.

Table 4 Distribution of residual 13C assimilates after main crop harvest in the plant–soil system at the full heading stage of the ratoon crop

According to the13C assimilate allotment of different ratoon crop accessions at the full heading stage,this further confirmed that SF had a positive carry-overeffect on the increases in the partitioning dosage of13C assimilates to the rhizosphere soil,root systems,sheaths of the remaining stubble,regenerated stems and regenerated panicles by 15.4,17.4,54.9,40.5,34.7,and 44.3% in the JFZ ratooning season,and by 9.5,44.3,137.6,181.6,108.3,194.0,and 304.3% in the YY2640 ratooning season,respectively,relative to NF treatment. Notably,not only could the SF regime significantly increase the partitioning of13C assimilates to the rhizosphere soil,root systems and the sheaths of the remaining stubble,as well as the regenerated stems,leaves and panicles of the ratoon crop,but it could also reduce the allocation rate of13C assimilates to the rhizosphere soil and the sheaths of the remaining stubble in the JFZ ratooning season relative to the NF treatment. The same was true in the YY2640 ratooning season. Further analysis of13C assimilate transport in the rice stubble from the ripening stage of the main crop to the full heading stage of the ratoon crop indicated that SF significantly increased the translocation rate of13C assimilates from the residual stubble of the JFZ and YY2640 main crops by 29.2 and 8.5%,respectively relative to the NF treatment (Fig.2). This implied that SF coupling with AWD could effectively improve the material export and transport ability from the temporarily stored compounds in the residual stubble to the stems,sheaths and panicles of the ratoon crop. These findings further suggested that the application of nitrogen fertilizer together with AWD water administration for root-vigor and tiller promoting could effectively control the translocation of13C assimilates from residual stubble to the belowground roots and soil,and significantly increase the partitioning rate and amount of13C assimilates to the ratoon crop,thereby providing an important material foundation for their growth and development. We conducted a three factorF-value analysis on the data in Tables 3 and 4,and found that for V×N there is no significant difference in the distribution rates of13C,but for V×S,N×S and V×N×S there are significant differences in the allocation of13C,which is due to the lower allocation rates of13C to soil,roots,stems,and leaves,but a higher allocation rate to the grains in ratoon rice (Table 5).

Table 5 The interactions of three factors: variety,nitrogen fertilizer,main crop,and ratoon crop on the contents and distribution rates of 13C in soil and the different organs1)

Contents of soluble sugar and starch in residual rice stubbleFurther analysis revealed that SF coupling with AWD for root-vigor preservation and tiller promotion could significantly enhance the contents of soluble sugar and starch in the residual rice stubble as shown in Fig.3,since that soluble sugar content increased by 12.5 and 185.4%,and starch by 58.2 and 362.4% in the stems and sheaths of the residual rice stubble of the JFZ main crop,respectively. In addition,the soluble sugar also increased by 12.5 and 151.1%,and starch by 26.1 and 113.3% in the two parts of the residual rice stubble of the YY2640 main crop,respectively,which further confirmed the13C assimilate allocation pattern as discussed above.Therefore,these findings suggested that the SF treatment coupled with AWD water administration could not only boost the metabolic activity of carbohydrates,but also significantly increase the reserved nutrient substances in the stems and sheaths of the residual stubble,which in turn improved the stubble standing quality,thereby increasing the regeneration rate and grain yield of the ratoon crop.

Phytohormone contents in roots and residual rice stubbleThe results indicated that the SF coupling with AWD water administration had a significant impact on the contents of ZR,GA,ABA and IAA in the roots and the rice stubble at the mature stage of the main crop (Fig.4). The results indicated that the SF treatment could significantly increase the contents of ZR,GA and IAA by 254.4,248.1,and 42.3% in the roots and by 53.2,10.0,and 198.5%in the rice stubble of the JFZ main crop,respectively,relative to the NF treatment. The same trend was also observed in the YY2640 main crop,which showed that the contents of ZR,GA and IAA were enhanced by 63.5,310.7,and 59.1% in the roots and by 46.3,92.3,and 597.9% in the rice stubble. However,the SF coupled with AWD treatment reduced the content of ABA by 71.9% in the roots and by 35.1% the rice stubble of YY2640,while the reverse was true in the JFZ main crop,which is ideal for maintaining the higher root-vigor and boosting the sprouting of the axillary buds in the residual rice stubbles of the two different types of rice accessions.

Polyamine contents in the roots and residual rice stubbleThe patterns of changes in the contents of free Put,Spd and Spm in the roots and residual rice stubble of the main crop under the treatment of SF coupled with AWD is shown in Fig.5. The results revealed that relative to the NF treatment,the SF regime could significantlyreduce the free Put contents by 16.8 and 5.0%,but increase the free Spd by 15.6 and 153.5%,and the free Spm by 12.4-and 1.8-fold in the roots and residual stubble of the JFZ main crop at the ripening stage,respectively.The SF treatment could also significantly reduce the free Put by 38.7% in the roots,but it increased the free Put by 99.2% in the residual stubble. Meanwhile,the SF treatment could also significantly increase the free Spd by 25.1 and 5.3% and the free Spm by 8.21-and 1.42-fold in the roots and the residual stubble of YY2640 at the ripening stage,respectively. Furthermore,the effects of nitrogen fertilizer (SF) coupled with the AWD treatment for root-vigor preservation and tilller promotion on the contents of combined or bound polyamines were the same as on the contents of free polyamines in the roots and residual stubble of the main crop at the maturity stage(Figs.6 and 7). This indicated that the SF treatment could significantly increase the bound Spd and Spm contents,but reduce the bound Put in the root systems and in the residual rice stubble of the main crop (Figs.6 and 7). This infers that the application of nitrogen fertilizer for root vigor preservation and tiller promotion in the middle and late stages of the main crop can help to delay root senescence and stimulate the sprouting and growth of dormant axillary buds in rice stubble by increasing the contents of Spd and Spm in the root systems and residual stubble of the main crop at the ripening stage.

Fig. 3 Effects of nitrogen fertilizer treatment coupling with alternative wetting and drying (AWD) for root-vigor preservation and tiller promotion on the contents of soluble sugar and starch in the stems and sheaths of the main crop stubble during the maturity stage. NF,no nitrogen fertilizer treatment;SF,supplying nitrogen fertilizer. Different lowercase letters indicate significant differences according to Duncan’s multiple range test (P<0.05).

Fig. 4 Effects of nitrogen on the contents of zeatin nucleoside(ZR),gibberellin (GA),abscisic acid (ABA) and auxin (IAA) in the roots and stubble of main crop rice during the maturity stage.NF,no nitrogen fertilizer treatment;SF,supplying nitrogen fertilizer. Bars mean SE (n=3). Different lowercase letters indicate significant differences according to Duncan’s multiple range test (P<0.05).

Fig. 5 Effects of nitrogen on the contents of free Put,Spd and Spm in the roots and stubble of main crop rice during the maturity stage. NF,no nitrogen fertilizer treatment;SF,supplying nitrogen fertilizer. Bars mean SE (n=3). Different lowercase letters indicate significant differences according to Duncan’s multiple range test (P<0.05).

4.Discussion

4.1.Current status and problems in the research on ratoon rice cultivation

Fig. 6 Effects of nitrogen on the contents of soluble conjugated Put,Spd and Spm in the roots and stubble of main crop rice during the maturity stage. NF,no nitrogen fertilizer treatment;SF,supplying nitrogen fertilizer. Bars mean SE (n=3). Different lowercase letters indicate significant differences according to Duncan’s multiple range test (P<0.05).

Fig. 7 Effects of nitrogen on the contents of soluble bound Put,Spd and Spm in the roots and stubble of main crop rice during the maturity stage. NF,no nitrogen fertilizer treatment;SF,supplying nitrogen fertilizer. Bars mean SE (n=3). Different lowercase letters indicate significant differences according to Duncan’s multiple range test (P<0.05).

Previous studies suggested that the restrictions in further improvement of the regenerated rice yield are related to improper management of water and fertilizer in the late stage of the main crop in addition to variety characteristics(Wanget al.2019;Zhenget al.2022). However,with the popularization and application of mechanized harvesting,the ease of crushing damage to the residual rice stubble is problematic,which seriously affects the realization of yield potential in the ratoon crop. Consequently,the field water management technology of drying the field soil twice was proposed for ratoon rice under mechanized cultivation,which in fact has been verified in practice (Wanget al.2019;Huanget al.2021). However,most of these results were obtained based on single factor experiments,and few studies on the interactions of multiple factors have been reported (Xuet al.2015;Wanget al.2019;Huanget al.2021;Zhenget al.2022). In particular,research is limited on the interactions of multiple factors such as variety characteristics,water management,nitrogen application and fertilization methods on axillary bud sprouting from mechanically low-cut stubble and yield attributes of regenerated rice as well as its underlying mechanism,thus restricting the rapid spread of the above-mentioned technology.

4.2.Interactions of water and fertilizer on axillary bud sprout and yield formation in the ratoon crop

In this study,the interactions of water and fertilizer were studied with respect to axillary bud sprouting,tiller occurrence,effective panicles and yield formation of the ratoon crop with a low stubble height. The underlying physiological mechanisms from the perspective of multifactor interactions of artificial crushing damage and nondamage of different main crops coupled with different water and fertilizer management patterns were also investigated. The results suggested that relative to CF,in the late growth period of the main crop,the timely implementation of AWD,especially at 7–10 d before harvesting and 3 d after harvesting the main crop,and applying nitrogen fertilizer for root-vigor preservation and tiller promotion,then coupled with moderate redrying of the field soil,significantly improved the number of tillers generated from the low position nodes of the residual stubble,which in turn increased the effective panicles per plant and grains per panicle,thus reducing the impact of crushing damage to the remaining stubble.As a result,there was no significant difference in yield of the regenerated rice between the crushing damaged area and the non-crushing damaged area,which resulted in high yields as a whole (Table 2). However,although fertilization under non-alternating wet and dry cannot fully compensate for the loss of yield,the yield of YY2640×SF×CF×CD was only 5.32% lower than that of YY2640×SF×CF×NC. There was no significant difference between the two treatments,while JFZ×NF×CF×CD was 13.04% lower than JFZ×NF×CF×NC. This indicates that under non-alternating wet and dry conditions,fertilization can partially alleviate the loss of yield caused by crushing damage,which is related to the promotion of axillary bud germination in the crushing damage area by fertilization.Therefore,our findings suggested that the action of any a single factor (fertilization or alternating wet and dry) could not achieve reasonable effectiveness relative to the multifactor interaction. Both varieties revealed that artificial harvesting can achieve high yield under fertilization conditions,while under artificial crushing damage conditions,the residual rice stubble is crushed into the soil without moderate drying,resulting in a yield loss of over 40%. However,when the soil moisture decreases to below 30% in the later stage of the main crop,artificial crushing damage does not crush the remaining rice stubble into the soil,and the number of tillers and yield can increase under fertilization conditions. Moreover,we also found that the newly regenerated tillers from the higher position nodes could independently form a new plant and tiller to produce more effective panicles in the mechanical crushing damage area relative to those in the non-crushing damage area. Furthermore,we also found that low stubble height with crushing damage could also promote the sprouting of axillary buds to produce more tillers and effective panicles to some extent(Table 2). This was attributed to the increases in JA signal transduction and the synthesis and accumulation of endogenous hormones (IAA and CTK) to promote axillary bud sprouting and growth in response to the double stresses of moderate field drought and crushing damage to the residual rice stubble (Wanget al.1997;Lukaset al.2020). These findings implied that whether the cut height of the remaining rice stubble is lower or not,the main reason for the reduction of axillary bud sprouts caused by mechanical harvester crushing damage,and the key to obtaining high and stable yield,is to determine the reasonable water-fertilizer interaction for high-yielding cultivation of rice ratooning.

4.3.Effect of alternation of dry and wet on the distribution of 13C photoassimilates

Further studies using a13C stable isotope tracer method revealed that under the AWD treatment,timely fertilizer application for root-vigor preservation and tiller promotion before and after harvesting the main crop was beneficial for promoting the delivery of13C photoassimilates to the vegetative organs at the mature stage of the main crop. A significant increase was further noted in the partitioning of photosynthetic products to the root systems,the remaining rice stubble and axillary buds. Our studies revealed that the timely application of nitrogen fertilizer for rootvigor preservation and bud promotion before harvesting the main crop could temporarily reserve more nutrient assimilate substances in the stem and sheath of the stubble at the mature stage of the main crop,which laid an important material foundation for the remobilization of photoassimilates from the residual rice stubble of the main crop to promote the sprouting of axillary buds and the generation of more tillers. Zhanget al.(1980) also reported that after harvesting the main crop,52–70% of the assimilates in residual rice stubble were transferred to the plants of the ratoon crop,and 30–48% remained in the stubble,which were mainly transported to the axillary buds at the second and third nodes of the stem base (Zhanget al.1980;Liuet al.2008). Meanwhile,we also found that the application of nitrogen fertilizer coupled with AWD treatment for root-vigor and tiller promotion significantly increased the translocation and exportation rates of13C assimilates from the whole rice stubbles of both the JFZ and YY2640 main crops,and effectively controlled the remobilization of13C assimilates from the rice stubble to the below-ground root systems and rhizosphere soil by enhancing the partitioning rate and amount of13C assimilates to the ratoon crop.This in turn allows for the rapid and abundant delivery of photosynthates to the grain pools,especially when the ratoon crop was at the middle and late grain-filling stage(Huanget al.2020). The present studies suggested that appropriately reducing soil moisture in the late growth period of the main crop can slow down the declining rate of photosynthetic intensity,respiratory intensity and chlorophyll content of the rice,which is ideal for growth promotion and development of the regenerated buds. Similar to our research results,the timely application of nitrogen fertilizer under AWD treatment before and after harvesting the main crop could effectively increase the content of non-structural carbohydrates in the residual stubble at the later stage of the main crop,especially after harvesting the main crop.This is the key to improving the quality of rice stubble,which in turn increases the intake,storage and transportation capacity of the photoassimilates in the regenerated rice,in turn promoting the sprouting of axillary buds and producing of more tillers from low position nodes of the stems in the remaining stubble. This,in turn,enhances the effective panicles,grains per panicle and harvest index,and finally achieves high yield in the ratoon crop.

4.4.Interactions of water and fertilizer on hormones and polyamines of the ratoon crop

Since higher plants consist of multiple organs with different functions and nutrient requirements,they rely on local and remote signaling pathways to coordinate the complex physiological and morphological plant-wide responses to balance the amount of nitrogen obtained from the soil with the demands for growth and development. The timely application of nitrogen fertilizer for root-vigor preservation and tiller promotion followed by moderate field drought before and after harvesting the main crop was also found to significantly increase the contents of ZR,GA and IAA,but reduce the content of ABA in the roots and the residual stubble of YY2640 at the maturity stage. The CK content could be significantly increased in the sap of maize roots and stems by nitrogen supplements (Takeiet al.2001;Takatoshiet al.2011). The relationship between nitrogen and CK is bidirectional,that is,N could regulate CKde novosynthesis,but nitrogen absorption could be regulated by CK (Takeiet al.2004;Sakakibaraet al.2006;Kudoet al.2010;Kyoheiet al.2021). ABA is generally considered to be a stress hormone involved in abiotic and biotic stress responses,but there is considerable evidence that ABA levels are associated with nitrogen status in several plants (Schussleret al.1984;Tsuneo 1993). Previous studies suggested that the expression of ABA was downregulated under high NO3–condition,while the reverse was true under low NO3–conditions,which explains the genetic and physioecological reasons for the differences in changing patterns of ABA between the two regenerated rice cultivars (Wilkinson and Davies 2002). YY2640 is anindica/japonicahybrid rice with a relatively long growth duration,which is relatively tolerant to drought but sensitive to nitrogen fertilizer,while JFZ is a conventionalindicarice variety with the opposite performance in growth duration and fertilizer tolerance.Therefore,premature senescence in the JFZ cultivar often occurs in the ratoon crop when soil nutrients are insufficient in the field. In this study,the addition of nitrogen fertilizer combined with moderate field drought before and after harvesting the main crop inevitably led to a change in the nitrate nitrogen content. Interestingly,the other root signaling molecule ABA also inevitably changed in response to the dual induction of drought and the root signaling molecule NO3–in the soil. Auxin has been considered a candidate for regulating the nitrogen signaling from stem to root because it is transported from above-to below-ground for the promotion of lateral root initiation and development (Takatoshiet al.2011). The timely application of nitrogen fertilizer combined with moderate field drought before and after harvesting the main crop could significantly increase the content of NO3–in the soil,induce the expression of auxin receptor gene AFB3,promote the growth of lateral roots and axillary buds,and increase the absorption of nitrogen (Fukaki and Tasaka 2009;Vidalet al.2010). The results of this study also found that after coupling fertilization and AMD,a higher IAA content was maintained,and the physiological process is conducive to maintaining higher root vitality,promoting the sprouting of regenerated axillary buds and producing more tillers from the low position nodes,thereby increasing the effective panicles and grain yield of the ratoon crop (Xuet al.1993;Wanget al.2020). We also found that the effects of nitrogen fertilizer application coupled with AWD before and after harvesting the main crop on the contents of combined and bound polyamines were consistent with those on free polyamines in the roots and rice stubble at the maturity stage of the main crop,indicating that it increased the contents of Spd and Spm,but reduced the contents of Put in the root system and rice stubble. Spd and Spm are known to resist aging and promote root growth and flowering (Abidet al.2018).

5.Conclusion

The results of this study suggested that varieties,water management,nitrogen application mode and rice stubble status have significant interactions on axillary bud sprouting from low-cut rice stubble and yield attribute performance of the ratoon crop. Any single factor could not play a role as significant as the interaction of multiple factors. Relative to continuous flooding(CF),the application of nitrogen fertilizer for root-vigor preservation and tiller promotion coupled with moderate field drought before and after harvesting the main crop could significantly promote the sprouting of axillary buds at the low position nodes of stems and produce more tillers,and hence increase the effective panicles and the grains per panicle,thus reducing the impact of crushing damage to the residual stubble. As a result,there was no significant difference in grain yield between the crushing damaged area and the non-crushing damaged area. On the contrary,it was difficult to recover the effects of rice stubble damage under mechanized harvesting even if nitrogen fertilizer was applied in time,but not followed by moderate drought of the field soil in time,andvice versa,which provides a basis for optimizing the cultivation technology program. Further study revealed that such favorable cultivation conditions could significantly promote13C assimilate translocation to the vegetative organs at the mature stage of the main crop,which could significantly optimize the photosynthetic product distribution in the root system,rice stubble and its axillary buds;and significantly increase the storage of non-structural carbohydrates in residual rice stubble to improve its standing quality.This also reduced the remobilization of13C assimilates remaining in the stems and sheaths at the post grainfilling stage of the ratoon crop to the below-ground root systems and rhizosphere soil,which lays a good material foundation for boosting the axillary bud sprouts and tillers,thereby producing more effective panicles and a high yield in the ratoon crop. These findings further help us to deeply understand the optimal water and fertilizer supply conditions of ratoon rice with low stubble height under mechanized harvesting.

Acknowledgements

This research was supported by the National Nature Science Foundation of China,the National Key Research and Development Program of China (302001109,2016YFD0300508,2017YFD0301602,2018YFD0301105),the Fujian and Taiwan Cultivation Resources Development and Green Cultivation Coordination Innovation Center,China(Fujian 2011 Project,2015-75),and the Natural Science Foundation of Fujian Province,China (2022J01142).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendicesassociated with this paper are available on https://doi.org/10.1016/j.jia.2023.05.038