Green manuring reduces cadmium accumulation in rice: Roles of iron plaque and dissolved organic matter.

In southern China, winter green manure is widely used in rice cropping systems for improving grain yields and soil fertility. Cd pollution has recently been reported in some of these paddy fields. Research on the in-depth understanding of how green manuring affects Cd absorption in rice is limited. This study aimed to investigate the impacts of different green manures, including single plantation and mixed plantation on the absorption of Cd by rice and explore the underlying mechanisms. Pot experiments demonstrated that compared with winter fallow-rice, green manuring treatments considerably decreased rice Cd content, promoted the conversion of bioavailable Cd fraction into a more stable form, induced the formation of iron plaque, and increased the content of humic-like fraction (HF) in soil dissolved organic matter (DOM). Treatment with mixed plantation resulted in a greater decrease in rice Cd content and an increase in HF and iron plaque contents than single plantation. Hydroponic experiments confirmed that both iron plaque and green manure-derived DOM significantly reduced the Cd content in rice seedlings. In conclusion, green manure incorporation is an efficient measure for the safe utilization of Cd-contaminated soil, and mixed plantation of different green manures exerts stronger effects.

Green manuring relocates microbiomes in driving the soil functionality of nitrogen cycling to obtain preferable grain yields in thirty years.

Fertilizers are widely used to produce more food, inevitably altering the diversity and composition of soil organisms. The role of soil biodiversity in controlling multiple ecosystem services remains unclear, especially after decades of fertilization. Here, we assess the contribution of the soil functionalities of carbon (C), nitrogen (N), and phosphorus (P) cycling to crop production and explore how soil organisms control these functionalities in a 33-year field fertilization experiment. The long-term application of green manure or cow manure produced wheat yields equivalent to those obtained with chemical N, with the former providing higher soil functions and allowing the functionality of N cycling (especially soil N mineralization and biological N fixation) to control wheat production. The keystone phylotypes within the global network rather than the overall microbial community dominated the soil multifunctionality and functionality of C, N, and P cycling across the soil profile (0-100 cm). We further confirmed that these keystone phylotypes consisted of many metabolic pathways of nutrient cycling and essential microbes involved in organic C mineralization, N2O release, and biological N fixation. The chemical N, green manure, and cow manure resulted in the highest abundances of amoB, nifH, and GH48 genes and Nitrosomonadaceae, Azospirillaceae, and Sphingomonadaceae within the keystone phylotypes, and these microbes were significantly and positively correlated with N2O release, N fixation, and organic C mineralization, respectively. Moreover, our results demonstrated that organic fertilization increased the effects of the network size and keystone phylotypes on the subsoil functions by facilitating the migration of soil microorganisms across the soil profiles and green manure with the highest migration rates. This study highlights the importance of the functionality of N cycling in controlling crop production and keystone phylotypes in regulating soil functions, and provides selectable fertilization strategies for maintaining crop production and soil functions across soil profiles in agricultural ecosystems.

Exploring site-specific N application rate to reduce N footprint and increase crop production for green manure-rice rotation system in southern China.

Milk vetch (Astragalus sinicus L.) is leguminous green manure (GM) which produces organic nitrogen (N) for subsequent crops and is widely planted and utilized to simultaneously reduce the use of synthetic N fertilizer and its environmental costs in rice systems. Determination of an optimal N application rate specific to the GM-rice system is challenging because of the large temporal and spatial variations in soil, climate, and field management conditions. To solve this problem, we developed a framework to explore the site-specific N application rate for the low-N footprint rice production system in southern China based on multi-site field experiments, farmer field survey, and process-based model (WHCNS_Rice, soil water heat carbon nitrogen simulator for rice). The results showed that a process-based model can explain >83.3% (p < 0.01) of the variation in rice yield, aboveground biomass, crop N uptake, and soil mineral N. Based on the scenario analysis of the tested WHCNS_Rice model, the simple regression equation was developed to implement site-specific N application rates that considered variations in GM biomass, soil, and climatic conditions. Simulation evaluation on nine provinces in southern China showed that the site-specific N application rate reduced regional synthetic N fertilizer input by 29.6 ± 17.8% and 65.3 ± 23.0% for single and early rice, respectively; decreased their total N footprints (NFs) by 23.4% and 49.3%, respectively; and without reduction in rice yield, compared with traditional farming N practices. The reduction in total NF was attributed to the reduced emissions from ammonia volatilization by 35.2%, N leaching by 28.4%, and N runoff by 32.7%. In this study, we suggested a low NF rice production system that can be obtained by combining GM with site-specific N application rate in southern China.

Synergistic effects of diazotrophs and arbuscular mycorrhizal fungi on soil biological nitrogen fixation after three decades of fertilization.

Biological nitrogen (N) fixation (BNF) via diazotrophs is an important ecological process for the conversion of atmospheric N to biologically available N. Although soil diazotrophs play a dominant role in BNF and arbuscular mycorrhizal fungi (AMF) serve as helpers to favor BNF, the response of soil BNF and diazotrophic communities to different long-term fertilizations and the role of AMF in diazotrophs-driven BNF are poorly understood. Herein, a 33-year fertilization experiment in a wheat-maize intercropping system was conducted to investigate the changes in soil BNF rates, diazotrophic and AMF communities, and their interactions after long-term representative fertilization (chemical fertilizer, cow manure, wheat straw, and green manure). We found a remarkable increase in soil BNF rates after more than three decades of fertilization compared with nonfertilized soil, and the green manure treatment rendered the highest enhancement. The functionality strengthening was mainly associated with the increase in the absolute abundance of diazotrophs and AMF and the relative abundance of the key ecological cluster of Module #0 (gained from the co-occurrence network of diazotrophic and AMF species) with dominant diazotrophs such as Skermanella and Azospirillum. Furthermore, although the positive correlations between diazotrophs and AMF were reduced under long-term organic fertilization regimes, green manuring could reverse the decline within Module #0, and this had a positive relationship with the BNF rate. This study suggests that long-term fertilization could promote N fixation and select specific groups of N fixers and their helpers in certain areas. Our work provides solid evidence that N fixation and certain groups of diazotrophic and AMF taxa and their interspecies relationship will be largely favored after the fertilized strategy of green manure.

Co-incorporation of Chinese milk vetch (Astragalus sinicus L.), rice straw, and biochar strengthens the mitigation of Cd uptake by rice (Oryza sativa L.).

Soil cadmium (Cd) contamination is becoming a widespread concern because of its threat to global ecosystem health and food security. Co-incorporation of Chinese milk vetch (MV) and rice straw (RS) is a common agricultural practice in Southern China; however, the effects of combining these two materials with biochar on Cd bioavailability remain unclear. This study investigated the effects of MV, RS, rape straw biochar (RB), iron-modified biochar (FB), and their combinations on Cd uptake by rice through incubation and field experiments. The results showed that compared with the control without material input (CK), MV + RS (MR), MV + RS + RB (MRRB), and MV + RS + FB (MRFB) considerably reduced the Cd concentration in brown rice by 61.20 %, 65.38 %, and 62.65 %, respectively. Furthermore, the treatments increased the formation of iron­manganese plaque (IMP) at different growth stages; MRRB and MRFB exhibited the highest increase rates among the treatments. Quantitatively, the Fe plaque and Mn plaque were increased by 20.61 %-47.23 % and 80.18 %-172.74 %, respectively. Compared with CK, the MRRB and MRFB treatments reduced the soil available Cd by 35.09 %-54.45 % and 38.20 %-50.20 %, respectively, at all stages. This decrease was substantially lower than that observed in the MV, RS, and MR treatments. Similar trends were observed in the incubation experiment. Additionally, the Community Bureau of Reference Sequential Extraction Analysis indicated that the MRRB and MRFB treatments converted the bioavailable Cd fractions into a stable form. Partial least squares path model and redundancy analysis revealed that pH was the major factor influencing Cd bioavailability. This study emphasized that the dual impact factors from the enhancement of Cd passivation capability and IMP formation jointly result in the reduction of Cd uptake by rice. Consequently, the co-incorporation of MV, RS, and biochar is promising for remediating Cd-contaminated paddy soils in Southern China.

The chromosome-level genome assembly of Astragalus sinicus and comparative genomic analyses provide new resources and insights for understanding legume-rhizobial interactions.

The legume species Astragalus sinicus (Chinese milk vetch [CMV]) has been widely cultivated for centuries in southern China as one of the most important green manures/cover crops for improving rice productivity and preventing soil degeneration. In this study, we generated the first chromosome-scale reference genome of CMV by combining PacBio and Illumina sequencing with high-throughput chromatin conformation capture (Hi-C) technology. The CMV genome was 595.52 Mb in length, with a contig N50 size of 1.50 Mb. Long terminal repeats (LTRs) had been amplified and contributed to genome size expansion in CMV. CMV has undergone two whole-genome duplication (WGD) events, and the genes retained after the WGD shared by Papilionoideae species shaped the rhizobial symbiosis and the hormonal regulation of nodulation. The chalcone synthase (CHS) gene family was expanded and was expressed primarily in the roots of CMV. Intriguingly, we found that resistance genes were more highly expressed in roots than in nodules of legume species, suggesting that their expression may be increased to bolster plant immunity in roots to cope with pathogen infection in legumes. Our work sheds light on the genetic basis of nodulation and symbiosis in CMV and provides a benchmark for accelerating genetic research and molecular breeding in the future.

Co-utilizing milk vetch, rice straw, and lime reduces the Cd accumulation of rice grain in two paddy soils in south China.

The danger posed by cadmium (Cd) pollution to rice production is continuously increasing. Co-utilizing milk vetch (Astragalus sinicus L.) and rice straw is a good practice for rice yield and soil fertility in south China. However, its effects on Cd availability in soil-rice systems remain unclear. A micro-plot trial of two typical paddy soils (alluvial sandy soil and reddish clayey soil) in south China was conducted to investigate the effects of milk vetch, rice straw, lime, and their combined application on Cd availability and the related mechanisms. Soil chemical properties, CaCl2-extractable Cd (CaCl2-Cd), total content of Cd (Total-Cd), Cd fractionation (BCR sequential-extraction method), and Cd accumulation in rice were measured. Results showed that the co-utilization of milk vetch, rice straw, and lime (GRFL) decreased the Cd content in rice grain by 91.43% and 15.63% in early rice of two soils, respectively. Cd was not detected in late rice grains. CaCl2-Cd decreased by 0.025 mg kg-1 in late rice of alluvial sandy soil, 0.057 and 0.044 mg kg-1 decreased in early and late rice of reddish clayey soil, and Total-Cd decreased by 19.4% and 9.1% for early rice of two soils, respectively. Co-utilizing milk vetch, rice straw, and lime changed the distribution of different chemical forms of Cd, decreased the content of bioavailable Cd in soil by reducing the Aci-Cd and RedCd, and benefited the formation of more stable residual fraction (ResCd). Redundancy analysis showed that the improvement in soil pH, dissolved organic matter (DOM), and other soil properties was the main cause of the transformation of Cd form. Among the soil properties, pH and DOM had the greatest impacts on Cd availability. In conclusion, co-utilizing milk vetch and rice straw can alleviate the danger of soil Cd in rice production, and this effect could be strengthened by applying lime.

Succession of fungal community and enzyme activity during the co-decomposition process of rice (Oryza sativa L.) straw and milk vetch (Astragalus sinicus L.).

The co-incorporation of rice straw (RS) and milk vetch (MV) into paddy fields has been increasingly applied as a sustainable farming practice in southern China. Our previous study revealed the contribution of bacteria to the co-decomposition of the RS and MV mixture, although additional underlying factors driving the co-decomposition process need to be clarified. The present study further determined the succession of fungal communities and enzyme activity in the co-decomposition process of the RS and MV mixture. The results showed that non-additive synergistic effects on biomass loss were observed in 55.6% of the sampled RS and MV mixture during the co-decomposition process, stimulating mixture decomposition. Overall fungal abundance was 19.6-30.6% higher in the RS and MV mixture throughout the study than in the single residue. Fungal diversity and community structure were mainly affected by the sampling date rather than the type of residue. Specifically, mixing RS and MV significantly increased the abundance of Peziza sp. and Reticulascus tulasneorum (lignocellulose- and lignin-decomposing fungi) and exhibited higher activities of C- and N-related hydrolases than monospecific residues. Random forest (RF) models showed that bacteria contributed more to the residue decomposition and activities of C-related hydrolases, N-related hydrolases, and oxidases than fungi. However, both RF and partial least squares path models revealed that fungal abundance and community structure directly or indirectly affected the residue decomposition rate. These findings showed that mixing RS and MV could stimulate their decomposition by enhancing C-related hydrolase activity and Peziza sp. and Reticulascus tulasneorum abundance.

Spectroscopic characteristics of water-extractable organic matter from different green manures1.

Water-extractable organic matter (WEOM) plays an important role in many chemical processes and in soil organic matter accumulation. Large amounts of WEOM can be released by green manure when being incorporated into the soil. However, the characteristics of WEOM extracted from different green manures (GMs) are unclear. In this study, WEOM samples were extracted from Chinese milk vetch (Astragalus sinicus L), radish (Raphanus sativus L), ryegrass (Lolium perenne L), hairy vetch (Vicia villosa L), February orchid (Orychophragmus violaceus) and rye (Secale cereale L) and their characteristics were studied by spectroscopic analysis. WEOM generated from legume GMs contained more water-extractable organic carbon (WEOC) than other GMs and Chinese milk vetch was the highest. UV-visible spectroscopy analysis revealed that all the samples were rich in C=C and C=O functional groups. Carboxylic acid, alcohol, phenol, fatty acids, aliphatic aromatic and amide compounds were found by FTIR spectroscopy in these WEOM samples. WEOM derived from Chinese milk vetch contained more carboxylic acid and inorganic sulphates only appeared in WEOM extracted from the radish. The aromaticity index, humification index, and FTIR absorption ratio 1650/2925 and 1650/2850 showed that WEOM extracted from Chinese milk vetch and hairy vetch had a relatively lower aromaticity and humification degree than other GMs. The fluorescence regional integration (FRI) analysis showed that Chinese milk vetch and hairy vetch had a higher portion of protein-like substance fractions than other GMs. We may deduce that WEOM generated from Chinese milk vetch and hairy vetch has higher chemical activity than other GMs.

Using milk vetch (Astragalus sinicus L.) to promote rice straw decomposition by regulating enzyme activity and bacterial community.

The present study determined the dynamic changes of enzyme activity and bacterial community in rice straw (RS) and milk vetch (MV) co-decomposing process. Results showed that mixing RS and MV promoted decomposition. The mixture enhanced ß-glucosidase and ß-cellobiohydrolase activities relative to its monospecific residue during the mid-late stage of decomposition. The mixture enhanced Enterobacteriaceae (monosaccharide decomposing bacteria) abundance during the initial stage of decomposition, and the abundance of Hydrogenispora, Bacteroides, Ruminiclostridium, and Acidobacteriaceae that could hydrolyze fiber during the mid-late stage of decomposition relative to single RS and MV, respectively, which would benefit mixture decomposition. Furthermore, more interconnected and competitive relations existed between the bacteria in the mixture. These results indicated that mixing RS and MV promoted residue decomposition by increasing hydrolytic enzyme activities and changing bacterial community. This study concluded that co-incorporating RS and MV may be recommended as a promising practice for the efficient utilization of RS resources.

[Effects of Green Manures on Soil Dissolved Organic Matter in Moisture Soil in North China].

Soil dissolved organic matter (DOM) plays an important role in the biogeochemistry of carbon, nitrogen, and phosphorus and in the transport of heavy metals and pesticides in soil. In moisture soil, green manures and soils were sampled in situ at the ploughed stage of green manures. A 56-day laboratory incubation experiment was conducted to simulate the dynamic changes of soil DOM influenced by the decomposition of green manures, the green manures were Hairy vetch (Vicia villosa Roth.), February orchid (Orychophragmus Violaceus L.), Rye (Secale cereale L.), the soil without green manure was used as a control (CK). The composition and ultraviolet-visible spectrum parameters of soil DOM were investigated at different incubation stages. Results showed that green manures could increase the dissolved organic carbon (DOC), the total organic acids (TOAs) and total carbohydrate (TCs) contents, and all treatments were reached a peak on the 1st day and decreased later. Hairy vetch affected DOC and TOAs most and were increased by 114.01% and 109.10% higher than CK respectively at the 1st and 14th day. Rye influenced the total carbohydrate (TCs) most and was maximumly 323.18% higher than CK at the 42nd day. Green manures could increase the dissolved organic nitrogen (DON) content, DON in all green manure treatments increased on the 1st day, decreased several days later and increased again after 20～30 d. Hairy vetch effected DON best and was 305.83% higher than CK at the 42nd day. All green manures increased in SUVA254, SUVA260, SUVA272, SUVA280 and SAUC240-400, while decreasing in A250/A365 and A240/A420. The PCA analysis of ultraviolet-visible spectrum parameters showed that SUVA254, SUVA260, SUVA272, SUVA280 had a high positive correlation between each other, and the same situation was found between A250/A365 and A240/A420. Among them, SAUC240-400 was a key factor parameter featuring the characteristics of DOM. The results suggested that green manures could increase the contents of DOM and its aromaticity, hydrophobic percentage, humification degree and average molecular weight, and could be increased and the stability of DOM could be enhanced accordingly. The ultraviolet-visible spectrum parameters could indicate the changes of characteristics of DOM in this study.

Diffusive Gradients in Thin Films as a Reference Method for Assessing Soil Phosphorus by Visual and Near-Infrared Spectroscopy.

Phosphorus (P) deficiency is a severe challenge in many agricultural areas around the globe, while at the same time, aquatic environments are threatened by leaching and runoff of excess P in other areas. Accurate, cheap, and rapid assessment of crop P needs and risk of P loss is therefore necessary to optimize the use of P fertilizer worldwide. The purpose of this study was to develop a method to predict soil P concentrations by visual and near-infrared spectroscopy using reference P concentrations determined by diffusive gradients in thin films (DGT); Olsen P results were included for comparison. The study was conducted on paddy soils from six main rice-producing ( L.) provinces in southern China. Using DGT P as a reference resulted in a better visual and near-infrared calibration to predict soil P concentrations, as compared with using Olsen P.

[Long-Term Different Fertilizations Changed the Chemical and Spectrum Characteristics of DOM of the Irrigation-Desert Soil in North-Western China].

By using Ultraviolet-visible Spectrometry, Fourier Transform Infrared Spectrometer and Elemental Analyzer, spectrum and chemical characteristics of soil DOM affected by long-term different fertilizations were investigated in irrigation-desert soil in North-western China based on an experiment started from 1988. Four different fertilization treatments were included, i. e., organic fertilizer (OF), green manure (GM), chemical fertilizer (CF) and a control of no fertilization (CK). The results showed that fertilization could increase the contents of DOM. Compared to CK, the treatments of OF, GM, CF increased the dissolved organic carbon (DOC) by 37%, 29%, 16%; increased the dissolved nitrogen (DON) by 334%, 257%, 182%; increased the total carbohydrate (TCs) by 90%, 25%, 2%; and increased the total organic acids (TOAs) by 195%, 116%, 58%; respectively. Furthermore, DOC, DON, TCs, and TOAs in the OF treatment were significantly higher than those in CK, they were also significantly higher in the GM and CF treatments except for TCs. The ultraviolet-visible analysis showed that fertilizations enhanced the SUVA(254), SUVA(260), SUVA(272) and SUVA(280) of DOM, indicating that fertilizations increased the aromatic and hydrophobic percentage, humification degree, and average molecular weight, and thus resulting in more stability of DOM. Same trends were showed for all the 4 ultraviolet spectrum absorption values in different fertilizations, i. e., the strongest effect was found in the OF treatment, and then was the GM treatment and CF treatment successively. From the results by the Fourier Transform Infrared Spectrometry, the characteristic peak of aromatic in the OF treatment was observed shifting from 1 625 to 1 649 cm(-1), which was close to the characteristic peak of humin, suggesting that the aromaticity of DOM in the OF treatment was higher than the other treatments. The characteristic peaks of C-O at 1 260-1 000 cm(-1) belonging to sugar, alcohol, and carboxylic acid were highest in the GM treatment, showing that the green manure could increase rich oxygen radicals. The highest characteristic peaks of N-H at 3 559, 3 419 and 1 456 cm(-1) were observed in the CF treatment, indicating that the chemical fertilizer could increase amine substances. The contents of C, O and N in the OF, GM, CF treatments were also increased respectively according to the elemental analysis.

[Temporal and spatial variations of soil NO(3-)-N in Orychophragmus violaceus/spring maize rotation system in North China].

The February orchid (Orychophragmus violaceus)-spring maize rotation system is established to resolve the problems caused by the expansion of fallow fields in North China. Based on a site-specific experiment, temporal and spatial variations of soil NO(3-)-N were investigated during the period from February orchid incorporation to maize harvest. The results showed that the nitrate content in soil profiles not only showed a temporal characteristic, i. e., increasing at the beginning of the maize season and decreasing then after, but also showed a spatial characteristic, i. e., the gradual occurrence of the peak of nitrate content from shallower to deeper layer with the growth season of maize. Meanwhile, incorporation of February orchid could affect temporal and spatial variations of soil NO(3-)-N. February orchid planting reduced the soil NO(3-)-N accumulation in the profile of 0180 cm. After incorporation of February orchid, similar characteristics were observed at the seedling and bell stages of maize, i. e., the soil NO(3-)-N mainly stayed in the profile of 0-20 cm, and NO(3-)-N concentrations in the treatments with February orchid were higher in 0-100 cm layer and lower in 100-180 cm layer than those of the treatments without February orchid. After tasseling stage, opposite phenomena were found, and the soil NO(3-)-N content was all relative low. Overall, incorporation of February orchid could increase the storage capacity of soil NO(3-)-N in the profile of 0-180 cm.