The field experiment was included four fertilizer treatments: chemical fertilizer alone (MF), rice straw and chemical fertilizer (RF), 30% organic manure and 70% chemical fertilizer (OM), and without fertilizer input as a control (CK). A randomized block design was adopted in the plots, with three replications of each treatment. And each plot size was 66.7 m2 (10.0 × 6.67 m). The field experiment were ensured that same total amount of N, phosphorus pentoxide (P2O5), potassium oxide (K2O) for MF, RF and OM treatments during early rice and late rice whole growth period, respectively. During the early rice and late rice whole growth period, the total amount of N, P2O5, K2O for MF, RF and OM treatments were 142.5, 54.0, 63.0 kg ha−1 and 157.5, 43.2, 81.0 kg ha−1, respectively. And the kind of organic manure for OM treatment was decomposed chicken manure. The kinds of chemical fertilizer were included urea, ordinary superphosphate, and potassium chloride, respectively. Before transplanting of rice seedling, air-dried rice straw were manually spread onto the soil surface and incorporated into the soil at a cultivation depth of 20 cm. During early rice and late rice whole growth period, 70% and 60% of N were applied at tillage before transplanting of rice seedling, respectively, and the remaining N were applied at top dressing stage of rice. All the P2O5 and K2O fertilizer were applied at tillage before transplanting of rice seedling. One-month-old of early rice and late rice seedling were transplanted with the density of 150,000 plants ha−1 in paddy field. Other more detail information about fertilizer management and filed arrangement were described as by Tang et al. (2018)19.
Soil sampling and samples preparation
There had ensured that permission to collect soil sampling in this experiment. Undisturbed soil samples were collected from each plot in 25 August, 2019, at the tillering stage of late rice. Three soil cores (10 × 10 × 15 cm) at the depth of 0–20 cm from each plot were collected and equally merged as representative soil samples for one replicate of each fertilizer treatment. Moist soil were gently broken apart along the natural breakpoints and passed through a 5-mm sieve to remove visible organic debris and crop root. After thorough mixing, different particle-size fractions were separated according to the method described as by Stemmer et al.20. In the present study, five particle-size fractions were obtained for each soil samples, such as > 2000 μm, 2000–200 μm, 200–50 μm, 50–2 μm and 2–0.1 μm. These particle-size fractions were then stored at room temperature for analysis of soil chemical characteristic, at 4 °C for soil extracellular enzyme analysis and − 80 °C for the molecular analysis. The SOC, total nitrogen (TN) contents and soil enzyme activity (β-glucosidase, β-cellobiohydrolase) of the soil samples were investigated according to the method described as by Zhang et al.21.
Soil laboratory analysis
Extraction and purification of soil humus
The extraction of different humus fractions from soil samples were performed with 5 g dry soil and 50 mL of 0.1 mol L−1 NaOH in 0.1 mol L−1 sodium pyrophosphate under a N2 atmosphere, and it were repeated several times until colorless supernatants were obtained. The suspensions were centrifuged at 5000 × g for 15 min and the pooled alkali extract were acidified to pH 2.0 with H2SO4, and kept for 24 h at room temperature. The soluble fulvic acid (FA) was separated from coagulation (humic acid (HA) fraction) by centrifugation9. The residue, which was the precipitate in the centrifuge tube, was collected to provide humin (HM). Total carbon (C) content of FA and HA were investigated by using multi B/C 3100 TOC/TN and C content of HM were measured by using a vario macro cube element analyzer (Elementar Analysensysteme GmbH, Hanau, Germany). The soil C content of humus (FA, HA and HM) of the soil samples were investigated according to the method described as by Jindog et al.8.
CPMAS solid-state 13CNMR spectroscopy
In the present study, chemical composition of purified FA and HA fractions of soil samples with MF, RF, OM and CK treatments were via CPMAS solid-state 13CNMR spectroscopy and expressed as the relative abundance of the major C type. The solid-state 13CNMR spectra were measured on a Bruker Avance III 400 NMR spectrometer (Germany) conducting at a spinning speed of 5 kHz and a contact time of 1 ms, with a 1H 90 °C pulse length of 4 μs and a recycle delay of 0.8 s. The chemical shift regions 0–45, 45–95, 95–165 and 165–200 ppm were referred to alkyl C, o-alkyl C, aromatic C and carboxylic C, respectively 21. The areas of the spectral regions were measured though the integration routine of the spectrometer and expressed as percentage of the sum of all spectral areas, respectively22. The degree of aromaticity (ARO%) and aliphaticity (AL%) of the FA and HA C were calculated according to the equations as following:
$$ {text{ARO}}left( % right) = frac{{{text{Aromatic C }}left( {{95} – {text{165 ppm}}} right)}}{{{text{C}};{text{signal }}left( {0 – {text{165 ppm}}} right)}} $$
(1)
$$ {text{AL}}left( % right) , = frac{{{text{Aliphatic C }}left( {0 – {text{95 ppm}}} right)}}{{{text{C}};{text{signal }}left( {0 – {text{165 ppm}}} right)}} $$
(2)
The alkyl C/o-alkyl C ratio were calculated and regarded as an indicator of the degree of organic matter decomposition23.
Deoxyribonucleic acid (DNA) extraction and quantitative polymerase chain reaction (qPCR) assay
Soil DNA was extracted from 0.5 g fresh soil by using the FastDNA SPIN Kit (MP Biomedicals, Illkirch, France) and a Fast Prep-24 Homogenization System (MP Biomedicals, Irvine, CA) according to the manufacturer’s instructions. Successful DNA extraction was characterized by using electrophoresis on 1% (wt/vol) agarose gels. The quantity and quality of DNA were checked by using Nanodrop spectrophotometer (Nanodrop, PeqLab, Germany).
Genes encoding fungal glycoside hydrolase family 7 cellobiohydrolase I gene (cbhI) and bacterial glycoside hydrolase family 48 (GH48) were selected as biomarkers of cellulolytic fungi and actinobacteria, respectively. All qPCR assays were carried out in an iCycler system (Bio-Rad, USA) by using SYBR Green I chemistry and the data were analyzed by using Bio-Rad iQ5 v2.0, quantitative PCR were investigated according to the method described as by Fan et al.24.
Statistical analysis
The statistical analyses of each investigate items in this manuscript were conducted by using SAS 9.3 software package25. The data of each particle-size fractions with all fertilizer treatments in this manuscript were compared by using Fisher’s significant difference at the p < 0.05 probability level. Pearson’s correlation analyses were performed to assess the linear correlation among soil properties, enzyme activity, C groups of FA and HA with abundance of cbhI and GH48 genes. The results of each investigate items were expressed as mean and standard error.
Statement on guidelines
There was not used live plant in the present experimental research and field study, and comply with relevant institutional, national, and international guidelines and legislation.
