Effects of Straw Returning on Soil Chemical Properties and Microbial Community Diversity under the Rice-Crayfish Integrated System

Abstract

This study presents an investigation of soil chemical properties and microbial community diversity by Biolog ECO analysis in a seven-year field experiment using winter flooded fallow + no straw returning (W), winter flooded fallow + straw returning (WS), and winter flooded fallow + straw returning + crayfish farming (WSC) at soil depths of 0–10 cm and 10–20 cm. Compared with the WS treatment, the WSC treatment had significantly higher total organic carbon (TOC) content in the 0–10 cm layer as well as greater available nitrogen (AN) and total nitrogen (TN) contents and acid phosphatase and sucrase activities in the 10–20 cm layer, while the pH value, total reducing substances, and Fe²⁺ content in the 0–20 cm layer were considerably lower. The WSC treatment improved the microbial species abundance in the 10–20 cm layer and the utilization rate of carbon sources in the 0–20 cm layer compared with the WS treatment. The soil microbial species abundance, microbial community diversity, and utilization rate of carbon sources in all of the layers examined were significantly higher in the WSC treatment than in the W treatment. The results indicate that straw returning under the rice-crayfish integrated system improves the contents of TOC, TN, and AN, decreases reducing substances properties, increases acid phosphatase and sucrase activities, and improves microbial community functional diversity, thereby contributing to the improvement of soil quality and the long-term sustainable development of the rice-crayfish integrated system.

1. Introduction

The double-cropping of crayfish (Procambarus clarkii) and rice (Oryza sativa) was practiced in Louisiana, USA, before being introduced to China in the early 21st century [1,2]. In the rice-crayfish integrated system, the crayfish are allowed to live in the paddy fields, where the rice straw serves as the food basis for crayfish after the rice is harvested. This system fully utilizes the shallow water environment and the winter idle period of rice fields. The combined planting and aquaculture systems have increased farmers’ incomes. Thus, the rice-crayfish integrated systems have significant great economic and social benefits. Currently, the rice-crayfish integrated system has become one of the primary cultivation models in the middle and lower reaches of Yangtze River, with approximately 1.4 × 10⁶ ha in 2021, mainly distributed in Hubei, Anhui, Hunan, and Jiangsu provinces in China. The soil chemical properties and the structure of the soil microbial community are significantly altered in the rice-crayfish integrated system [3,4]. Si et al. [5] showed that the rice-crayfish integrated system increased soil carbon level and strongly affected the microbial community composition and structure in the deeper layers of soil. Yuan et al. [6] showed that crayfish aquaculture in rice fields significantly improved the soil quality.

It has been reported that the total output of straw in China ranks first in the world (~1.04 billion tons), with a large straw returning area and wide regional distribution [7,8]. Straw returning, a major method for utilizing straw resources, can be applied to the soil either directly or after stacking and decomposing [9]. Several studies have demonstrated the impact of straw returning on soil chemical properties, the structure of the soil microbial community, and soil microbial activity [10,11,12]. Straw returning changes soil nutrient contents and enhances the rice yield [13,14]. Yan et al. [15] showed that rice straw returning significantly increased total organic carbon content, active soil organic carbon fractions, and soil microbial richness but did not affect the soil microbial community diversity. In addition, Bu et al. [16] found that straw returning had a dominant effect on the bacterial community composition in a 12-year rice-rice-rape rotation system. Studies have also shown that straw returning had significant effects on the activity levels of soil enzymes [17,18]. Wu et al. [19] showed that the straw returning increased the levels of urease, phosphatase, and invertase activities over a 5-year period. Zhang et al. [20] observed that a fertilizer plus residues treatment led to higher potential activities of β-glucosidase, lignin peroxidase, and manganese peroxidase enzymes, whereas the same treatment reduced the activities of laccase enzymes in a 10-year fertilization experiment.

Compared with the conventional straw returning, the rice straw under the rice-crayfish integrated system is returned to the field in the condition of long-term flooding that can facilitate the cultivation of crayfish. In the rice-crayfish integrated system, the crayfish can feed on plant debris, and relevant microbes are produced during the decomposition of the straw. Meanwhile, the rapid decomposition of straw can be promoted by the excrement produced by crayfish during growth. Therefore, there may be alterations of soil chemical properties and microbial community diversity. At present, studies related to rice straw returning in China are primarily based on rice cropping rotation or double rice cropping [21,22]. Some studies have also focused on the effect of gas emissions (e.g., CH₄, N₂O, and NH₄) in the integrated rice-crayfish farming system [23,24]. However, there are few reports concerning the effects of Procambarus clarkii activities on the straw returning in the integrated rice-crayfish farming system. We hypothesized that soil microbial communities respond to the straw returning under Procambarus clarkii activities, and these responses are different at different soil depths. The main objectives of this study were: (a) to determine and compare the functional diversity of microbial community for W, WS, and WSC treatments, and (b) to comprehensively evaluate the differences in soil chemical and microbial properties among W, WS, and WSC treatments, providing scientific guidance for improving soil fertility in the rice-crayfish integrated system and maintaining a healthy and sustainable agricultural ecosystem.

2. Materials and Methods

2.1. Study Area and Experimental Design

The study was conducted on a 7-year-old rice-crayfish integrated system in waterlogged paddy fields at Immigrant Village, Houhu Farm, Qianjiang City, Hubei Province, China (112°41′32.5″E, 30°22′41.2″N). This area is part of the low lake areas of Jianghan Plain, belonging to the humid climate zone of the north subtropical monsoon region, with the static groundwater level of 40–60 cm in winter. The average annual temperature is 16.1 °C, with a frost-free period of 246 days. The average annual rainfall is 1100 mm. The soil type is a fluvo-aquic paddy soil developed from lacustrine deposits.

The field experiment started in 2014, and three treatments were set up: winter flooded fallow + no straw returning (W), winter flooded fallow + straw returning (WS), and winter flooded fallow + straw returning + crayfish farming (WSC). Each treatment was set with three replicates, each with a cell area of 100 m².

The rice cultivar used in the three treatments was Jianzhen 2. The method for the straw returning was high stubble followed by irrigation and rotary tillage before rice planting. The stubble height was about 40 cm, and the return amount was 1900 kg·ha⁻¹. Crayfish larvae (weighing 5 ± 2 g) were stocked at a density of 9 × 10⁴ larvae·ha⁻¹, and the crayfish self-propagated inside the rice paddies. Then, a proper amount of brood stock was added at this time of the year according to the actual situation. The crayfish feed was added from March to May every year. The average feed input was 2.7 × 10³ kg·ha⁻¹. The crayfish were released into the flooded field on 25 October 2014, exactly 20 days after the rice harvest. Mature crayfish were harvested in June of the second year, and immature crayfish migrated to the peripheral trenches before re-entering the rice field after field puddling, seedling planting, field drying, and rewatering. In the second season, mature crayfish were harvested before the rice harvest. To prevent the crayfish from escaping, ditches with a width of 0.4 m and a depth of 1.0 m were set in each community, while crayfish ditches with a width of 3.0–4.0 m and a depth of 0.8–1.0 m were set at one side of the community, and nylon nets were also set around the field.

The fertilizer amounts of the three treatments were N 150 kg·ha⁻¹, P₂O₅ 36 kg·ha⁻¹, and K₂O 120 kg·ha⁻¹. The contents of total nitrogen (TN), total phosphorus (TP), and total potassium (TK) in the crayfish feed were 46.6 g·kg⁻¹, 11.0 g·kg⁻¹, and 10.5 g·kg⁻¹, respectively.

Before the experiment in 2014, the basic physical and chemical properties of 0–20 cm topsoil were as follows: pH 7.12, total organic carbon (TOC) 15.33 g·kg⁻¹, TN 2.36 g·kg⁻¹, TP 0.45 g·kg⁻¹, TK 19.50 g·kg⁻¹, available nitrogen (AN) 129.50 mg·kg⁻¹, available phosphorus (AP) 9.13 mg·kg⁻¹, and available potassium (AK) 178.67 mg·kg⁻¹.

2.2. Soil Sampling and Storage

Soil samples were collected on the 11 November 2020 after the harvest using a sample auger at depths of 0–10 cm and 10–20 cm. Sampling was conducted from five different sites within each replicate plot, and the five samples were combined to prepare a composite sample for the plot. Immediately after sampling, visible root fragments and stones were manually removed, and the samples were mixed well and divided into two portions. One portion of fresh soil was passed through a 2 mm sieve and stored in a refrigerator at 4 °C until biological analysis, and the other portion was air-dried and filtered in preparation for chemical characteristics analysis.

2.3. Soil Chemical Properties Analysis

Soil chemical properties were assayed according to the methods described by Bao [25]. Soil pH was measured in a soil water mixture (1:2.5 w/v) using a pH meter. The TOC content was determined by oxidation with potassium dichromate and titration with ferrous ammonium sulfate. The TN was determined by the Kjeldahl digestion method. The TP and TK were extracted and determined by the perchloric acid digestion methods using spectrophotometer protocols. The soil AN was converted to NH₄⁺ under alkaline conditions, collected in H₃BO₃ solution, and then determined by titration with standard 0.01 mol·L⁻¹ H₂SO₄. The AP was determined using the molybdenum blue method with a spectrophotometer after extraction with 0.5 mol·L⁻¹ NaHCO₃ at pH 8.5. The AK was determined using the ammonium acetate extraction method.

2.4. Soil-Reducing Substances Properties Analysis

Potassium periodate colorimetry was used for Mn²⁺, phenanthroline colorimetry was used for Fe²⁺, and the Al₂(SO₄)₃ leaching-potassium dichromate volumetric method was used for the total amount of reducing substances [26].

2.5. Soil Enzyme Activity Analysis

Enzyme activity was assayed according to the methods described by Guan [27], and acid phosphatase activity was estimated by determining the amount of phenol released after incubating the samples with phenyl disodium phosphate (0.5% w/v) for 24 h at 37 °C. Urease activity was measured by determining the amount of NH₄⁺ released from a hydrolysis reaction after incubating the samples with urea (10% w/v) for 24 h at 37 °C, and sucrase activity was measured by determining the amount of glucose released after incubating the samples with sucrose (8% w/v) at 37 °C for 24 h.

2.6. Soil Functional Diversity of the Microbial Community Analysis

Soil functional diversity of the microbial community was determined using the Biolog-ECO method [28]. Ten grams of fresh soil were passed through a 2 mm sieve, added to 100 mL 0.85% (w/v) sterile NaCl solution, and agitated for 30 min. Under bacteria-free condition, the mixture was diluted to 10⁻³ with sterile 0.85% NaCl solution, and an eight-channel pipette was used to add 150 μL of the diluted suspension to each well of the Biolog ECO plate. Each soil sample was analyzed in triplicate. Cultures were grown at a constant temperature of 25 °C. The absorbance at 590 nm was measured for each well after 24, 48, 72, 96, 120, 144, 168, 196, and 240 h.

(1) Average absorbance (AWCD) can measure the total ability of the microbial community to utilize carbon source.

$$\mathrm{AWCD}=\frac{{\displaystyle \sum \left(A_i-A_{A1}\right)}}{31}$$

(1)

In this equation, A_i is the relative absorbance at 590 nm of the i-th reaction well; A_A1 is the relative absorbance of well A1; wells with an A_i − A_A1 value of < 0 are set to 0 for the purpose of the calculations so that all values of A_i − A_A1 are greater than or equal to 0.

(2) The Shannon index (H′) was used to evaluate richness.

$$H{}^{\prime }=-{\displaystyle \sum P_i\times \ln \left(P_i\right)}$$

(2)

Here, P_i is the ratio of the relative absorbance of the ith well to the total relative absorbance of the entire sample plate.

(3) Simpson’s index (D) was used to evaluate species dominance, and its variant, the Gini index, is often used to evaluate diversity.

$$D=1-{{\displaystyle \sum \left(P_i\right)}}^2$$

(3)

Here, P_i is the ratio of the relative absorbance of the i-th well to the total relative absorbance of the entire sample plate.

(4) McIntosh’s index (U) is a diversity measure based on multi-dimensional spatial distance of community species, i.e., it is a measure of consistency.

$$U=\sqrt{\left({\displaystyle \sum n_i{}^2}\right)}$$

(4)

Here, n_i is the relative absorbance of the i-th well.

2.7. Statistical Analysis

The data were analyzed using Excel 2010, and SPSS software (version 22.0). Treatment means were compared using the least significant difference test at p < 0.05. Pearson’s correlation analyses were conducted to investigate relationships between soil chemical properties and biological parameters.

3. Results

3.1. Soil Chemical Properties Analysis

As shown in

Table 1. Soil chemical properties under different treatments.

Soil Depth		pH	Total Organic Carbon	Available N	Available P	Available K	Total N	Total P	Total K
(cm)			(g·kg−1)	(mg·kg−1)	(mg·kg−1)	(mg·kg−1)	(g·kg−1)	(g·kg−1)	(g·kg−1)
0–10	W	6.81 ± 0.06a	24.14 ± 0.19b	114.88 ± 4.42b	11.16 ± 0.83a	197.05 ± 4.88a	2.50 ± 0.01a	0.67 ± 0.02a	23.01 ± 0.16a
	WS	6.84 ± 0.04a	25.34 ± 0.64b	127.00 ± 3.45ab	12.79 ± 0.16a	205.52 ± 5.08a	2.56 ± 0.01a	0.68 ± 0.00a	23.12 ± 0.21a
	WSC	6.42 ± 0.03b	26.90 ± 0.35a	136.04 ± 4.24a	12.01 ± 0.75a	194.09 ± 12.25a	2.55 ± 0.07a	0.67 ± 0.02a	23.00 ± 0.50a
10–20	W	6.98 ± 0.01a	19.88 ± 0.51b	105.12 ± 4.95ab	10.61 ± 0.24a	171.23 ± 5.08a	2.18 ± 0.03b	0.61 ± 0.01a	23.18 ± 0.30a
	WS	6.91 ± 0.03a	21.21 ± 0.19a	89.43 ± 2.07b	10.29 ± 0.64a	178.00 ± 2.96a	2.21 ± 0.04b	0.62 ± 0.01a	23.14 ± 0.32a
	WSC	6.73 ± 0.05b	21.28 ± 0.27a	108.93 ± 5.44a	11.05 ± 0.85a	185.62 ± 1.53a	2.62 ± 0.15a	0.64 ± 0.01a	23.03 ± 0.17a

Note: W, winter flooded fallow + no straw returning; WS, winter flooded fallow + straw returning; WSC, winter flooded fallow + straw returning + crayfish farming. Means with different letters for the same property in the same soil layer indicate significant differences at p < 0.05. Values are means ± standard errors.

, the soil total organic carbon (TOC), available nitrogen (AN), available phosphorus (AP), available potassium (AK), and the total P (TP) content decreased with increasing soil depth under the three treatments. There were no significant differences in the pH value, AN, AP, AK, TN, TP, or TK contents at a depth of 0–20 cm between the W and WS treatments, but the TOC content in the 10–20 cm layer was significantly higher in the WS treatment than in the W treatment. Compared with the WS treatment, the TOC content in the WSC treatment was significantly increased in the 0–10 cm layer. The AN and TN contents of the 10–20 cm layer were significantly higher in the WSC treatment than in the WS treatment by 21.8% and 18.6%, respectively. In addition, the AN content in the WSC treatment was significantly increased compared with the W treatment in the 0–10 cm layer. The pH value was considerably lower in the WSC treatment than in the WS treatment in all of the layers examined.

3.2. Soil-Reducing Substances Properties Analysis

Soil total reducing substances, Fe²⁺, and Mn²⁺ contents increased with increasing soil depth (Figure 1). There were no significant differences in the Fe²⁺ or Mn²⁺ contents of the 0–20 cm layer between the W and WS treatments. However, the total reducing substances of the 0–10 cm layer was significantly higher by 28.5% in the WS treatment than in the W treatment. The Fe²⁺ content and total reducing substances were considerably lower in the WSC treatment than in the WS treatment in all of the layers examined. The Mn²⁺ content in the WSC treatment was significantly decreased by 22.1% compared with the WS treatment in the 0–10 cm layer.

3.3. Soil Enzyme Activity Analysis

Soil enzyme activity decreased with increasing soil depth (Figure 2). Soil acid phosphatase and urease activities showed an increasing trend in the WS treatment compared with the W treatment in all of the layers examined, but the differences were not statistically significant (p > 0.05). Soil sucrase activity of the 0–10 cm and 10–20 cm layer was significantly higher in the WS treatment than in the W treatment by 60.6% and 29.9%, respectively. Soil acid phosphatase and sucrase activities showed an increasing trend in the WSC treatment compared with the WS treatment at a depth of 0–20 cm, and the difference was significant in the 10–20 cm layer. In addition, soil acid phosphatase activity of the 0–10 cm layer was increased by 30.2% in the WSC treatment compared to the W treatment.

3.4. Soil Functional Diversity of the Microbial Community Analysis

The soil average well color development (AWCD) value is one of the indices to judge the total carbon utilization ability of the microbial community, because it reflects the soil microbial activity and the diversity of physiological functions of the microbial community [29]. With the increasing duration of culture (Figure 3), the utilization degree of different carbon sources gradually increased. In the 0–20 cm layer, the slope of the AWCD curve was the highest at 24–120 h of culture, indicating that carbon source metabolic activity of soil microbes was the highest at this stage, and then entered a stable period. During 72–192 h (Figure 3), the soil AWCD value in the WS treatment was higher than that in the W treatment in the 0–20 cm layer and the AWCD value of the WSC treatment was higher than that in the WS treatment in the 10–20 cm layer. In addition, the AWCD value of the WSC treatment was higher than that in the W treatment in the 0–20 cm layer. These results showed that the straw returning improved the microbial activity in the 0–20 cm layer. The straw returning + crayfish farming significantly increased the microbial activity in the 10–20 cm layer compared with the WS treatment.

The Shannon index reflects the diversity of the bacterial community, with a higher index indicating greater diversity of the bacterial community [30]. The Simpson index reflects the changes in the population of each species, with a higher index indicating the position of the dominant species being more prominent [31]. The McIntosh index is used to measure the community species consistency, with a higher index indicating an increased degree of carbon source utilization [32].

Table 2. Functional diversity index of the soil microbial community under different treatments.

Soil Depth (cm)		AWCD of 96 h	Shannon Index	Simpson Index	McIntosh Index
0–10	W	0.87 ± 0.06b	2.89 ± 0.03b	0.939 ± 0.002b	6.68 ± 0.10c
	WS	1.27 ± 0.02a	3.11 ± 0.05a	0.951 ± 0.003a	8.71 ± 0.08b
	WSC	1.42 ± 0.03a	3.21 ± 0.02a	0.957 ± 0.001a	9.08 ± 0.04a
10–20	W	0.51 ± 0.09b	2.50 ± 0.20b	0.897 ± 0.006c	4.81 ± 0.48b
	WS	0.69 ± 0.10b	2.73 ± 0.17ab	0.922 ± 0.004b	5.40 ± 0.65b
	WSC	1.23 ± 0.13a	3.16 ± 0.04a	0.954 ± 0.002a	8.10 ± 0.69a

Note: W, winter flooded fallow + no straw returning; WS, winter flooded fallow + straw returning; WSC, winter flooded fallow + straw returning + crayfish farming. Means with different letters for the same property in the same soil layer indicate significant differences at p < 0.05. Values are means ± standard errors.

shows that at 96 h of culture in the Biolog ECO microplate, those indices were significantly different for the AWCD value in the 0–20 cm layer among the W, WS, and WSC treatments. The Shannon and McIntosh indices of the 0–10 cm layer were significantly higher in the WS treatment than in the W treatment by 7.6% and 30.4%, respectively. The Simpson index of the 0–20 cm layer was significantly increased in the WS treatment compared to the W treatment. Compared with the WS treatment, the Simpson index in the WSC treatment was significantly increased in the 10–20 cm layer. The McIntosh indices of the 0–10 cm and 10–20 cm layer were significantly increased in the WSC treatment compared to the WS treatment by 4.2% and 50%, respectively. Moreover, the Shannon index in the WSC treatment was increased by 26.4% compared with the W treatment.

3.5. Analysis of Soil Microbe Carbon Source Utilization

Based on the types of carbon sources in the Biolog ECO microplate, the 31 carbon sources were divided into carbohydrates, carboxylic acids, amino acids, phenol acids, amines and polymers. The changes in the AWCD values of microbial carbon source utilization at 96 h of three treatments were analyzed.

As shown in Figure 4, with increasing soil layer depth, the soil microbial utilization rates of carbohydrates, carboxylic acids, amino acids, phenol acids, amines, and polymers showed a gradual decreasing trend among three treatments. The utilization rates of carbohydrates, amino acids, and amines in the WS treatment were increased by 52.4%, 45.7%, and 34.1%, respectively, compared with the W treatment in the 0–10 cm layer. The utilization rate of carboxylic acids and polymers was significantly higher in the WS treatment than in the W treatment in the 0–20 cm layer. Compared with the WS treatment, the utilization rates of carbohydrates, carboxylic acids, amino acids, and amines in the WSC treatment were significantly increased by 109.9%, 31.0%, 105.9%, and 97.2%, respectively, in the 10–20 cm layer. The utilization rate of phenol acids in the 0–10 cm and 10–20 cm layer was higher in the WSC treatment than in the WS treatment by 82.3% and 51.6%, respectively. These results indicated that the straw returning had a strong influence on the utilization rates of carbon sources in the 0–10 cm layer. The straw returning + crayfish farming was more affected by the utilization rates of carbon sources in the 10–20 cm layer.

3.6. Interaction of Chemical and Reducing Substances Properties with Soil Enzyme Activity and Microbial Community Diversity Indices

As shown in

Table 3. Correlation coefficient of soil chemical properties, reducing substances properties, enzyme activity, and microbial community diversity indices.

	AWCD of 96 h	Acid Phosphatase	Urease	Sucrase	McIntosh Index	Simpson Index	Shannon Index
pH	−0.719 **	−0.812 **	0.317	−0.644 **	−0.697 **	−0.702 **	−0.654 **
TOC	0.503 *	0.507 *	−0.31	0.256	0.498 *	0.483 *	0.514 *
AP	0.537 *	0.466	0.013	0.429	0.565 *	0.442	0.421
AK	0.712 **	0.654 **	0.006	0.435	0.752 **	0.778 **	0.685 **
AN	0.721 **	0.641 **	−0.321	0.454	0.727 **	0.588 *	0.583 *
TK	−0.259	−0.07	0.014	−0.081	−0.258	−0.221	−0.346
TN	0.748 **	0.644 **	−0.416	0.637 **	0.762 **	0.789 **	0.668 **
TP	0.616 **	0.572 *	−0.151	0.404	0.629 **	0.651 **	0.501 *
Fe2+	−0.766 **	−0.723 **	0.373	−0.556 *	−0.767 **	−0.781 **	−0.688 **
Mn2+	−0.132	−0.212	0.093	−0.011	−0.148	−0.028	−0.034
Total Reducing Substances	−0.772 **	−0.780 **	0.147	−0.540 *	−0.770 **	−0.890 **	−0.773 **

Note: * Significant at p < 0.05. ** Significant at p < 0.01.

, the pH value, Fe²⁺ content, and total reducing substances had highly significant negative correlations with the AWCD at 96 h as well as with acid phosphatase, sucrase, and the Shannon, Simpson, and McIntosh indices. The TOC, AK, AN, and TP contents had significant positive relationships with the AWCD at 96 h, acid phosphatase, and the Shannon, Simpson, and McIntosh indices. The TN content also had significant positive relationships with the AWCD at 96 h, acid phosphatase, sucrase, and the Shannon, Simpson, and McIntosh indices. In addition, the AP content had a significant direct correlation with the AWCD at 96 h and McIntosh index.

4. Discussion

Previous studies have reported that the straw returning and the rice-crayfish integrated system increased the concentrations of elements such as N, P, and K [33,34]. In the present study, the straw returning and straw returning + crayfish farming increased the TOC content in the 0–20 cm layer compared with the no straw returning treatment. The straw is an abundant carbon resource. After straw returning, the straw becomes decomposed, thereby increasing the soil TOC content [35]. Compared with the straw returning treatment, the straw returning + crayfish farming significantly increased AN and TN contents in the 10–20 cm layer, but decreased the pH value in the 0–20 cm layer. In the rice-crayfish integrated system, the farmers often provide excessive high-protein feed to Procambarus clarkii to increase profits. The uneaten feed, as well as the molts, excretions, and dead crayfish accumulated, thereby increasing the soil N content. In the condition of long-term flooding, these substances were fermented and decomposed under anoxic conditions, producing a large number of organic acids, thus decreasing the pH value.

Soil reducing substances are important indicators of soil redox status, a factor that has significant impacts on crop growth and yield [36]. Our study showed that the total reducing substances in the straw returning treatment was higher than that in the no straw returning treatment under winter flooded fallow conditions. After straw returning, the straw was ploughed and decomposed, quickly depleting the oxygen in the soil. This caused the anoxic conditions and decreased the soil redox potential (Eh), so increasing the total reducing substances [3,36]. In our study, compared with the straw returning treatment, the straw returning + crayfish farming decreased the Fe²⁺ content, and total reducing substances in the 0–20 cm layer and the Mn²⁺ content in the 0–10 cm layer. The results were inconsistent with the findings of Yuan et al. [3] and Noellemeyer et al. [37], where the conventional rice paddy was regarded as a pilot area. In our study, the experimental site belonged to a typical gleying paddy in the Jianghan Plain. The digging activity of crayfish penetrated the surface and base layers of the rice paddy soil, affecting water and air circulation [5], thus alleviating the anoxic conditions which made the Fe²⁺ and Mn²⁺ oxidized to Fe³⁺ and Mn⁴⁺, respectively, and decreasing the amounts of soil reducing substances.

Soil enzyme activity is often considered an important indicator of soil quality [38]. Urease, acid phosphatase, and sucrase are related to the N, P, and C cycles [39]. In our study, soil sucrase activity of the 0–20 cm layer was significantly higher in the straw returning and straw returning + crayfish farming treatments than in the no straw returning treatment. The soil sucrase activity was correlated with soil organic carbon content [17]. In our study, the straw returning and straw returning + crayfish farming increased the soil TOC content. Large amounts of rice residue remaining in the soil may have accelerated the catalysis of soil enzyme activities related to C cycling [40]. The acid phosphatase activity was negative related to the pH value [41]. In this study, the straw returning + crayfish farming increased acid phosphatase activity in the 0–20 cm layer. The chief reason was the decrease of pH value that enhanced soil acid phosphatase activity.

Soil microbial community functional diversity is an indicator of soil microbe community structure and function, and as such it reflects the ecological characteristics of soil microbes [42]. The Biolog ECO method is a sensitive system for detecting functional changes in soil microbial community structure, and at present, it is broadly used to assess soil microbial community functional diversity [43]. Many studies have indicated that the straw returning improved the structure and function of the soil microbial community, thus enhancing the metabolic capacity of the community [44,45]. In this study, compared with the no straw returning, the straw returning treatment increased the soil microbial community diversity, the ability to utilize carbon sources in the 0–10 cm layer, and the species abundance of the bacterial community in the 0–20 cm layer. These results were in good agreement with those of Yu et al. [45]. The straw returning + crayfish farming significantly increased the microbial utilization rate of carbon sources in the 0–20 cm layer compared with the no straw returning and straw returning treatments. On the one hand, the straw returning + crayfish farming increased the TOC content, which can used as the carbon metabolism substrates. On the other hand, the crayfish activities increased soil permeability and affected the growth environment of soil microbes, thus improving the microbial carbon metabolism.

Research on the utilization of the different carbon sources by soil microbes can reveal detailed information concerning microbial community metabolism [46]. In this study, the straw returning under winter flooded fallow conditions had a strong influence on the utilization rates of carbon sources in the 0–10 cm layer. The reason was that the straw was ploughed, becoming distributed on the soil surface and thus increasing the carbon sources utilization rate of the 0–10 cm layer. The straw returning + crayfish farming increased the utilization rates of all carbon sources under winter flooded fallow conditions in the 10–20 cm layer compared with the straw returning treatment, particularly increasing the utilization rates of phenol acids in the 0–20 cm layer. The phenol acids were considered to be allelochemicals related to rice autointoxication [47]. Chou and Liu [48] found that rice residues submerged in soil could release phenol acids substances during the decomposition period, especially under waterlogged conditions. The straw returning + crayfish farming increased the utilization rates of phenol acids, so promoting rice growth.

Soil microbial community diversity was affected by the changes in the soil environment [49]. The correlation analysis showed that the soil pH value, AK, TN, Fe²⁺ contents, and total reducing substances were significantly correlated with the microbial community diversity indices. Meanwhile, compared with the other two enzymes, acid phosphatase activity was more affected by soil chemical and reducing substance properties.

5. Conclusions

The straw returning + crayfish farming increased the contents of total organic carbon, total nitrogen, and available nitrogen, improved the soil chemical properties, and enhanced the acid phosphatase and sucrase activities, but decreased Fe²⁺ content and total reducing substances, so alleviating soil gleying. The straw returning + crayfish farming harbors the greatest functional diversity in microbial community in the 10–20 cm layer, whereas the straw returning is closely related to relationship with the microbial functional diversity in the 0–10 cm layer. The straw returning + crayfish farming accelerated the utilization rate of carbon sources in 0–20 cm layer, especially the utilization of phenolic acids. Therefore, the straw returning under the rice-crayfish integrated system improves the utilization rate of straw resources, promotes the soil nutrient cycle, and provides scientific guidance for further popularizing the rice-crayfish integrated system.