Effects of long-term green manure application on the content and structure of dissolved organic matter in red paddy soil

GAO Song-juan , GAO Ju-sheng , CAO Wei-dong , ZOU Chun-qin HUANG Jing , BAl Jinshun DOU Fu-gen

1 College of Resources and Environmental Sciences, Center for Resources, Environment and Food Security/Key Laboratory of Plant-Soil Interactions, Ministry of Education, China Agricultural University, Beijing 100193, P.R.China

2 Key Laboratory of Plant Nutrition and Fertilizer, Ministry of Agriculture/Institute of Agricultural Resources and Regional Planning,Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China

3 Red Soil Experimental Station in Hengyang, Chinese Academy of Agricultural Sciences, Qiyang 426182, P.R.China

4 Soil and Fertilizer Institute, Qinghai Academy of Agriculture and Forestry Sciences, Qinghai University, Xining 810016, P.R.China

5 Texas A&M AgriLife Research Center at Beaumont, Texas 77713, USA

Abstract Dissolved organic matter (DOM) plays important roles in soil biogeochemistry activity and nutrients transportation in soils,but studies regarding the long-term effects of green manures on the content and structure of DOM in red paddy soil have not been reported yet. A long-term green manure experiment established in 1982 was utilized to test the DOM contents in different treatments, and the spectral characteristics of DOM were investigated by using ultraviolet-visible (UV-Vis)spectrometry and Fourier transform infrared (FTIR) spectrometry. The experiment included four cropping systems: ricerice-milk vetch (RRV), rice-rice-rape (RRP), rice-rice-ryegrass (RRG) and rice-rice-winter fallow (RRF), among them, milk vetch, rape, and ryegrass are popular winter green manure species in southern China. The results showed that the content of dissolved organic carbon (DOC), which is widely used to estimate the concentration of DOM, was significantly promoted after the incorporation of green manures compared with the other sampling stages. The contents of aromatic groups and the degree of humification of DOM increased in RRV and RRP, suggesting more complex compositions of the soil DOM after long-term application of milk vetch and rape. The contents of phenol, alcohol and carboxylic acid group at the mature stage of early rice were significantly higher than those at the stage of after green manures turned over, especially for the RRV treatment. The absorption ratio of FTIR indicated that winter plantation of rape increased the aromatic-C/aliphatic-C ratio, and ryegrass increased the aromatic-C/carboxyl-C ratio. In conclusion, long-term planting of milk vetch and rape as green manures increased the degree of aromaticity, humification and average molecular weight of DOM, and made the DOM more stable in red paddy soil.

Keywords: green manure, red paddy soil, dissolved organic matter, ultraviolet-visible spectra, Fourier transform infrared spectra

1. lntroduction

Dissolved organic matter (DOM) is one of the most active compositions in soils, which can affect the transport and decay of soil pollutants, and the availability of soil nutrients(C, N, P, S, etc.) (Liuet al.2013; Zhuet al.2013). In addition,DOM is an important factor to influence soil microbial growth and metabolism, as well as the turnover of soil organic matter (Guppyet al.2005). DOM composes of both low molecular compound, i.e., free amino, carbohydrate and organic acid, and high molecular compound, i.e., enzyme,amino sugar, humus and polyphenols, etc. Most of its composition is bioavailable and can be easily absorbed and utilized by soil microorganisms, and hence the quantity and quality of DOM are main indicators of soil quality (Cook and Allan 1992; Kalbitzet al.2003a, b; Marschner and Kalbitz 2003). As reviewed by Kalbitzet al.(2000), the turnover of soil DOM was emphasized as major pathways of biogeochemical cycling in agricultural ecosystem.

The study of DOM mainly focus on their contents, the functional groups, compositions and properties, which are considered to reflect the transformation of soil nutrient.Spectral analysis is widely applied as a fast way to measure DOM content without destroying the samples (Fuet al.2016). Ultraviolet-visible (UV-Vis) spectrum (Hunt and Ohno 2007; Heet al.2011; Uyguner-Demirelet al.2011) and Fourier transform infrared (FTIR) spectrum (Blodauet al.2009; Heet al.2012) are widely used in the investigation of DOM. The UV-Vis parameters and absorption ratio of FTIR(1 650/2 930, 1 650/2 850, 1 650/1 050, etc.) and spectral peaks are the common indicators of functional groups and structure of DOM (Blodauet al.2009; Heet al.2012). By using these spectral methods, we can study deeply about the composition changes of DOM under different management practices and soil conditions.

Numerous biotic and abiotic factors affect the content and structure of soil DOM, including soil physiochemical properties, climate, crop types and agricultural management practices (Longet al.2015; Shanget al.2015; Smebyeet al.2016). The fertilization and crop residue management are the prominent management factors which had profound influences on soil DOM (Qiuet al.2015; Shanget al.2015). The turnover of DOM directly improved soil organic matter content and soil nutrients supply, and increased the aggregate of soil and the capacity of water and nutrients through appropriate tillage practices (Guggenberger and Kaiser 2003). The exogenous carbon input had different influences on the structure and composition of soil DOM.Zhanget al.(2017) found that the addition of biochar increased the content of DOM, but the composition was not changed. The incorporation of green manures bring large amount of plant residues into soils, and the decomposition of plant residues may cause the change of soil DOM compositions and SOC sequestration.

The usage of winter green manures in paddy soil is a traditional and effective cultivation pattern in southern China for maintaining soil fertility. A 30-year long-term experiment documented that winter green manure increased 18.8–28.8% of crop yield compared with winter fallow (Gaoet al.2013). Previous study reported that the main sources of soil DOM included soil organic matter (SOM), plant litter, root exudations and microbial metabolism (Kalbitzet al.2000). In agricultural ecosystem, organic matter was often considered the main DOM sources of cropland. The application of green manures bring lots of fresh organic matter into soils, and could promote the renewal of soil organic matter and soil nutrients efficiently. The decomposition of green manures enhanced the growth and activity of soil microbial (Elfstrandet al.2007), and changed the structure and compositions of soil DOM (Wershaw 2004). In southern China, milk vetch,winter rape and ryegrass were commonly used as winter green manure in rice cropping system. By now, how the DOM is affected by long-term application of different kinds of green manure in paddy soil is still unknown. Therefore,the objectives of this study were to evaluate the effects of green manures on DOM contents, and to investigate their structure and composition in red paddy soil, by using UV-Vis and FTIR spectrometry methods.

2. Materials and methods

2.1. Field description

The field experiment is located at Hengyang Red Soil Experimental Station (26°45′N, 111°52′E; elevation 150 m)of Chinese Academy of Agricultural Sciences in Hunan Province. The local average annual temperature is 18.3°C,the accumulated temperature above 10°C is 5 600°C, the frost-free period is 300 days, and the annual precipitation is 1 250 mm. The soil is classified as Ferralic Cambisol based on FAO classification system (FAO 2006), which is a typical red paddy soil in southern China, and originally developed from quaternary red clay. Before the experiment started,the soil pH was 6.5, the SOM, total nitrogen (TN), total phosphorus and total potassium were 20.1, 0.94, 0.66 and 11.5 g kg–1, respectively, the available nitrogen, available phosphorus (AP) and available potassium (AK) were 156,7.2 and 176 mg kg–1, respectively.

2.2. Experimental design and soil sampling

The cropping system of the experiment is double rice-green manure or double rice-winter fallow, and started from 1982.The experiment was set up as a completely randomized block design in triplicate with the plot size of 2.5 m×15.0 m. Four treatments were rice-rice-milk vetch (RRV), rice-rice-winter rape (RRP), rice-rice-ryegrass (RRG), and rice-rice-winter fallow (RRF, nothing is planted in winter as control). The same rates of N, P2O5, and K2O fertilizer were applied for both early and late rice, at 153, 84, and 129 kg ha–1, respectively.Rice straw was removed from the plots after rice harvesting.Seeding rates of milk vetch (Astragalus sinicusL.), winter rape (Brassica napusL.) and ryegrass (Lolium multiflorum)were 37.5, 7.5 and 15.0 kg ha–1, respectively. No fertilizer was applied during the growth of winter green manures.The green manure crops were incorporated into soilin situat a depth of 20 cm at their full-bloom stage. Early rice was transplanted in late April, usually 15 days after incorporation of green manures. In 2013, green manures were incorporated into soil on 30 March, and the early rice was transplanted on 25 April and harvested on 13 July. Aboveground biomass samples of each winter green manure were collected before incorporation. Biomass yields (fresh weight) and nutrient contents of green manures were listed in Table 1. The early rice yields in 2013 of RRV, RRP, RRG and RRF were 7 657,7 773, 7 453 and 5 683 kg ha–1, respectively. Soils were sampled before green manures incorporation (S0, 29 March,the full-bloom stage of milk vetch and winter rape), 22 days after green manures turned over (S1, 21 April, before the transplantation of early rice) and at the mature stage of early rice (S2, 11 July). The topsoil of 0–20 cm was collected from 5 points of each plot, with plant materials and stones removed.Part of the collected soil sample was immediately stored at 4°C for the measurement of DOM and inorganic nitrogen, and the other was air-dried for soil chemical analysis.

2.3. Chemical analysis

Soil pH was measured with a soil to water ration of 1:2.5 (Lu 2000). SOM was determined with potassium dichromate oxidation method and TN was determined with the Kjeldahl method (Lu 2000). AP extracted using 0.5 mol L–1NaHCO3,AK extracted using 1 mol L–1CH3COONH4, and the measurements of AP and AK were following the methods of Lu (2000).-N extracted by 2 mol L–1KCl, and measured with a continuous flow analyzer (AA3, SEAL, Germany).The chemical properties of the soils are listed in Table 2.

2.4. DOM extraction and characterization

To extract the soil DOM, 10 g of fresh soil sample was mixed with 100 mL of distilled water and then shaken for 1 h at a speed of 200 r min–1. Then the solution was centrifuged at a speed of 4 000 r min–1for 1 h. After centrifugation the supernatant was vacuum-filtered through 0.45 μm millipore filter. The extracted DOM was partly stored at 4°C for the measurement of dissolved organic carbon (DOC) and UV-Vis spectroscopy, and the left was freeze-dried for a Fourier transform infrared analysis. The content of DOC was determined on a TOC analyzer (multi N/C 2100 TOC/TN, Analytikjena, Germany). UV-Vis absorption from 200 to 800 nm of DOM was measured using a UV-Vis spectrophotometer (UV-2100, Rayleigh, Beijing, China).The characteristic ultraviolet spectrum absorption values at 254 nm (SUVA254) and 280 nm (SUVA280) were calculated as absorbance divided by DOC concentration. The ratios of the absorbance at 254 and 436 nm (E254/E436), 254 and 365 nm (E254/E365), 365 and 470 nm (E365/E470) were calculated. The SUVA254value is positively correlated with humification degree of DOM (Chenet al.2003; Swietliket al.2004). The SUVA280value is used as an index to evaluate the abundance of aromatic groups, and the higher SUVA280value indicating the more complex structure of soil DOM(Chinet al.1994). E254/E436is used for helping estimate the sources of DOM (Jafféet al.2004; Huret al.2006). E254/E365is used as a surrogate for average molecular weight and aromaticity, relatively low value indicating higher average molecular weight of DOM (Doradoet al.2003; Hiriart-Baeret al.2008). E365/E470is used for characterizing the functional group in the UV-Vis range (Uyguner and Bekbolet 2005).The FTIR spectra were obtained from DOM samples mixed with KBr (1 mg of freeze-dried DOM and 100 mg of dried spectrometry-grade KBr) using the FTIR spectrometer(VERTEX70, Bruker, Germany). The spectra were recorded in 4 000–400 cm–1range at 4 cm–1resolution, and 32 scans were performed on each acquisition.

Table 1 Fresh biomass yields and nutrient contents of dry matter of green manures

Table 2 Soil chemical properties in different treatments at different sampling stages

2.5. Data analysis

Microsoft Excel 2013 and SAS 8.1 were used for data analysis; Sigmaplot 10.0 was used for making figures.

3. Results and discussion

3.1. Concentration of DOC

The DOC content is widely used to estimate the concentration of DOM (Filep and Rékási 2011). DOC plays important roles in element cycling, especially in the cycling of soil organic carbon in agricultural soils (Shanget al.2015). In this study,the DOC content had no significant differences among treatments at S0, S1 and S2 stages (Fig. 1-A). After turned over of green manures (S1 stage), the average content of DOC was 711.10 mg kg–1, which was significantly higher than that before the application of green manures (S0 stage)(the average DOC content was 428.50 mg kg–1) and the mature stage of early rice (S2 stage) (the average DOC content was 412.79 mg kg–1) (Fig. 1-B). Furthermore, the lowest DOC content after incorporation of green manures was even higher than the highest one at the other two stages. Kalbitzet al.(2000) reviewed that plant residues,root exudates and organic C decomposition are the main sources of soil DOM in farmland. The addition of organic matter into soil may accelerate mineralization and enhance the release of DOC in the soil (Kalbitzet al.2005). Green manure incorporated into soil as fresh biomass can be an important source of DOC in the field. The soil DOC content increased significantly after the incorporation of green manures, indicating the contribution by the decomposition of green manures. The decomposition of plant residues may promote the release of native soil organic carbon, and the priming effect may be the main reason of the promotion(Kuzyakov 2010). Longet al.(2015) reported that manure application resulted in a seasonal variation of soil DOC, and the production and decomposition of soil DOM influenced by temperature and humidity. The soil condition varied a lot among the three sampling stages, and the effects of rice growth on soil properties also changed in different stages.The characteristics of DOM were closely correlated with soil properties (Fanget al.2014). Besides the variation among sampling stages, the DOC content had no significant difference among 4 treatments in our study, indicating that the sampling stage played more profound influences on DOC content than treatments.

3.2. UV-Vis spectra of DOM

Fig. 1 The dissolved organic carbon (DOC) content in paddy soil under the effects of different green manures (A) and the difference among the 3 sampling stages (B). RRV, rice-rice-milk vetch; RRP, rice-rice-rape; RRG, rice-rice-ryegrass; RRF, rice-rice-winter fallow. S0, before green manures incorporation; S1, after green manures turned over; S2, the mature stage of early rice. Different lowercase letters and capital letters in the figure represent significant difference at P＜0.05 and P＜0.01 (Duncan’s test), respectively.Bars in Fig. 1-A indicate SE, and the horizontals in Fig. 1-B represent the maximum, upper quartile, median, lower quartile and minimum values, respectively.

Fig. 2 Ultraviolet-visible (UV-Vis) spectrum of soil dissolved organic matter under the effects of green manures at S1 stage (A)and S2 stage (B) in red paddy soil. RRV, rice-rice-milk vetch; RRP, rice-rice-rape; RRG, rice-rice-ryegrass; RRF, rice-rice-winter fallow. S1, after green manures turned over; S2, the mature stage of early rice.

All the samples had wide UV-Vis absorption bands in our study (Fig. 2). The trends of the absorbance were almost the same; decreasing along with the increase of wavelength.The absorbance of each treatment right after green manure incorporation (S1 stage) was obviously higher than the corresponding one collected at the mature stage of early rice (S2 stage) in the ultraviolet range (wavelength range from 200 to 400 nm), indicating that soil DOM had strong hyperchromic effect in the ultraviolet range after green manures incorporated, and the composition and structure of the functional groups were different at the 2 sampling stages. At S1 stage, RRV and RRP had higher absorption than RRG and RRF, and RRV had the highest absorption among the four treatments at S2 stage.

The characteristic ultraviolet spectrum absorption values and absorption ratios at the special wavelengths in UV-Vis range are important parameters to reveal the feature and composition of soil DOM (Ilinaet al.2014). The ultraviolet characteristics had no significant difference between the S1 and S2 stages (Table 3). RRG increased the E254/E365value at S1 stage. While no significant difference was found at S2 stage. The results illustrated that long-term application of milk vetch and rape make soil DOM more complex through improving the aromaticity and humification degree. Opposite to milk vetch and rape, winter plantation of ryegrass decreased the aromaticity and average molecular weight of soil DOM. RRV and RRG increased the E254/E436value,thus revealed that utilization of milk vetch and ryegrass changed the sources of soil DOM. The three kinds of green manures in this study had different plant characteristics.The different C/N ratio and material composition may lead to different decomposition process and resulted in various effects on soil DOM. Don and Kalbitz (2005) found that during litter decomposition, the lignin-derived compoundscontributed more to the source of produced DOC. While the inner relationship between green manure decomposition and DOM composition need further investigation.

Table 3 Characteristic ultraviolet spectrum absorption values and absorption ratios of soil dissolved organic matter under the effects of different green manures at the two sampling stages in red paddy soil

The UV-Vis parameters of the 4 treatments varied before the transplantation of early rice (S1 stage) while had no difference at the mature stage of early rice (S3 stage),showing that not only the content of DOM decreased, but also the properties became similar along with the growth of early rice. Previous study found that application of green manures in moisture soil made soil DOM more stable by increasing the aromaticity, hydrophobic percentage,humification degree and average molecular weight (Changet al.2017), and the results were consistent with our study.As one kind of high efficiency tillage practice in paddy soil,winter green manuring had noticeable influences on the structure and transformation of soil carbon by changing the characteristics of DOM.

3.3. FTlR spectra of DOM

FTIR spectrometry is a qualitative tool for monitoring functional groups and bands of the chemical compounds (Fuet al.2016). The assignments of the major FTIR spectral bands are formulated as follows (Lguiratiet al.2005; Smidtet al.2005; Madariet al.2006). The absorption peak at 3 274–3 420 cm–1reflect the O-H vibrations of the hydroxyl groups of phenols, alcohols and carboxyl functions and N-H vibrations from amides and amines. The band at 2 920–2 966 cm–1is contributed by symmetric C-H stretching in -CH3and -CH2- of aliphatic chains. The band at 1 649–1 680 cm–1is caused by C=C vibration in aromatic and C=O vibration in quinones, ketonic acids and primary amides. The absorption peak at 1 405–1 414 cm–1reflect the anti-symmetric COO–stretching, O-H deformation, C=O stretching of phenols and aliphatic C-H deformation. The band at 1 040–1 200 cm–1reflect the C-O-C stretching of carbohydrates, C=O stretching of cellulose or hemicellulose.The FTIR spectra of all the samples showed that the position of infrared characteristic absorbing peaks was similar among the 4 treatments but the peak intensity changed (Fig. 3),indicating that winter green manuring changed the amount of functional groups of soil DOM. The peak intensity also differed in different sampling stages. At the mature stage of early rice (S2 stage), the peak intensity at 3 405 cm–1was much higher than that after the incorporation of green manures (S1 stage), especially the RRV treatment. This kind of change showed that the contents of phenol, alcohol and carboxylic acid were accelerated along with the growth of early rice.

The structure changes of DOM were also monitored by the absorption ratio of FTIR (Heet al.2012). The absorption ratio 1 650/2 925 and 1 650/2 850 (aromatic-C/aliphatic-C ratio), 1 650/1 568 and 1 650/1 414 (aromatic-C/carboxyl-C ratio), 1 650/1 080 (aromatic-C/carbohydrate-C ratio) were selected to evaluate the changes of FTIR spectra after the application of different green manures in this study (Table 4).Similar to ultraviolet characteristics, the FTIR intensity ratios varied among different treatments after the turned over of green manures (S1 stage), but had no significant difference at the mature stage of early rice (S2 stage). The 1 650/2 925 and 1 650/2 850 values were significantly higher in RRP than those in RRG, while RRG had significantly higher 1 650/1 568 value than RRF. The results indicated that winter plantation of rape increased the aromatic-C/aliphatic-C ratio,and ryegrass increased the aromatic-C/carboxyl-C ratio.

Fig. 3 Fourier transform infrared (FTIR) spectra of soil dissolved organic matter under the effects of different green manures at the 2 sampling stages in red paddy soil. RRV, rice-rice-milk vetch; RRP, rice-rice-rape; RRG, rice-rice-ryegrass; RRF, ricerice-winter fallow. S1, after green manures turned over; S2,the mature stage of early rice.

As belonging to different families, the biochemical characteristics of the plant residues of milk vetch, rape and ryegrass vary from each other. In this study, longterm utilization of milk vetch improved the aromaticity and molecular weight of DOM, showing that soil DOM in the RRV treatment had more complicated compositions, while it was contrary for the application of ryegrass. Previous study reported that the change of soil conditions under different land use and management practices contributed to DOM dynamics of the terrestrial ecosystem (Chantigny 2003). Joneset al.(2014) found that the quality of DOM was the key factor to indicate the soil quality rather than the quantity of DOM. Although long-term application of winter green manure had no significant effect on DOM content,the composition and structure of DOM were changed by green manures in paddy soil. These findings could partially explain the yield difference of rice crops under different green manures in the studied cropping systems in southern China(Gaoet al.2013).

4. Conclusion

This study showed that long-term application of green manures changed the composition of soil DOM in red paddy soil. The difference among treatments showed that the long-term utilization of milk vetch and rape made the soil DOM more stable and more complex in compositions. The changes of DOM properties caused by application of greenmanures may indicate the mechanisms of green manures on soil fertility and sustainability.

Table 4 Fourier transform infrared (FTIR) intensity ratios of soil dissolved organic matterunder the effects of different green manures at the 2 sampling stages in red paddy soil

1)RRV, rice-rice-milk vetch; RRP, rice-rice-rape; RRG, rice-rice-ryegrass; RRF, rice-rice-winter fallow.

Values are means±SE (n=3); different letters within a column at the same stage represent significant difference between treatments(Duncan’s test,P＜0.05).

Acknowledgements

This study was supported by the earmarked fund for China Agriculture Research System (2013–2017) and the Chinese Outstanding Talents Program in Agricultural Sciences.