Growth of tandem long-mat rice seedlings using controlled release fertilizers: Mechanical transplantation can be more economical and high yielding

HE Wen-jun , HE Bin , WU Bo-yang , WANG Yu-hui , YAN Fei-yu, DING Yan-feng , LI Gang-hua #

1 Sanya Institute of Nanjing Agricultural University/Jiangsu Collaborative Innovation Center for Modern Crop Production/Key Laboratory of Crop Physiology Ecology and Production Management, Nanjing Agricultural University, Nanjing 210095, P.R.China

2 National Engineering and Technology Center for Information Agriculture, Nanjing 210095, P.R.China

3 China-Kenya Belt and Road Joint Laboratory on Crop Molecular Biology, Nanjing 210095, P.R.China

4 School of Life Sciences and Food Engineering, Huaiyin Institute of Technology, Huai’an 223003, P.R.China

Abstract The traditional soil-based rice seedling production methods for mechanical transplanting are resource-intensive, time consuming and laborious.The improvement and optimization of nutrient management in soil-less nursery raising methods like tandem long-mat seedlings (TLMS) are necessary for the resource-efficient cultivation of rice.In the present study, a controlled-release fertilizer (CRF)-polymer-coated compound fertilizer with 3 months release period(PCCF-3M) was applied as seedling fertilizer (SF), and five different dosages of SF (SF-0, SF-10, SF-20, SF-30, and SF-40) were compared with an organic substrate as the control (CK).Among all SF treatments, the best results were obtained with the application of 20 g/tray of SF (SF-20), as the seedling quality and machine transplanting quality were comparable to those of CK.In contrast, the lower dosages (SF-0 and SF-10) resulted in low nitrogen content and reduced shoot growth, while the higher dosages (SF-30 and SF-40) resulted in toxicity (increased malondialdehyde accumulation) and inhibited the root growth.Similarly, SF-20 increased panicle number (5.6-7.0%) and yield (4.3-5.3%)compared with CK, which might be related to the remaining SF entangled in the roots supporting the tiller growth of rice seedlings in the field.Moreover, SF-20 reduced the seedling block weight (53.1%) and cost of seedling production(23.5%) but increased the gross margin, indicating that it was easy to handle and economical.Taken together, our results indicate that SF-20 is a cost-effective way to promote the growth and transplanting efficiency of rice seedlings.To our knowledge, this study is the first to determine the optimum dosage of CRF for the soil-less production of rice seedlings.

Keywords: machine-transplanted rice, tandem long-mat seedlings, controlled release fertilizer, seedling quality, yield

1.Introduction

Rice is the major staple crop that is consumed by a large proportion of the population in China.Nursery transplanting is still one of the predominant rice production methods in China (Penget al.2009).In recent years,a rural labor shortage has occurred due to fast-paced urbanization and industrialization in China.Consequently,farmers are shifting towards mechanical transplanting methods to deal with the labor shortage and to save their time (Bhushanet al.2007; Knightet al.2011).However,the conventional nursery production methods used for machine-transplanted rice are resource intensive, time consuming and laborious.For instance, seedbed soil fertilization is considered a key measure in most seedling production methods, but it uses the topsoil of cultivated land, erodes the soil structure and subsequently results in reduced crop yields (Leiet al.2017).In order to limit the dependence on soil-based seedling production,researchers have utilized a mixture of inorganic,valuable and non-renewable resources, such as quartz sand, perlite and vermiculite with soil and other organic substances, to prepare their nursery production matrix(Chen and Avnimelech 1986; Robertson 1993).However,with increasing environmental awareness, concerns related to the depletion of these non-renewable resources have been raised due to the large-scale cultivation of rice in China and worldwide (Zhanget al.2012; Altland and Locke 2017; Perez-Cabreraet al.2021).As a result,rice researchers have focused on the development of renewable and eco-organic substrates for the full or partial replacement of pre-existing materials that are nonrenewable substrates for rice seedling production (Shaoet al.2014; Renet al.2020; Liet al.2021).

In recent years, long-mat seedling technology for rice seedling production has been developed by using the existing soil-less cultivation methods.Similarly, based on Japanese long-mat seedling cultivation technology(Tasaka 1999; Oikawaet al.2005; Wanget al.2008b), the technologies of hydroponically grown long-mat seedlings(HLMS) (Liet al.2016, 2019, 2020) and tandem longmat seedling (TLMS) (Liet al.2021) are being applied in China to reduce costs and improve work efficiency.These methods utilize rice husk, non-woven fabric or bast fiber as an economical and permeable matrix for rice nursery production.Moreover, these methods also reduce the labor cost of handling the matrix and seedling trays after sowing in the process of seedling cultivation because of the lighter weight matrix.Notably, HLMS utilizes non-woven fabrics and there are environmental concerns regarding their persistence and degradability.In contrast, TLMS employs a degradable bast fiber instead of non-woven fabric, making it more eco-friendly than HLMS.Additionally, the seedling tray of TLMS is lighter and cheaper than that of HLMS.Previous studies with soil-less media have focused on HLMS and studies on the growth performance and transplanting efficiency of TLMS are very few.Moreover, the nutrient management in these soil-less techniques needs a nutrient solution circulation system that is expensive, and the intermittent requirement for normal water-soluble fertilizer also makes it inconvenient to operate and affects the stability of seedling quality (Liet al.2021).Therefore, further research is necessary to develop a simple, efficient and durable nutrient management method that is suitable for these technologies, in order to remove the constraints that might limit the development and large-scale application of these seedling production technologies.

In recent years, the use of slow-releasing fertilizers,often called “controlled-release fertilizer (CRF)”, has been increasing in seedling producing nurseries, especially for raising crop or forest plants.These CRFs are able to supply sufficient nutrients for an extended period of time,that not only minimizes the chance of toxicity due to overfertilization but also reduces the labor needed for multiple fertilizations (Jacobset al.2005; Riyajanet al.2012).Previous studies on the effects of CRF on the seedling morphology and mineral nutrition status in nurseries have shown that CRF can optimize the nutrient delivery(Haaseet al.2006), enhance the seedling quality (Kloosteret al.2012; Zamunéret al.2012), and promote seedling survival in the nursery (Fanet al.2004; Olietet al.2004;Irinoet al.2005; Fuet al.2017).Moreover, the use of CRF for rice seedling growth results in increased nitrogen use efficiency of the transplanted rice (Yanget al.2013a,b; Zhanget al.2016).However, previous studies on the application of CRFs in rice nurseries are limited to traditional soil-based nursery production methods for rice.Therefore, whether the use of CRFs is suitable for growing the seedlings by mechanical transplanting based on a soil-less substrate remains unclear.Moreover,studies are also required to determine the suitable dosage of CRFs in order to optimize its use in the soil-less production of rice nurseries.

Considering the limited research on TLMS and the need for optimizing nutrient management in soil-less production of rice nurseries, the present study was designed with the two objectives of improving the nutrient supply in the TLMS method and determining the suitable dosage of CRFs that could result in the better growth and transplanting efficiency of TLMS.The results of the present study can lay a theoretical foundation and practical reference for the large-scale application of mechanized rice cultivation.

2.Materials and methods

2.1.Experimental location

We conducted this study at the research base of our lab (Danyang Experimental Base of Nanjing Agricultural University), which is in Danyang, Nanjing, Jiangsu Province, China (31°54´N, 119°28´E).For all the seedlings of either TLMS or CK (local conventional seedling raising methods) were carried out in a climate control chamber with the following parameters: average temperature of 25°C, light duration of 13 h d-1, luminous intensity of 300 μmol m-2s-1, and CO2concentration of 400 ppm.The soil of the experimental field was a clay loam with 86.4, 13.2 and 98.8 mg kg-1as available nitrogen (N), phosphorus (P) and potassium (K),respectively.

2.2.Experimental design

The growth media used to raise the rice seedling in the tandem nursery trays contained 40 g of bast fiber film and 1 cm of rice husks (Liet al.2021).A tandem nursery tray is shown in Appendix A.The bast fiber film was obtained from Harbin Jinzhu Trading Co., Ltd.(Harbin, China),while rice husks were obtained from the Guorong Rice Mill in Yanling Township (Danyang City, Jiangsu, China).The width of the nursery raising tray was 28 cm and the length of the nursery could be adjusted by changing the number of trays.

Through a pre-experiment on the appropriate amount of N for each leaf age (1.1, 2.1 and 3.1) of TLMS and the release rates of various fertilizers with different release periods at the seedling stage (including 1, 2, 3 and 4 months), the polymer-coated compound fertilizer(with contents of N, P2O5, K2O each at 15%) and a 3-month release period (PCCF-3M, 15-15-15, Taizhou Woye Ecological Agriculture Co.Ltd., Jiangsu, China)were selected as the seedling fertilizer (SF) of TLMS.In present study, five different dosages of PCCF-3M were set: SF-0, SF-10, SF-20, SF-30, and SF-40 (where the numbers are the dosages of NPK per tray).The SF was evenly spread between the bast fiber film and rice husk (Fig.1).For CK, rice seedlings were grown on the conventional seedling substrate of rice (with contents of N,P2O5, K2O that were 49, 67 and 166 mg kg-1, respectively)(Jiahexing Agricultural Biotechnology Co.Ltd., Jiangsu Province, China) in seedling trays with a 58-cm length, a 28-cm width and a 3-cm depth.

Fig.1 The application position of seedling fertilizer (SF) and the technology for cultivating tandem long-mat seedlings (TLMS).

In this experiment, a localjaponicarice Ninjing 8 was used as the experimental material.After 2 days of soaking in water, seeds were kept in the dark at 30°C for 1 day, and the germinated seeds were transferred in the nursery tray.Three replications were set in each treatment, with 10 trays per replication (six trays for seedling quality measurements and four trays for transplanting).The nursery management adopted for seedling cultivation employed the application of microirrigation for the hard land seedling raising technique.

Subsequently, 15 days after sowing (when the leaf age was 3.1), seedlings were transplanted with a mechanical transplanter (30 cm×13 cm).The size of each test plot was 14 m×3.6 m with three replications for each treatment.In each TLMS treatment, the remaining fertilizer entangled in the roots was brought to the field through mechanical transplanting, as shown in Appendix B.The fertilizers were applied in the paddy field according to the local traditional methods, in which the total N (300 kg ha-1)was applied in three splits using 30, 20, and 50% at each stage (basal fertilizer, 1 day before transplanting; tillering fertilizer, 7 days after transplanting; and topdressing, at the panicle initiation (PI) stage).The nitrogen (N as urea),phosphorus (P as P2O5) and potassium (K as K2O) were in a ratio of 1:0.5: 0.8.Among NPK, P was applied once as basal fertilizer and K fertilizer was applied in two splits, i.e.,half as the basal fertilizer and half at the PI stage.All the irrigation and pest management practices were performed according to the local traditions in the area.

2.3.Sampling and measurements

SF release characteristics in the nursery stageFertilizer weighing (10±0.01) g was placed into a nylon mesh bag(12 cm×8 cm) with a hole diameter of 1.0 mm, and spread on the nursery tray.Three bags were sampled at the leaf ages of 1.1, 2.1 and 3.1.The remainder was buried 3 cm below the soil surface near the root in the field and three bags were sampled at the tillering stage (about 30 days after transplanting), panicle initiation stage (about 50 days after transplanting) and heading stage (about 80 days after transplanting).The collected samples were washed with distilled water to remove impurities, and then placed in an oven for drying at 30°C until a constant weight.They were then weighed and the loss was calculated.

Shoot and root parametersBefore transplanting, the shoot parameters including the leaf age, shoot height,stem diameter and root morphology (scanned by an Epson Perfection V700 Photo, Seiko Epson Co., Naganoken, Japan) were analyzed from 30 seedlings that were randomly selected from each treatment.Moreover, the number of seedlings and emergence rate in a 100-cm2block of seedlings in each treatment were calculated.For dry matter accumulation, we collected 100 seedlings from each treatment and divided them into two portions,i.e., aboveground and belowground, and dried them in an oven at 105°C for 30 min and then at 80°C until they reached a constant weight.Then the dry samples were ground into powder for the determination of starch and N contents.

Rooting ability and root vigorBefore transplanting,30 seedlings with uniform growth in each treatment were selected and all the roots were cut off.Then the seedlings were fixed on foam boards and placed in plastic boxes filled with water.After 5 days, the total length of the roots of each plant was measured as the rooting ability(Ren 2007).Root vigor was measured with a plant root vigor detection kit that used the TTC colorimetric method(r30351-2, Shanghai Yuanye Bioengineering Institute,Shanghai, China).

Root entanglement forceBefore transplanting, a seedling block (28 cm×29 cm) was cut out from each treatment and placed on a flat and smooth glass plate.Then, one end of the seedling block was fixed to the glass plate, while the other end was connected to the hook of a spring dynamometer and pulled until the block was completely broken.The reading on the spring dynamometer was taken as the entanglement force (Liet al.2016).

Chloroplast pigment contentBefore transplanting,100 mg fresh leaves were collected and homogenized in ethanol (10 mL, 95% pure) (Arnon 1949), and then the leaf extract was centrifuged at 10 000×g for 10 min.Finally, the absorbance was measured at 663 and 645 nm to estimate the chlorophyll content as follows:

Chlorophyll=7.18×OD663+17.32×OD645

Starch contentA total of 0.1 g of dry sample was extracted twice with 5 mL of 80% ethanol solution in a water bath at 80°C, and the extract was decolorized with activated carbon.Subsequently, 3 mL of anthrone was added to 10 μL of supernatant, then bathed in boiling water for 10 min and cooled for 15 min.The residue was placed in boiling water to release the starch, then hydrolyzed with 9.2 mol L-1HClO4, and the supernatant was retained after centrifugation.Anthrone reagent was added, the absorbance at 620 nm was measured and a standard curve was generated using glucose (Seifteret al.1950).

Nitrate reductase (NR) activityThe frozen samples(0.50 g) collected before transplanting were homogenized in the extraction buffer containing Tris-HCl (100 mmol L-1), MgCl2(1 mmol L-1), EDTA (1 mmol L-1) and β-mercaptoethanol (10 mmol L-1).Then the homogenized samples were centrifuged (10 000×g) followed by the addition of phosphate buffer (pH 7.5; 100 mmol L-1KH2PO4) and NADH (0.4 mL of 2 mg mL-1), and placed in a water bath at 25°C.Subsequently, the reaction was terminated by adding sulfanilamide (1%) solution and naphthyl ethylene amines (0.02%) (Yuet al.1998).Finally, after centrifuging the respective mixtures of NR at 4 000 r min-1, the absorbance was recorded at 540 nm.

Nitrogen contentWe employed the Kjeldahl apparatus to estimate the nitrogen content in the samples, according to Barbanoet al.(1991).

Antioxidant enzyme activity and malondialdehyde(MDA) contentThe enzyme solution was extracted from 0.5 g of fresh leaf samples collected before transplanting, using an extraction buffer composed of ethylenediaminetetraacetic acid (0.2 mmol L-1),hepes (25 mmol L-1), ascorbate (2 mmol L-1) and PVP(polyvinylpyrrolidone, 2% w/v).The homogenate was centrifuged (14 000×g, 15 min, 4°C) and the supernatant was used as the enzyme extract for determining the activities of antioxidant enzymes.Subsequently, for measuring the specific activities of superoxide dismutase(SOD), catalase (CAT), and peroxidase (POD), the reaction mixtures were prepared accordingly and absorbance values were obtained to estimate each enzyme activity as elaborated by Chenet al.(2017).

The MDA was extracted from 0.1 g fresh leaf samples collected before transplanting using the thiobarbituric acid solution.Then, after centrifugation at 5 000×g for 10 min at room temperature, the absorbance of the supernatant was checked at 450, 532, and 600 nm.The MDA was quantified as follows:

MDA=[6.45×(OD535-OD600)-0.56×OD450]×V/W

where V is extract volume (mL) and W is fresh weight (g)

Mechanical transplantation indicatorsThree days after seedling transplantation, six rice lines (one operational width of the mechanical rice transplanter) were observed in each plot.Each line was monitored for 100 consecutive hills and the data for missing hills, floating or damage seedlings and seedlings per hill were recorded to check the efficiency of the mechanical transplanter.

Tiller numbersThe tiller numbers from 60 hills in each plot were investigated.The data were recorded at 10, 20,30, 40, 50, 80, and 125 days after transplanting.

Yields and yield componentsAt plant maturity, 60 hills per plot were sampled randomly and the number of effective tillers was obtained from each sample.Subsequently, based on the average effective panicles,the data for spikelets per panicle and seed-filling rate were derived.The theoretical yield was estimated using the 1 000-grain weight.

2.4.Statistical analysis

The economic benefits of the different seedling raising methods were analyzed according to Sarangiet al.(2015).The total cost included the production input(excluding land rent) and labor input.The total benefit was calculated based on the harvested grain yield and the current market price.

All the data were recorded in at least three biological replicates and analyzed by SPSS 18.0 for the ANOVA and Duncan’s multiple range test (P<0.05).The data are presented here as mean±SD.Furthermore, Pearson’s correlation analysis was used to reveal the relationships among various indicators.The figures were drawn using Graphpad Prism 8 (Graphpad Software Inc., San Diego,USA).

3.Results

3.1.SF cumulative release rate

The SF release rate increased with increasing leaf age,and the cumulative release rate of fertilizer was about 6.9% at the nursery stage (Fig.2-A).Correspondingly,the cumulative releases of SF-0, SF-10, SF-20, SF-30 and SF-40 were 0, 0.69, 1.38, 2.07 and 2.76 g/tray,respectively.Additionally, the remaining SF was released rapidly within 30 days after transplanting, at cumulative release rates of 63.9 and 68.4% in 2020 and 2021,respectively (Fig.2-B).

Fig.2 Seedling fertilizer (SF) cumulative release rates in nursery trays (A) and in the field (B).The error bars represent the standard error of the means (n=3).

3.2.Shoot growth parameters

Significant phenotypic differences were observed between CK and the SF treatments (Fig.3-A).Compared to the control, SF-0, SF-10 and SF-30 showed leaf yellowing,while SF-40 significantly inhibited the growth of rice seedlings.The leaf age, shoot height, stem diameter and shoot dry weight showed upward trends with increases in the SF dosage (Fig.3-B and E).For the emergence rate, however, the SF-30 was significantly lower than the other SF treatments (Fig.3-F).Notably, there were no significant differences between SF-20 and CK in any of the parameters, indicating that the use of SF-20 in TLMS may help to replace the conventional methods for raising seedlings.

Fig.3 Effects of different dosages of seedling fertilizer (SF) on the shoot growth of seedlings.A, phenotype.B, leaf age.C, shoot length.D, stem diameter.E, shoot dry weight.F, emergence rate.CK, conventional seedling substrate; SF-0-30, 0, 10, 20, and 30 g of polymer-coated compound fertilizer with 3 months release period (PCCF-3M), respectively.The error bars represent the standard error of the mean.Different letters above the columns indicate the statistically significant differences at P<0.05 (Duncan’s range test) between different treatments.

3.3.Root growth parameters

The number of root tips, root length and surface area declined significantly with an increasing SF dosage(Fig.4-A-C).However, the highest root dry weight, root vigor and rooting ability among all SF treatments were observed in SF-20 (Fig.4-E-G).Compared with CK, SF-20 significantly increased the root dry weight, while the other parameters at SF-20 had no significant differences with CK.

Fig.4 Effects of different dosages of seedling fertilizer (SF) on the root growth of seedlings.A, root tips.B, root length.C, surf area.D, average diameter (avg diam).E, root dry weight.F, root vigor.G, rooting ability.CK, conventional seedling substrate; SF-0-30, 0, 10, 20, and 30 g of polymer-coated compound fertilizer with 3 months release period (PCCF-3M), respectively.The error bars represent the standard error of the mean.Different letters above the columns indicate the statistically significant differences at P<0.05 (Duncan’s range test) between different treatments.

3.4.Physiological characteristics of carbon and nitrogen

The contents of chlorophyll and starch increased with an increasing dose of SF, and peaked in the SF-20 treatment(Fig.5-A and B).However, the SF-30 treatment led to reduced pigment contents of the rice seedlings, indicating that a higher SF dosage may result in toxicity for the rice seedlings.In addition, the NR activity and N content increased with an increasing SF dosage (Fig.5-C and D).

3.5.Protective enzyme activities and MDA content

With an increase in the SF dosage, the activities of CAT and POD increased significantly (Fig.6-B and C), while the content of MDA showed the opposite trend (Fig.6-D).Notably, the SF-20 treatment showed the highest activities of CAT and POD, and lowest MDA content as compared to the other SF treatments, indicating that SF-20 is the most suitable dosage for avoiding transplanting injury in TLMS seedlings.There were no significant differences between the SF-20 treatment and CK for any of the parameters, suggesting that SF-20 could be a potential treatment to replace the conventional nursery raising methods with TLMS.

3.6.Root entanglement force and seedling block weight

The root entanglement force decreased significantly with an increase in the SF dosage, but the SF-0, SF-10 and SF-20 treatments were all significantly higher than CK(Fig.7-A).All of the SF treatments significantly reduced the seedling block weight by 50.6-71.6% as compared with CK.Altogether, these results indicate that seedlings raised by the TLMS method are easy to carry, and combined with SF, they have the potential to replace the conventional seedling production methods (Fig.7-B).

3.7.Correlation matrix and multivariate analysis

The N content was significantly and positively correlated with leaf age, shoot length, stem diameter, shoot dry weight and root average diameter, but significantly and negatively correlated with root length and root tips.This indicated that a higher N content of the plant promoted aboveground growth, but inhibited root extension.In addition, there were significant and negative correlations between the MDA content and the SOD/CAT activities (Fig.8).

Fig.8 Pearson’s correlations of all seedling parameters.The color gradient is proportional to the Pearson’s correlation coefficient value.Blue and red colors denote positive and negative correlations, respectively.NR, nitrate reductase; SOD, superoxide dismutase; CAT, catalase; POD, peroxidase; MDA, malondialdehyde.Not marked, P≥0.05; ＊, P<0.05.

3.8.Cost of raising seedlings

All the SF treatments significantly reduced the cost of raising seedlings, mainly due to the reduced costs for seedling medium and labor.The costs of raising seedlings for SF-0, SF-10, SF-20 and SF-30 were 49.6,36.4, 23.5 and 10.7% lower than that of CK, respectively.These results suggest that even with the use of SF,TLMS is still more economical than conventional methods (Table 1).

Table 1 Effects of different dosages of seedling fertilizer (SF) on the cost of raising seedlings (CNY m-2 seedling field)

3.9.Mechanical transplantation quality

The different SF treatments had no significant differences in missing hill rate or damaged seedling rate, but there were significant differences in the floating seedlings rate and the number of seedlings per hill in the two years.With an increase in the SF dosage, the floating seedling rate showed a decreasing trend, and SF-0 was significantly higher than SF-20 and SF-30.The lowest value for seedlings per hill was observed in SF-30 compared to the other treatments.In addition, there were no significant differences between the SF-20 treatment and CK, except for the floating seedling rate in 2021 (Table 2).

Table 2 Effects of different seedling raising methods on the mechanical transplantation quality

3.10.Tiller dynamics

The trend of tiller growth for each treatment was consistent over time in the two years.With an increasing SF dosage, the number of tillers generally increased at first and then decreased in each period, and SF-20 remained at the highest level after transplanting.It is worth noting that the number of initial seedlings and the tillering in the early stage of SF-30 were significantly lower than those of CK, while the tiller number did not vary significantly between SF-30 and CK in the end.This dynamic change is also reflected in the SF-10 (Fig.9),suggesting that SF plays an important role in promoting tillering growth.

Fig.9 Effects of different seedling raising methods on the tiller numbers.CK, conventional seedling substrate; SF-0-30, 0, 10,20, and 30 g of PCCF-3M, respectively.The error bars represent the standard error of the mean (n=3).

3.11.Yield and yield components

The different SF dosages showed significant effects on yield during the two years.Overall, the yield increased with an increasing SF dosage, except for the SF-30 treatment that had a reduced yield compared to the other SF treatments.The highest yield was noted in the SF-20 treatments, which could be attributed to the higher panicle numbers.Moreover, the yield in SF-20 was 4.3 and 5.3%higher than in CK in the two years, respectively (Table 3).

Table 3 Effects of different seedling raising methods on yield and yield components

3.12.Economic benefits

The trend of economic benefits was consistent in the two years (Table 4).The cost increased with increasing SF dosage, and the costs of all SF treatments were lower than that of CK.However, the gross returns increased at first and then decreased with increasing SF dosage, and SF-20 was significantly higher than the other treatments.Similarly, SF-20 was significantly higher than the other treatments in terms of gross margin, which indicated that SF-20 is more economical.

Table 4 Effects of different seedling raising methods on economic benefits

4.Discussion

4.1.Effects of different SF dosages on seedling quality

The nutrient management in a rice nursery is very important for growing healthy rice seedlings which can withstand the normal operation of a mechanical transplanter and give high yields (Penget al.2011).Macro-elements like nitrogen, phosphorus and potassium are the most important in maintaining the normal physiological metabolism of rice, and their deficiency orexcess can cause negative effects on rice growth (Vanceet al.2003; Fideliset al.2011).Research has shown that rice seeds start absorbing nutrients after germination and rooting, even though the roots are weak (Juliaet al.2018).In addition, it is necessary to provide the nutrients needed for the growth of young seedlings in time, when the seedlings is at the stage of nitrogen weaning (about 1.1 of leaf age), otherwise the growth of the seedlings might be inhibited.The seedling-raising fertilizer used in this experiment is a resin-coated compound fertilizer, which can provide the three kinds of macro-elements for the growth of seedlings.These three nutrients are released slowly after sowing, which ensures that the seedlings can absorb the nutrients immediately when rooting and avoids poisoning the seedlings.Moreover, the fertilizer release is accelerated after the 1.1 leaf age (Fig.2) and provides sufficient nutrients for seedling growth.This characteristic timing of nutrient release is similar with the nutrient requirements of rice seedlings.

Many studies on nursery nutrient management have shown that within a certain range, the plant height, stem diameter, leaf area, chlorophyll and nitrogen content of the rice seedlings increased with an increase in nitrogen application, which is conducive to reducing transplanting shock and promoting tillering (Sharma and Ghosh 1999; Subedi 2013).However, an excessive nitrogen dosage leads to an excessive leaf area index of seedlings, deterioration of the growth environment and reduction in the seedling rate, and these factors affect the establishment of stronger rice seedlings (Huiet al.2006).Appropriate amounts of phosphorus and potassium fertilizer can increase the stem diameter,promote the root growth of seedlings, make a stronger blanket, and easily cultivate robust seedlings that are suitable for machine transplanting (Guoet al.2003;Wanget al.2008).This study showed that treatments of SF-10, SF-20 and SF-30 significantly increased leaf age, plant height, stem diameter, shoot dry weight and N content compared with SF-0 (Figs.3-B-E and 5-D).Moreover, the N content was significantly and positively correlated with leaf age, shoot length, stem diameter and shoot dry weight (Fig.8).These results indicated that a higher SF dosage is more conducive to the shoot growth of seedlings, which is consistent with previous research.This improved growth is related to the increases in NR activity and chlorophyll content by SF, which promoted nitrogen absorption and photosynthetic accumulation of the plants (Fig.5-A-D).However, the number of root tips and root length both showed downward trends with an increase in the SF dosage (Fig.4-A and B), and the N content was significantly and negatively correlated with root length and root tips (Fig.8), most likely because (i)there are physical barriers which prevent the root from growing downward after the SF was spread in the trays;and (ii) higher N rates lead to toxicity and inhibition of root growth, especially in SF-30 and SF-40.This also resulted in a significant reduction in the root entanglement force (Fig.7-A).However, a proper SF dosage increases root dry weight and root vigor (Fig.4-E and F), which is beneficial to the uptake of nutrients and accumulation of substances in the plants.The rooting ability of rice seedlings is often used to test the quality of seedlings,which is mainly determined by the length or dry weight of new roots (Ren 2007).This study found that the rooting ability was higher in high SF dosage treatments (SF-20 and SF-30) compared to the low SF dosage treatments(SF-0 and SF-10) (Fig.4-G), which may be related to the higher dry weights and starch contents of the shoots in the former (Renet al.2007).

Environmental stresses and physical injuries are well-known factors that cause oxidative damage to plants and induce lipid peroxidation and cellular death(Takahashiet al.2017).Similarly, MDA is a product of lipid peroxidation in plant cells, and it is regarded as an important biomarker for assessing the stress physiology of plants.However, plants also possess several counter mechanisms to prevent severe lipid oxidation amid the over production of reactive oxygen species.One of these systems is the antioxidative defense mechanisms of plants,that include important antioxidant enzymes like SOD, POD,CAT and others.These enzymes remove excess reactive oxygen species, protect the cell membrane structure and reduce the damage caused by reactive oxygen species,thereby enhancing the ability of the plants to adapt or resist external adversity (Kadiogluet al.2011; Naharet al.2016; Yoon-Haet al.2017).This study found that the low and high SF dosage treatments exhibited higher MDA levels (Fig.6-D), which indicates that the seedlings are under stress, including by poisoning and the physical obstruction caused by excess fertilizer or lack of fertilizer.In addition, there were significant and negative correlations between the MDA content and SOD/CAT activities (Fig.8).Correspondingly, both low and high SF dosage treatments significantly reduced the CAT and POD activities, resulting in an inability of the plants to scavenge reactive oxygen species and accumulate MDA (Fig.6-B-D).Higher levels of MDA will damage the membranes or cells and affect the normal physiological metabolism, which is unfavorable for seedling growth (Yanet al.2021).This may also be the reason for the higher seedling vigor of CK and SF-20.Altogether, the results indicate that an optimal supply of SF is necessary to produce seedlings in a better growth state and improve the seedling quality.

4.2.Effects of different SF dosages on mechanical transplantation quality

The vigor of nursery-seedlings is a key factor that determines the post-transplantation rice population and the growth and yield of transplanted rice seedlings(Sasaki 2004; Nagasakaet al.2007).From this perspective, our results showed that SF treatments significantly affect various agronomic attributes,including plant height, dry weight, seedling rate etc.,and impact the effectiveness and quality of mechanical transplantation.Without the addition of any SF (SF-0),the floating seedling rate increased, which may be related to the lower dry weight.Moreover, the higher applications of SF significantly reduced the number of seedlings per hill (Table 2), which was mainly related to the lower seedling rate (Fig.3-F).The HLMS previously developed by a Japanese group showed inferior quality attributes for mechanical transplanting as compared to the traditional soil-grown rice seedlings, and studies found that the damaged seedling rate ranged between 30-50%, ultimately limiting the rice yield (Tasakaet al.1997; Tasaka 1999).In contrast, our TLMS showed a damage rate of only 1.28-1.74% following mechanical transplantation, and the rates of floating seedlings and missing hills were both lower, mainly due to the use of a novel seedling cultivation matrix and an optimized seedling cultivation management method based on HLMS.Notably, among the SF treatments, the seedling quality and mechanical transplantation quality of SF-20 were comparable to CK and better than the other SF treatments.However, the yield of SF-20 was higher than that of CK, which could be related to the residual SF that was entangled in the roots.Moreover, the strong entanglement between the root system of TLMS and the nursery substrate containing SF reduced the damage that occurred during manual handling and mechanical processing in the process of transplanting the rice seedlings.These attributes have significant value for mechanical transplantation, particularly for hybrid rice with a low seeding rate and higher seed price (Chauhanet al.2011).

4.3.Effects of different SF dosages on yield and economic benefits

A sufficient supply of nitrogen fertilizer in the rice nursery may help rice seedlings to produce more tillers and a higher yield after mechanical transplantation.For example, the application of 100 kg ha-1of urea resulted in a post-transplantation increase in the number of tillers and grain yield (Gomosta 2004).Similarly, Pandaet al.(1991) reported that the rice yield increased up to 2.5-fold due to an increase in grain weight, depending upon the different doses of nitrogen that were supplied to the rice nursery plants before their transplantation.Our results also showed that, compared with SF-0,the other SF treatments significantly increased the tiller numbers after transplanting, thereby increasing the number of panicles and yield at maturity, but the various SF dosages had no significant effect on grain weight (Table 3).This is related to the role of SF in improving the seedling quality and providing nutrients for root absorption after transplanting, thereby promoting the early growth of the rice seedlings.In addition, burying the fertilizer near the roots is conducive to its absorption by rice roots, thereby promoting the initial growth of the rice after transplanting and increasing the yield (Liuet al.2017; Menget al.2017).This study found that the tiller numbers of SF-10 and SF-30 were less than that of CK due to the poor quality of seedlings and machine-transplanting at the early stage after transplanting.However, there were no significant differences in the tiller numbers between the two treatments and CK at the later stage of tillering, and the yields were also comparable to that of CK (Fig.9).These results could be related to the remaining SF that was entangled in seedling roots, which would have been released quickly during the tillering stage and supplied nutrients for seedling growth (Fig.2-B), thereby promoting the tillering,especially in the SF-30 treatment.Compared with CK,SF-20 had significantly increased numbers of tillers and panicles, thereby increasing the yield, which might also be related to the promotion of nutrient absorption and growth by SF during the initial growth of the transplanted rice seedlings.Moreover, the reduction of material and labor in the seedling cultivation costs in the SF treatments made it more profitable (Tables 1 and 4), which indicates that the application of 20 g/tray PCCF-3M in TLMS is an economical and efficient method of seedling cultivation.

5.Conclusion

The results of the present study show that the use of CRFs is an effective and economical strategy for improving soil-less seedling production by TLMS for the mechanical transplantation of rice.The results showed that SF-20 could prove very effective in promoting the seedling growth and yield.Moreover, with reduced production costs, the gross margin of SF-20 was better than that of CK.Taken together, the present study addressed an important problem related to nutrient management in the current soil-less nursery production methods.Our future research will systematically study the influences of various seedling cultivation methods (such as seedling carrier, water management, and medium)on seedling quality, and the relationships between them,in order to further explore the technology of soil-less seedling production and promote the growth and yield of machine-transplanted rice.

Acknowledgements

This work was funded by the National Natural Science Foundation of China (31871573), the Key Research and Development Program of Jiangsu Province, China(BE2017369) and the Jiangsu Agriculture Science and Technology Innovation Fund, China (JASTIF) (CX(18)1002).

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.2023.05.007