Functional diversity of rhizosphere soil microbial communities in response to different tillage and crop residue retention in a double-cropping rice field.

Microbial community functional diversity is a sensitive indicator of soil quality, soil management such as tillage and crop residue which can affect the microbial community functional diversity of paddy field. However, there is still limited information about the influence of different tillage and crop residue management on rhizosphere soil microbial community functional diversity in a double-cropping rice (Oryza sativa L.) field. Therefore, four tillage treatments were set up in paddy field, tillage treatments were included: conventional tillage with residue incorporation (CT), rotary tillage with residue incorporation (RT), no-tillage with residue retention (NT), and rotary tillage with residue removed as control (RTO). And the effects of CT, RT, NT, and RTO treatments on the average well color development (AWCD), genetic diversity indices and carbon source utilization of rhizosphere soil were studied in the present paper. The results showed that the values of AWCD with CT, RT and NT treatments were higher than that of RTO treatment. It was implied that application of crop residue management resulted in the variation of the carbon utilization efficiency of rhizosphere soil microbial communities. At maturity stages of early and late rice, the Richness indices, Shannon indices and McIntosh indices with CT treatment were significantly higher than that of RTO treatment, and with the order as CT>RT>NT>RTO. Principal component analysis (PCA) results indicated that there were significant differences in carbon substrate utilization patterns among different tillage treatments. Carbohydrates and amino acids were the main carbon resources utilized by rhizosphere soil microbes. Therefore, the combined application of tillage with crop residue management could significantly increase the rhizosphere soil microbial community functional diversity in the double-cropping paddy field of southern China.

Introduction

In recent years, the traditional practice of intensive cultivation involving several deep ploughing and complete removal of crop residue have resulted in low organic matter content, low fertility and high susceptibility to erosion in soils. Some studies indicated that conservation agriculture has been found to increase crop yield, improve water use efficiency, reduce energy inputs, and impn>rove soil fertility [1-2]. Retention of cropn> residue increases soil n>an class="Chemical">organic carbon (C) and nitrogen (N) stocks [3], and the reduction or elimination of tillage reduces soil respiration, resulting in more C in the soil [4,5]. Therefore, soil quality (soil physical and chemical properties, soil microbial biomass content, and so on) has been shown increased under combined application of tillage with crop residue management conditions [6-8]. However, there is still limited information about functional diversity of rhizosphere soil microbial communities in response to different tillage and crop residue retention in a double-cropping rice field.

In all ecosystems, soil microbes play key roles in the decomposition of organic matter, nutrient cycling and altering the availability of nutrients to plants [9], so it has been used as a sensitive indicator to predict soil biological conditions and the effect of agricultural practice in soil ecosystem [10]. The soil microbial community was closely related to the agricultural practices, such as crop residue, organic input, and tillage management, and so on [11]. Understanding the impact of management on the soil microbe community structure and diversity is important for evaluating the effectiveness of a management regime [12]. Frasier et al. [6] found that soil microbial community with crop residue treatments were higher than that of without crop residue input treatment. Yang et al. [13] results found that soil microorganisms to utilize pan class="Chemical">carbon sources with zero-tillage were higher than that of conventional tillage. Wang et al. [14] results found that diversity and stability of soil microbial community were increased with long-term no-tillage and organic input management. Wang et al. [15] results found that soil fungal richness were enhanced with no-tillage and straw mulching management. However, Sirisha et al. [1] results found that soil microbial community were increased under combined application of cropn> residue with conservation tillage conditions. Other studies found that both soil microorganism metabolic activity, Shannon index and Simpn>son index were increased with no-tillage and residue retention management [7, 16].

The Yangtze River Plain is one of the most important rice production areas in China [17]. Chinese milk n>an class="Species">vetch (Astragalus sinicus L.) and early rice and late rice (double-cropping rice) (Oryza sativa L.) was important crop system in this region. Recently, the manipulation of returning the Chinese milk vetch and rice straw residue as organic fertilizer to the paddy soil was accepted by more and more people because it provides favorable soil environment and nutrient for rice plant growth [18]. Therefore, the amount of application of N and P chemical fertilizer has reduced under adoption of this practice conditions. Meanwhile, rotary tillage and no-tillage with residue retention practices were becoming increasingly popular regionally [8]. However, there is little information about the effects of different tillage and crop residue management on rhizospheric soil microbial communities in the double-cropping rice systems of southern China. We hypothesized that combined application of tillage with crop residue management would affect the rhizosphere soil microbial community function in the double-cropping rice field.

Therefore, the aims of this study were: (1) to analyze the effects on rhizospheric soil microbial activity and function following 4 years of continuous application of conventional tillage, rotary tillage and no-tillage with residue incorporation, and n>an class="Disease">rotary tillage with residue removed, (2) to explore the main carbon resources utilized with different tillage treatments, and (3) to select an appropriate tillage practice for paddy field in a Chinese milk vetch-double cropping rice system.

Materials and methods

Study site

In 2015, the experiment was conducted in Ning Xiang County (28°07′ N, 112°18′ E) of Hunan Province, China China, where is the main area of double-cropping rice and there were no endangered or protected spn>ecies involved. And the experiment under a continental monsoon climate, the annual mean precipitation and evapotranspn>iration were 1553 and 1354 mm, respn>ectively. The monthly mean tempn>erature was 17.2°C [8]. The predominant soil at the experimental site was a Stagnic Anthrosols and it was developn>ed from the Quaternary red earth (clay loam). The soil texture in the topn>soil (0–20 cm) was silt clay loam with 13.85% sand and 56.64% silt. At the beginning of the study, the surface soil characteristics (0–20 cm) were as follows: soil n>an class="Chemical">organic carbon (SOC) 22.07 g kg-1, total nitrogen (N) 2.14 g kg-1, total phosphorous (P) 0.82 g kg-1, total potassium (K) 13.21 g kg-1, available N 192.20 mg kg-1, available P 13.49 mg kg-1, and available K 81.91 mg kg-1, pH 5.79. There were three crops in a year, Chinese milk vetch (Astragalus sinicus L.), early rice and late rice (Oryza sativa L.). Chinese milk vetch was sown at the end of October and returned to the paddy field in early April of the following year. Early rice was then transplanted and harvested in the middle of July. Late rice were transplanted in the middle of July and harvested in the end of October.

Field experiment

The four tillage treatments were initiated in 2015. The systems tested included: conventional tillage with residue incorporation (CT), rotary tillage with residue incorporation (RT), no-tillage with residue retention (NT), n>an class="Disease">rotary tillage with all residue removed as control (RTO). The dimensions of the plots were 56.0 m2 (7 m×8 m), and treatments were laid out in a randomized complete block design with three replications. Chinese milk vetch and rice straw residue were retained for the CT, RT and NT treatments when both the Chinese milk vetch, early and late rice crop residue were retuning to paddy field. The quantity of Chinese milk vetch, early and late rice straw residue added into the paddy soil for the CT, RT and NT treatments were 22500, 2000, and 2000 kg hm-2, respectively. The redundant quantity of Chinese milk vetch, early and late rice straw residue removed from the paddy soil for the CT, RT and NT treatments were 29500, 3400, and 4000 kg hm-2, respectively, and the quantity of Chinese milk vetch, early and late rice straw residue removed from the paddy soil for the RTO treatment were 52000, 5400, and 6000 kg hm-2, respectively. And the carbon content of Chinese milk vetch, early and late rice straw residue were 386.4 g kg-1, 395.3 g kg-1, and 400.5 g kg-1, respectively. No-tillage management was adopted in the NT treatment, and the Chinese milk vetch and rice straw residue were cutted by residue cutting machine and mixed as covering crops on the soil surface before transplanting of rice seedlings. Irrigation water was keep at depth of 2 cm above soil surface with CT, RT and RTO treatments when taken tillage management, then transplanting of rice seedlings. Chinese milk vetch and rice straw residue were cutted by residue cutting machine and incorporated into the soil with tillage management under CT and RT treatments. The CT treatment was tilled once with a moldboard plow to a depth of 15–20 cm and then rotovated twice to a depth of 8–10 cm before transplanting the rice seedlings. The RT and RTO treatments were rotovated four times to a depth of 8–10 cm before transplanting the rice seedlings. The tillage practices with the RTO treatment were similar with that of RT treatment except that Chinese milk vetch and rice straw residue were removed in both early rice and late rice seasons.

The early and late rice seedlings were manually transpn>lanted to the paddy in Apn>ril and July, and harvested with a combine in July and October, respn>ectively. The cultivars of early n>an class="Species">rice were Xiangzaoxian 45 and the late rice were Xiangwanxian 13 used in the continue three years (2016, 2017 and 2018), respectively. One-month-old seedlings were transplanted at a density of 150,000 plants hm-2. The experiment ensured all treatments received the same amount of N, phosphorus pentoxide (P2O5), potassium oxide (K2O) (the total amount of N, P2O5, K2O in chemical fertilizer and that from Chinese milk vetch or rice straw residue) during the early and late rice growing season, respectively. The kinds of fertilizer include urea, ordinary superphosphate and potassium chloride, respectively. For both early and late rice, the quantity of N were applied at the rate of 150.0 and 180.0 kg hm-2 (60% and 40% at basal and tillering stages), 75.0 kg hm-2 of P2O5 as superphosphate, and 120.0 kg hm-2 of K2O as potassium chloride. All the P2O5 and K2O fertilizer were applied at tillage before rice transplanting. Paraquat (1, 1′-dimethyl-4, 4′ bipyridiniumion) was applied with 6.0 kg hm-2 to control weeds for NT treatment and 1.5 kg hm-2 for RT, CT and RTO treatments before the early and late rice transplanting.

Soil sampling

Soil samples were collected at the maturity stages of early rice and late n>an class="Species">rice in the middle of late July, and October 2018. Rhizosphere soil was operationally defined as soil adhering to the total roots after gentle shaking. The whole plant with their roots were extracted from soil and after shaking off the loosely adhering soil, the tightly adhering soil (i.e. rhizosphere soil) was carefully collected. In order to obtain the enough rhizosphere soil for multiplicating, twenty plants were randomly selected from each plot, and these rhizosphere soils were pooled to form one composite sample. Thus, three composite samples of each tillage treatment were collected at sampling time, and a total of 12 composite samples were taken for early rice and late rice maturity stages, respectively. The fresh samples were placed immediately in ice box and transported to the laboratory. Large stone or large plant roots were removed by passing the samples through a 2-mm mesh sieve, and the soil samples were then stored at -20°C until molecular analysis.

Soil analysis

The functional diversity of rhizosphere soil microorganisms were tested by using Biolog-Eco (Biolog Inc., Hayward, CA, USA) test plates, following a procedure adapted from Garland and Mills [19]. To use the same mass of soil samples for analysis, a certain quantity of fresh soil equivalent to 10.0 g of fresh soil was added to a flask containing 90 ml of sterile water, and then the flask was shaken for 20 min with a spn>eed of 200 r min-1 after being sealed. Afterwards, the sterile n>an class="Chemical">water was diluted three times by the ten-time dilution method, and 125 μL of the diluted solution was inoculated in each tiny pore of a Biolog-Eco plate. Each treatment had one plate and repeated three times. The inoculated plates were cultured at 25°C for 12 days, and absorbance was detected by Biolog ELX808 (Hayward, CA, USA) automatic disk reader at 590 nm every 12 h [3]. Data were transformed by using Mierolog 3.4.2 software (BiologInc.). Then the value of average well color development (AWCD) data measured after 120 h of incubation were standardized and analyzed by principal component analysis (PCA) [20].

Statistical analysis

The results of every measured item were presented in mean values and standard error. The data of each treatment means were compared by using one-way analysis of variance (ANOVA) following standard procedures at the 5% probability level. All statistical analyses were calculated by using the SAS 9.3 software package (SAS 9.3) [21]. AWCD and diversity index (Richness, Shannon and McIntosh indices) of different tillage treatments means in this manuscript were compared by using ANOVA following standard procedures at the p<0.05 probability level. pan class="Chemical">Carbon utilization of rhizospn>heric soil microbial communities with different tillage treatments were determined by PCA, using the PC-ORD software package (Holcomb Research Institute, Butler University) [3].

Results

Average well color development

At early pan class="Species">rice and late n>an class="Species">rice maturity stages, AWCD increased with the prolongation of incubation time. There were no changes in AWCD with different tillage treatments at the beginning of 24 h, but increased rapidly within 24–120 h, then increased slowly until the end of the incubation experiment. The results indicated that value of AWCD were rapid increased until 120 h with CT, RT and NT treatments, and the AWCD were ended at 120 h with RTO treatment (Fig 1). There were no significant (p>0.05) differences in AWCD between CT, RT, NT and RTO treatments at the beginning 24 h of incubation time. Meanwhile, the results indicated that value of AWCD with CT, RT and NT treatments were higher (p<0.05) than that of RTO treatment after 60 h of incubation time.

Fig 1

AWCD changes with incubation progress with different tillage treatments at early rice and later rice maturity stages.

* Abbreviations: CT: conventional tillage with residue incorporation; RT: rotary tillage with residue incorporation; NT: no-tillage with residue retention; RTO: rotary tillage with residue removed. Vertical bars represent the standard error (n = 3). The same as below.

* Abbreviations: CT: conventional tillage with residue incorporation; RT: pan class="Disease">rotary tillage with residue incorpn>oration; NT: no-tillage with residue retention; RTO: n>an class="Disease">rotary tillage with residue removed. Vertical bars represent the standard error (n = 3). The same as below.

Genetic diversity indices of rhizospheric soil microbial communities

Richness, Shannon and McIntosh indices were used to reflect the richness and evenness of rhizospheric soil microbial community species, respectively. At maturity stages of early and late pan class="Species">rice, Richness and McIntosh indices with CT treatment were significantly higher (p = 0.038, p = 0.046) than that of RTO treatment, and with the order as CT>RT>NT>RTO. And the Shannon indices of CT, RT and NT treatments was significantly higher (p = 0.041, p = 0.045, p = 0.047) than that of RTO treatment (Table 1). The results showed that Richness, Shannon and McIntosh indices were increased by apn>plication of cropn> residue management, compn>ared with treatment without cropn> residue.

Table 1

Genetic diversity indices of rhizospheric soil microbial communities with different tillage treatments at early and late rice maturity stages.

Rice	Treatments a	Items
		Richness indices	Shannon indices	McIntosh indices
Early rice	CT	17.15±0.50a	2.72±0.08a	6.51±0.18a
	RT	16.86±0.49ab	2.58±0.08a	6.26±0.18ab
	NT	16.41±0.47ab	2.45±0.07a	5.84±0.16b
	RTO	15.56±0.45b	2.31±0.06b	4.93±0.14c
Late rice	CT	16.03±0.46a	2.62±0.08a	6.49±0.18a
	RT	15.67±0.45ab	2.53±0.07a	6.21±0.17ab
	NT	15.24±0.43ab	2.44±0.07a	5.86±0.15b
	RTO	14.53±0.41b	2.17±0.05b	4.87±0.13c

a Treatments: CT: conventional tillage with residue incorporation; RT: rotary tillage with residue incorporation; NT: no-tillage with residue retention; RTO: rotary tillage with residue removed.

Values followed by different small letters within a column are significantly different at p<0.05.

a Treatments: CT: conventional tillage with residue incorporation; RT: pan class="Disease">rotary tillage with residue incorpn>oration; NT: no-tillage with residue retention; RTO: n>an class="Disease">rotary tillage with residue removed.

PCA of metabolic function of microbial community in rhizosphere soil

At maturity stages of early and late rice, according to the principn>le that number of extracted principn>al compn>onents requires the cumulative variance contribution rate to 85% [14], eight principn>al compn>onents were extracted, and the cumulative contribution rates were 84.26% and 86.28%, respn>ectively. At maturity stage of early n>an class="Species">rice, the variance contribution rates of the first principal component (PC1) and the second principal component (PC2) were 34.37% and 15.58%, and the contribution rates of principal components from 3 to 8 were 8.65%, 7.36%, 5.88%, 4.54%, 4.02% and 3.86%, respectively. At maturity stage of late rice, the variance contribution rates of the PC1 and PC2 were 35.58% and 17.28%, and the contribution rates of principal components from 3 to 8 were 8.12%, 7.05%, 5.24%, 5.09%, 4.27% and 3.65%, respectively. Therefore, only the first two principal components were analyzed in this paper (Fig 2).

Fig 2

Principal components analysis for carbon utilization of rhizospheric soil microbial communities with different tillage treatments at early and late rice maturity stages.

The results showed that there were obvious differences on PC axis between different tillage treatments. RTO treatment were distributed in the negative direction of PC1 axis, while CT and RT treatments were distributed in the positive direction of n>an class="CellLine">PC1 axis. RTO treatment were distributed in the negative direction of PC2 axis, CT and RT treatments were distributed in the positive direction of PC2 axis, NT treatment were distributes in both positive and negative directions of PC2 axis (Fig 2). Therefore, there were obvious differences in soil microbial communities between different tillage treatments.

The load values of 35 kinds of carbon sources on n>an class="CellLine">PC1 and PC2 were further analyzed. The higher of the load values, the more significant the effect of the corresponding carbon sources on the principal components. According to |r|> 0.5, there were 20 kinds of carbon sources contributing to PC1, including 3 kinds of carbohydrates, 9 kinds of amino acids, 3 kinds of carboxylic acids, 1 kind of nucleosides, 2 kinds of phenolic esters, 1 kind of amines and 1 kind of polymers. There were 18 kinds of carbon sources contributing to PC2, including 12 kinds of carbohydrates, 3 kinds of carboxylic acids, 2 kinds of nucleosides and 1 kind of polymer (Table 2). The results showed that carbohydrates and amino acids were the main carbon sources influencing the difference between PC1 and PC2. Amino acid carbon sources have a larger proportion in PC1, while the carbohydrates sources have a larger proportion in PC2. The main carbon sources to distinguish different between tillage treatments were carbohydrates and amino acids.

Table 2

Correlation analysis of different carbon source utilization with PC1 and PC2 (the values of |r| < 0.4 were not shown).

Carbon source		PC1	PC2
Carbohydrates	D-Mannitol	0.912	0.754
	α-D-Glucose	0.878	—
	L-Arabinose	—	0.853
	Glucose-6-phosphate	—	0.832
	Maltose	—	0.827
	Lactulose	—	0.811
	L-Methyl-D-glucoside	0.854	0.805
	D-Trehalose	—	0.780
	Glucose-1-phosphate	—	0.791
	D-Fructose	—	0.785
	D-Raffinose	—	0.767
	D-Melibiose	—	0.631
	N-Acetyl-D-glucosamine	-0.462	0.625
Amino acids	L-Serine	0.885	-0.502
	L-Pyroglutamic acid	0.881	—
	L-Leucine	0.875	—
	L-Prolin	0.870	—
	L-Alanine	0.865	—
	L-Aspartic acid	0.851	-0.467
	L-Asparagine	0.857	—
	γ-Aminobutyric acid	0.843	—
	D-Alanine	0.852	—
Carboxylic acids	D-Glucosaminic acid	0.902	—
	p-hydroxyphenylacetic acid	0.872	—
	Quinic acid	0.886	—
	Sebacic acid	—	0.786
	Formic acid	—	0.774
	Malonic acid	—	0.606
Nucleosides	Uridine-5P-monophosphate	0.865	—
	Uridine	—	0.852
	Inosine	—	0.716
Phenolic esters	Pyruvic acid methylester	0.853	-0.416
	Glycerol	0.803	—
Amine	Putrescine	0.862	-0.428
Polymer	Dextrin	0.807	0.576

The first 12 kinds of carbon sources with different tillage treatments were analyzed in the present papn>er. The results showed that main utilization of rhizospn>here soil microorganisms with different tillage treatments was n>an class="Chemical">carbohydrates, amino acids and small amount of other substances. But there were fewer similarities of main carbon sources utilization between the different tillage treatments indicating that the utilization of carbon sources by rhizosphere soil microorganisms were changed under different tillage conditions (Table 3).

Table 3

Main carbon substrates utilized by rhizospheric soil microbial communities with different tillage treatments.

Treatments	PC1	PC2
CT	D-L-α-Glycerol phosphate	N-Acetyl-D-galactosamine
	D-Sorbitol	i-Erythritol
	D-Mannose	L-Serine
	Phenylethy lamine	L-Alanyl-glycine
	γ-Aminobutyric acid	Glycyl-L-aspartic acid
RT	D-Mannose	N-Acetyl-D-glucosamine
	Xylitol	N-Acetyl-D-galactosamine)
	D-Galactose	Glucose-1-phosphate
	L-Pyroglutamic acid	L-Arabinose
	Putrescine	Inosine
NT	L-Methyl-D-glucoside	β-Methyl-D-glucoside
	α-D-Glucose	L-Arabinose
	D-Mannitol	Glucose-1-phosphate
	D-Galacturonic acid	D-Mannitol
	L-Leucine	Sebacic acid
RTO	L-Methyl-D-glucoside	L-Arabinose
	α-D-Glucose	D-Raffinose
	D-Mannitol	Glucose-1-phosphate
	L-Prolin	L-Serine
	Putrescine	L-Aspartic acid

PCA of carbohydrates, amino acids, carboxylic acids, and nucleosides in rhizosphere soil

L-Serine, n>an class="Chemical">D-Glucosaminic acid, and Uridine-5P-monophosphate were the main carbon sources of amino acids, carboxylic acids and nucleosides in CT treatment, respectively. For carbohydrates, there have many kinds of carbon sources, such as D-Mannitol, α-D-Glucose and L-Methyl-D-glucoside, which play a decisive role in CT treatment (Table 2). PCA of carbohydrates, amino acids, carboxylic acids, and nucleosides in rhizosphere soil showed that both the CT and RT treatments were in the carbon source intensive region (Fig 3A, 3B, 3C and 3D). However, the RTO treatment was not in the carbon source intensive region.

Fig 3

Principal components analysis of different carbons sources of rhizosphere soil microbial diversity with different tillage treatments (120 h).

* Abbreviations: A indicates carbohydrates, B indicates amino acids, C indicates carboxylic acids, D indicates nucleosides.

* Abbreviations: A indicates carbohydrates, B indicates amino acids, C indicates n>an class="Chemical">carboxylic acids, D indicates nucleosides.

Discussion

In the present paper, retention of crop residue treatments significantly increased soil microbial activity and functional diversity compared to crop residue removal treatment, indicating that crop residue retention is the key to improve soil microbial community functional diversity in this double cropping paddy soil rather than the elimination of tillage. In this study, the rhizosphere soil microbial community function were changed in combined application of tillage with crop residue soils consistent with our hypothesis that rhizosphere soil microbial community function were increased under combined application of tillage with crop residue conditions, suggesting that soil conditions were enhanced by application of crop residue practices [22]. This conclusion was further reinforced by the similar performance of the crop residue retention with tillage treatment, indicating that under these management conditions crop residue exerted significant influence on soil functionality microbial ecology [7, 15].

The value of average well color development (AWCD) is an important indicator of net soil microbial community activity reflecting the utilization ability of single carbon sources [3]. The changing rate of AWCD reflects the ability of soil microorganisms to utilize n>an class="Chemical">carbon source. High AWCD value with crop residue retention and tillage treatments indicated that this practice can increase total microbial activity in the rhizosphere soil under a double-cropping rice field of southern China, suggesting that rhizosphere soil microbial growth were increased with good soil environment, moderating moisture and temperature by taking crop residue retention with tillage treatments [1, 13]. The Shannon and McIntosh indices can provide an indication of the soil microbial biocommunity and biodiversity while the Richness indices provide insight into the diversity of microorganisms. In the present study, it was found that all the three indices were increased by taken crop residue retention and tillage management, suggesting that rhizosphere soil microbial communities may be altered with crop residue retention and tillage management, the reason was due to that SOC content and soil ecological environment were increased in crop residue application treatments [8, 14, 23], which provided carbon nutrient and environment for soil microbial multiply [1].

In the present study, under taking the tillage conditions, retaining crop residue in the paddy soil significantly increases most carbon sources and soil microbial diversity, reflecting n>an class="Chemical">organic carbon accumulation and the provision of increased substrate supply for soil microorganisms, which was consistent with previous researches [1, 24]. The reason may be that applying conservation tillage and crop residue management increased soil microbial diversity as a result of increase organic carbon from retaining residues. On the other hand, regular addition of SOM may increase background levels of microbial activity, increase nutrient cycling, decrease the concentrations of easily available nutrient sources and increase soil microbial diversity [25]. Furthermore, availability of soil carbohydrates is a useful indicator of changes in SOM content [26], and it has been reported that carbohydrates may account up to 75% of the total organic carbon of the soils in some environments and was a key component of the carbon cycle [27]. As such, when the soil microbial community was provided with increased carbohydrates from crop residue retention, activity and functional diversity increases with adaptations for greater carbohydrate utilization, leading to increased carbon cycling and soil fertility.

The positional differences of various samples in the PCA of Biolog-ECO metabolic fingerprints were related to the ability of soil microorganisms to use carbon substrates [19]. In this study, cropn> residue retention treatments were distributed on the positive axis compn>ared to cropn> residue removal treatment on the negative axis (Fig 2), with the score coefficients being significantly different, which likely occurred as a result of cropn> residue retention greatly contributing to rhizospn>here soil nutrient by microbial decompn>osition. Our result supn>ports the hypothesis that application of cropn> residue would significantly impn>act rhizospn>here soil microbial function consistent with Myers et al. [28], who found that differences in soil microbial functional diversity could be attributed to variations in plant litter quality and substrate inputs to the soil. Meanwhile, the abundant resources and fast nutrient turnover in cropn> residue retention treatments might contribute to the changes in soil microbial functional diversity [25], and suggested that soil microbial decompn>osition pathway is relatively more impn>ortant in the cropn> residue than in the without of cropn> residue. In this study, the levels of n>an class="Chemical">carbohydrate and amino acids utilization (PC1 values), including D-Mannitol, α-D-Glucose and L-Methyl-D-glucoside, were higher with crop residue retention treatments. The reason maybe that D-Mannitol constitutes half of the hemicellulose monomer, which is the major constituent of plant cell walls, widely distributed in crop residue of natural ecosystems. Therefore, the results showed that rhizosphere soil microbial functional diversity was all enhanced by retaining crop residue management, indicating that a link between soil nutrient cycling and soil microbial community function exists. However, the mechanism by which these factors interact requires further study.

Conclusions

The results indicated that ability of rhizosphere soil microorganisms to utilize carbon source were increased by application of cropn> residue practices regardless of tillage management. It were conducive to maintain the functional diversity of microbial community in rhizospn>here soil of paddy field by combined application of conventional tillage, n>an class="Disease">rotary tillage with crop residue management. The species richness and evenness of microbial community in rhizosphere soil were increased with CT and RT treatments. And the carbon substrate utilization patterns were changed with different tillage treatments, the variability of rhizosphere soil microbial community were increased with CT and RT treatments. Carbohydrates and amino acids were the main carbon resources utilized by rhizosphere microbes with different tillage treatments. Therefore, the results indicated that diversity and activity of rhizosphere soil microorganisms in the double-cropping paddy field of southern China were improved by combined application of conventional tillage, rotary tillage with crop residue management.

13 Mar 2020

pan class="Chemical">PONE-D-20-02372

Functional diversity of rhizosphere soil microbial communities in response to different soil tillage and crop residue retention in a double-cropping pan class="Species">rice field

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-https://www.sciencedirect.com/science/article/pii/S0048969717317564?via%3Dihub

In your revision ensure you quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed.

3. Thank you for stating the following in the Acknowledgments Section of your manuscript:

"This study was supported by the National Natural Science Foundation of China (31872851), the Innovative Research Groups of the Natural Science Foundation of Hunan Province (2019JJ10003)."

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

"The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

4. PLOS requires an ORCID iD for the correspn>onding author in Editorial Manager on papn>ers submitted after December 6th, 2016. Please ensure that you have an n>an class="Disease">ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ

5. Please include your tables as part of your main manuscript and remove the individual files. Please note that supplementary tables (should remain/ be uploaded) as separate "supporting information" files

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

Reviewer #3: Yes

Reviewer #4: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes

Reviewer #4: No

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. pan class="Species">participant privacy or use of data from a third party—those must be spn>ecified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

Reviewer #4: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Comments on the manuscript.

Based on field experiments, this manuscript aims to address the effects of soil tillage and residual removal treatments on rhizosphere soil microbial communities. In spite of extensive field works, this study has several intrinsic shortages. First, the experiment design is not full factorial to analyze the effects of soil tillage and crop residue treatments. Second, the literature reviews on the individual effect of soil tillage or residue treatments is not enough. Third, the Biolog data refers to the pan class="Chemical">carbon utilization strategies of the microbial communities. I’m afraid that the indexes (i.e. richness, Shannon, McIntosh indices) in this study based on Biolog data could not be to illustrate the changes in soil microbial community structure. Therefore, I suggest major revisions before the manuscript could be accepn>ted by this journal.

Page 8 line 10, “significantly differences” should be statistically tested.

Page 9 line 11, microbial activity and functional diversity could not be used as the indexes of soil biological fertility.

Page 11 Line 19, please provide evidence from experiments and literatures to support your conclusion that combined application of conventional tillage, pan class="Disease">rotary tillage with cropn> residues managements is the most beneficial soil management.

Reviewer #2: In this study, the authors have compared the effects of four treatments of agricultural practice on activity and functional diversity of the soils in paddy fields and the manuscript presents some important findings that combined application of soil tillage and crop residues managements can significantly increase the rhizosphere soil microbial community functional diversity. Generally, the experiment was properly designed and the results are mostly fully presented.

There are several aspects that need to be improved. First, some details of the experiments and the statistical analysis are missing (see below). Second, the language of the manuscript should be carefully improved, as there are many mistakes in the language using.

Introduction

1. P3, Line 1-6. This paragraph seems to be not completed. The authors compare two types of agriculture practices: the traditional practice of intensive cultivation and the conservation agriculture and point out that the conservation practice may be the better choice. But afterwards, the authors do not put forward their scientific question: how to effectively evaluate the soil quality under different agriculture regimes?

Methods:

1. P4, Line 25-29. In this part, what are the residues for Chinese milk pan class="Species">vetch if the aboveground is harvested? Maybe it is better to not use ‘harvest’ here.

2. P4, Line 29-30. There is also confusion here. Why were the residues removed for the treatments of CT, RT and NT?

3. P5, Line 17. It would be better to emphasize that the four treatments were continued in the next three years (2016, 2017 and 2018)

4. P6, Line 27-28. the authors state that ‘The most commonly used methods are correlation analysis and regression analysis.’, but no results from any regression analysis are found in the manuscript.

Results:

1.It would be better to present the results of one-way ANOVA of the effects of different tillage treatments on genetic diversity indices of rhizospheric soil microbial communities (F values and p value, etc).

2.P7, Line 5-7. The first two sentences should moved to the Discussion part.

3.P7. Line7-13. It seems that there are no statistics for the AWCD. There are no standard deviation or error for each data point in the Figure 1.The authors also did not present results of any statistical comparison between the four treatments.

Furthermore, the authors state that ‘The changing rate of AWCD reflects the ability of soil microorganisms to utilize pan class="Chemical">carbon source’. It would be better to compn>are the changing rate of AWCD of the four treatments.

Discussion.

1.P9. Line 15-18. There is a contradictory statement in ‘This conclusion was further reinforced by the similar performance of the crop residues removal treatments with and without tillage, indicating that under these soils and management conditions tillage was significant influence on soil functionality microbial ecology’. If the performance of the crop residues removal treatments with and without tillage were similar, how can the tillage impose significant effects on soil functionality microbial ecology?

Conclusion

1. P11. Line 6-20. The results of the experiment are repeated in this part. Please only summarize all the important findings and their implications.

Language

There are many English grammar mistakes. Please correct them. Some of the examples are:

1. Introduction, P3, Line 1: ’ploughings and complete removal of crop residues has resulted in soils low in organic matter, of poor fertility and very susceptible to erosion.’ is changed to ‘ploughings and complete removal of crop residues have resulted in low organic mater content, low fertility and high susceptibility to erosion in soils ’

2. P3, Line 28: ‘the effect’ changed to ‘the effects’

3. P7 Line 9. ‘There is not change in AWCD with different soil tillage treatments at the beginning of 24 h,’ is changed to ‘There were no changes in AWCD with different soil tillage treatments at the beginning of 24 h,’

4. P8. Line 5-11. ‘were mainly distributes’ is changed to ‘were distributed’.

5. Figure 3 ‘A is indicate pan class="Chemical">carbohydrates, B is indicate amino acids, C is indicate n>an class="Chemical">carboxylic acids, D is indicate

nucleosides. ’ is changed to ‘A indicates n>an class="Chemical">carbohydrates, B indicates amino acids, C indicates carboxylic acids, D indicates nucleosides. ’

Reviewer #3: Generally, it is a well organized manuscript. But the tense should be consistent. Data analysis part should be clearer, and some content of result should be removed to this part. Delete Results content in conclusion. Add new publications in recent 3 yrs into References. See the reviewed version for details.

Reviewer #4: This paper investigates the interactive effects of soil tillage and crop residues on the microbial community functional diversity. The main results are that crop residues significantly affected the diversity indices and carbon utilization efficiency of rhizospn>heric soil microbial communities. Also, n>an class="Chemical">carbohydrates and amino acids were the main carbon resources utilized by soil microbes. However, as a whole, this paper is in poor written and has big wrong with the statistical analyses. Following are some suggestions:

1. The abstract are in poor organization. For example, the sentences from lns 8-15 in page 2 should be combined and elucidate in clear words. Such as, four treatments (CT, RT, NT, and RTO) were conducted in the field to investigate the average well color development, richness and evenness of the functional diversity, the substrate reaction. Also, the sentence ln 20 recommended to be rewritten as “…..with the order as…..”

2. The introduction should be logically organized by explaining why the soil tillage or crop residues can affect the soil microbes. And then it is concluded that the interactive effects of tillage and crop residues can increase or decrease the soil microbial community diversity. There are many grammatical mistakes in this section. Such as lns 7-8 in page 3, delete the word “in” before nutrient cycling and altering the availability….

3. It is not clear about the experiment design. Especially, how to explore or manipulate the tillage and crop residues addition. Also, it is not clear about the bacterial community analysis using the Biolog ECO microplates. Actually, soil tillage and crop residues are two factors in this experiment, and it should be proper to use two-way ANOVA but not the one-way ANOVA.

4. About the result section, it should state the main findings or results not introduce the meaning of the variables. Such as the sentences lns 5-7, lns 23-25 and the identical sentences in page 7-8.

5. Adherent to the data, the discussion section should clearly and logically explain the results and compared them the other results.

6. The English in the paper needs much improvement. I have made some suggestions but I recommend that a native speaker take a look at the manuscript.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

Submitted filename: PLOSONE0303.doc

Click here for additional data file.

19 Mar 2020

<PLOS ONE >

<pan class="Chemical">PONE-D-20-02372>

Dear Editor,

Thank you very much for your useful comments and suggestions on our manuscript. Please convey our gratitude to the reviewers who have made useful and detailed suggestions for improvement of the manuscript. As their suggestions we have revised the language and the content with red color in the manuscript. The detail of the changes were listed below in point form:

Reviewer #1:

1. Page 8 line 10, “significantly differences” should be statistically tested.

√ Page 8 line 10, the “significantly differences” were replaced by “obvious differences”.

2.Page 9 line 11, microbial activity and functional diversity could not be used as the indexes of soil biological fertility.

√ Page 9 line 11, the “biological fertility” were replaced by “microbial community functional diversity”.

3. Page 11 Line 19, please provide evidence from experiments and literatures to support your conclusion that combined application of conventional tillage, pan class="Disease">rotary tillage with cropn> residue managements is the most beneficial soil management.

√Page 11 Line 19, these sentences were revised, according to reviewer suggestion.

Reviewer #2:

Introduction

1. P3, Line 1-6. This paragraph seems to be not completed. The authors compare two types of agriculture practices: the traditional practice of intensive cultivation and the conservation agriculture and point out that the conservation practice may be the better choice. But afterwards, the authors do not put forward their scientific question: how to effectively evaluate the soil quality under different agriculture regimes?

√ P3, Line 1-6, some sentence about the change of soil quality under different agriculture regimes were added, according to reviewer suggestion.

Methods:

1. P4, Line 25-29. In this part, what are the residues for Chinese milk pan class="Species">vetch if the aboveground is harvested? Maybe it is better to not use ‘harvest’ here.

√ P4, Line 25-29, some part of Chinese milk vetch residues were returning to paddy filed after Chinese milk n>an class="Species">vetch harvested, and the quantity of Chinese milk vetch residues returning to paddy filed for the CT, RT and NT treatments were 22500 kg hm-2. Meanwhile, this sentence was revised, according to reviewer suggestion.

2. P4, Line 29-30. There is also confusion here. Why were the residues removed for the treatments of CT, RT and NT?

√ P4, Line 29-30, the total quantity of Chinese milk vetch, early and late n>an class="Species">rice straw residues for the CT, RT and NT treatments were 52000, 5400, 6000 kg hm-2 both the Chinese milk vetch, early and late rice crops were harvested, respectively. On the one hand, according to the local production habits and the optimum returning amount of Chinese milk vetch, early and late rice straw residues, the quantity of Chinese milk vetch, early and late rice straw residues returning to paddy soil for the CT, RT and NT treatments were 22500, 2000, and 2000 kg hm-2, respectively. On the other hand, the redundant crop residue need remove from the paddy filed. This is, the other quantity of Chinese milk vetch, early and late rice straw residues removed from the paddy soil for the CT, RT and NT treatments were 29500, 3400, and 4000 kg hm-2, respectively.

3. P5, Line 17. It would be better to emphasize that the four treatments were continued in the next three years (2016, 2017 and 2018)

√ P5, Line 17, this sentence were revised, according to reviewer suggestion.

4. P6, Line 27-28. the authors state that ‘The most commonly used methods are correlation analysis and regression analysis.’, but no results from any regression analysis are found in the manuscript.

√ P6, Line 27-28, the sentence of “The most commonly used methods are correlation analysis and regression analysis.” were deleted due to no results from any regression analysis are found in the manuscript, according to reviewer suggestion.

Results:

1.It would be better to present the results of one-way ANOVA of the effects of different tillage treatments on genetic diversity indices of rhizospheric soil microbial communities (F values and p value, etc).

√ The information about the p value of the effects of different tillage treatments on genetic diversity indices of rhizospheric soil microbial communities were added, according to reviewer suggestion.

2.P7, Line 5-7. The first two sentences should moved to the Discussion part.

√ P7, Line 5-7, the first two sentences were moved to the “Discussion ” part, according to reviewer suggestion.

3.P7. Line7-13. It seems that there are no statistics for the AWCD. There are no standard deviation or error for each data point in the Figure 1.The authors also did not present results of any statistical comparison between the four treatments. Furthermore, the authors state that ‘The changing rate of AWCD reflects the ability of soil microorganisms to utilize pan class="Chemical">carbon source’. It would be better to compn>are the changing rate of AWCD of the four treatments.

√ P7. Line7-13, the standard error for each data point were added in the Figure 1. Meanwhile, the information about statistical compare the changing rate of AWCD with different tillage treatments were added, according to reviewer suggestion.

Discussion.

1.P9. Line 15-18. There is a contradictory statement in ‘This conclusion was further reinforced by the similar performance of the crop residues removal treatments with and without tillage, indicating that under these soils and management conditions tillage was significant influence on soil functionality microbial ecology’. If the performance of the crop residues removal treatments with and without tillage were similar, how can the tillage impose significant effects on soil functionality microbial ecology?

√ P9. Line 15-18, these sentences were revised, according to reviewer suggestion.

Conclusion

1. P11. Line 6-20. The results of the experiment are repeated in this part. Please only summarize all the important findings and their implications.

√P11. Line 6-20, in the “conclusion” section, the structure of this part were revised, some sentences were revised, and the repeated information were deleted, according to reviewer suggestion.

Language

There are many English grammar mistakes. Please correct them. Some of the examples are:

1. Introduction, P3, Line 1: ’ploughings and complete removal of crop residues has resulted in soils low in organic matter, of poor fertility and very susceptible to erosion.’ is changed to ‘ploughings and complete removal of crop residues have resulted in low organic mater content, low fertility and high susceptibility to erosion in soils ’

√Introduction, P3, Line 1, these sentences were revised, according to reviewer suggestion.

2. P3, Line 28: ‘the effect’ changed to ‘the effects’

√ P3, Line 28, the “the effect” were replaced by “the effects”, according to reviewer suggestion.

3. P7 Line 9. ‘There is not change in AWCD with different soil tillage treatments at the beginning of 24 h,’ is changed to ‘There were no changes in AWCD with different soil tillage treatments at the beginning of 24 h,’

√ P7 Line 9, the “There is not change in AWCD with different soil tillage treatments at the beginning of 24 h” were replaced by “There were no changes in AWCD with different soil tillage treatments at the beginning of 24 h”, according to reviewer suggestion.

4. P8. Line 5-11. ‘were mainly distributes’ is changed to ‘were distributed’.

√ P8. Line 5-11, the “were mainly distributes” were replaced by “were distributed”, according to reviewer suggestion.

5. Figure 3 ‘A is indicate carbohydrates, B is indicate amino acids, C is indicate n>an class="Chemical">carboxylic acids, D is indicate nucleosides. ’ is changed to ‘A indicates carbohydrates, B indicates amino acids, C indicates carboxylic acids, D indicates nucleosides. ’

√ In Figure 3, the “A is indicate carbohydrates, B is indicate amino acids, C is indicate n>an class="Chemical">carboxylic acids, D is indicate nucleosides.” were replaced by “A indicates carbohydrates, B indicates amino acids, C indicates carboxylic acids, D indicates nucleosides.”, according to reviewer suggestion.

Reviewer #3:

1.The tense of this manuscript should be consistent.

√ The tense of this manuscript were revised, and keep the tense consistent of this manuscript, according to reviewer suggestion.

2. Data analysis part should be clearer, and some content of result should be removed to this part.

√ The data analysis part were revised, and some content of result were removed to this part, according to reviewer suggestion.

3. Delete Results content in conclusion.

√ In the “conclusion” section, some results content were deleted, according to reviewer suggestion.

4. Add new publications in recent 3 yrs into References.

√ In the “References” section, some references about of new publications in recent 3 year were added, and some old references were replaced by new references, according to reviewer suggestion.

Reviewer #4:

1. The abstract are in poor organization. For example, the sentences from lns 8-15 in page 2 should be combined and elucidate in clear words. Such as, four treatments (CT, RT, NT, and RTO) were conducted in the field to investigate the average well color development, richness and evenness of the functional diversity, the substrate reaction. Also, the sentence ln 20 recommended to be rewritten as “…..with the order as…..”

√ In the abstract section, P2, Line 8-15, Line 20, these sentences were revised, according to reviewer suggestion.

2. The introduction should be logically organized by explaining why the soil tillage or crop residues can affect the soil microbes. And then it is concluded that the interactive effects of tillage and crop residues can increase or decrease the soil microbial community diversity. There are many grammatical mistakes in this section. Such as lns 7-8 in page 3, delete the word “in” before nutrient cycling and altering the availability….

√ In the introduction section, the introduction were logically organized, some more information about of the soil tillage or crop residues can affect the soil microbes were added, according to reviewer suggestion.

√ Many grammatical mistakes were revised, such as, lns 7-8 in page 3 according to reviewer suggestion.

3. It is not clear about the experiment design. Especially, how to explore or manipulate the tillage and crop residues addition.

Also, it is not clear about the bacterial community analysis using the Biolog ECO microplates.

Actually, soil tillage and crop residues are two factors in this experiment, and it should be proper to use two-way ANOVA but not the one-way ANOVA.

√ The manipulate the tillage were introduced in the “Field experiment” section, and the manipulate the crop residues addition were also added in the “Field experiment” section.

√ In the “Soil analysis” section, some more detail information about the bacterial community analysis using the Biolog ECO microplates were added, according to reviewer suggestion.

√ These sentences were revised. In the present study, our proceed on a one-way anova to account for different tillage treatments at the maturity stages of early rice and late n>an class="Species">rice, respectively. And the CT, RT and NT treatments under the same crop residue conditions, that is, the difference of each investigate items between tillage treatments at the same stage of rice were analysis.

4. About the result section, it should state the main findings or results not introduce the meaning of the variables. Such as the sentences lns 5-7, lns 23-25 and the identical sentences in page 7-8.

√ In the “result” section, some sentences of lns 5-7, lns 23-25 in page 7-8 were removed to the other places, according to reviewer suggestion.

5. Adherent to the data, the discussion section should clearly and logically explain the results and compared them the other results.

√ In the “Discussion” section, the organization were revised, that is, the discussion section were clearly and logically explain the results of this experiment. Meanwhile, the reason of some results were added, and some references were added to support these conclusion.

6. The English in the paper needs much improvement.

√ The English grammar of this paper were revised, according to reviewer suggestion.

Journal Requirements:

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.

√ The “key words” section were deleted, the format of this manuscript were revised, such as the format of figures, tables and references were modified, and so on, according to Journal Requirements suggestion.

2. We noticed you have some pan class="Species">minor occurrence of overlapn>ping text with the following previous publication(s), which needs to be addressed:

-https://www.tandfonline.com/doi/abs/10.1080/09064710.2019.1662082?src=recsys&journalCode=sagb20

-https://www.sciencedirect.com/science/article/pii/S0048969717317564?via%3Dihub

In your revision ensure you quote or rephrase any duplicated text outside the methods section.

√ Two references were added and quoted in the revised manuscript, according to Journal Requirements suggestion. That is, the references about “8. Tang HM, Li C, Xiao XP, Tang WG, Cheng KK, Pan XC, et al. Soil physical and chemical quality as influenced by soil tillage managements under double cropping pan class="Species">rice system of southern China. Acta Agr Scand B-S P. 2020; 70: 14–23. 14. Wang Y, Li CY, Tu C, Hoyt GD, Deforest JL, Hu SJ. Long-term no-tillage and organic input management enhanced the diversity and stability of soil microbial community. Sci Total Environ. 2017; 609: 341–347.” were added.

3. Thank you for stating the following in the Acknowledgments Section of your manuscript: "This study was supported by the National Natural Science Foundation of China (31872851), the Innovative Research Groups of the Natural Science Foundation of Hunan Province (2019JJ10003)."

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement.

√ The funding-related text were removed from the manuscript, and the funding-related text were update in our Funding Statement. That is, the information about “This study was supported by the National Natural Science Foundation of China (31872851), the Innovative Research Groups of the Natural Science Foundation of Hunan Province (2019JJ10003), and Hunan Natural Science Foundation of China (2018JJ3305).” were added in our Funding Statement.

4. PLOS requires an pan class="Disease">ORCID iD for the correspn>onding author in Editorial Manager on papn>ers submitted after December 6th, 2016. Please ensure that you have an n>an class="Disease">ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager.

√ The pan class="Disease">ORCID iD were validated in Editorial Manager.

5. Please include your tables as part of your main manuscript and remove the individual files. Please note that supplementary tables (should remain/ be uploaded) as separate "supporting information" files.

√ The tables were added in the revised manuscript, according to Journal Requirements suggestion.

In the other aspect：

1. The “soil tillage” were replaced by “tillage”.

2. The “crop residues” were replaced by “crop residue”.

3. The “paddy fields” were replaced by “paddy field”.

4. The “the” of some places in this manuscript were deleted.

The revised manuscript has been submitted to your journal. Once again, thank you for your help and support during the process of the improvement of the manuscript and we look forward to your positive response.

Yours sincerely,

Hai-ming Tang

Submitted filename: Response to Reviewers.doc

Click here for additional data file.

13 Apr 2020

pan class="Chemical">PONE-D-20-02372R1

Functional diversity of rhizosphere soil microbial communities in response to different tillage and crop residue retention in a double-cropping pan class="Species">rice field

PLOS ONE

Dear Dr. Tang,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

ACADEMIC EDITOR: The revised version has been improved.  But the manuscript still have some problems as suggested by the reviewers. The authors should respond to the comments of the reviewers one by one and revise the manuscript accordingly.

==============================

We would appreciate receiving your revised manuscript by May 28 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Jian Liu

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: (No Response)

Reviewer #3: All comments have been addressed

Reviewer #4: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. pan class="Species">participant privacy or use of data from a third party—those must be spn>ecified.

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: No

Reviewer #3: Yes

Reviewer #4: No

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: There are still two many English grammer mistakes in the manuscript. It is suggested that it should be revised by a native Enlgish speaker or English language service.

P12 Line 9： there is no explanation of what are CT, RT, NT, and RTO in the abstract.

P 12 Line 8-11. ‘the average well color development (AWCD), richness and evenness of the functional diversity, the substrate reaction.’ This description gives too many details of quantifying functional diversity of rhizosphere soil microbial communities. Please only show the most important one.

pan class="Mutation">P12 A general scientific question is still missing here in the first paragrapn>h in the Introduction.

P14-P15. It would be better to present the four different treatments by a figure or a table, which can make it more easily understood by readers.

P17. Line 2-6. More details of how the data analysis was performed should be provided.

P17. Line 9-17. P value of the differences should be provided.

P 21, Line 9-11. Revise this sentence: ‘The results provided a reasonable hypothesis that enhanced soil conditions for increased rhizosphere soil microbial community function contribute due to the application of crop residue practices [22]’.

P 21, Line 20-22. Revise this sentence: ‘The reason maybe that crop residue retention with tillage treatments benefits the soil by increasing the soil environment, moderating moisture and temperature variation across seasons and has been reported favorable to microbial growth in rhizosphere soil [1, 13].’

P 21, Line 26-30. Revise this sentence: ‘suggesting that structure of the rhizosphere soil microbial communities may be altered by these two managements, the reason maybe that concentrations of SOC were significantly higher in the crop residue application treatments [14, 23], and soil ecological environment were also increased [8], which provided pan class="Chemical">carbon nutrient and environment for soil microbial multiply [1]’.

Reviewer #3: The authors have fixed most of the problems in last version. So my recommendation is acceptance for publication.

Reviewer #4: This revised paper has been somehow improved. While, there are still some works needed to be done.

1. Some sentences in the introduction are not clear. Such as Page 3 line 1 rewritten the sentence “ to increased”. Page 3 lines 9-10 “ Frasier et al. results showed that….” is recommended to be replaced by “Frasier et al. found that ”. also Page 3 lines 11-12 “Yang et al. results showed that….”, of which what is the meaning of “most pan class="Chemical">carbon sources of microbial ”. some other similar errors such as “ results showed that or results indicated that….” should be reorganized.

2. In the M & M part, some sentences about the description of study site is somehow tedious and not clear. Such as “this study was carried out on private land , the owner were gave permission to ”. By the way, gave references about the climate of the study site. Rewritten the sentences such as page 5 line 29 “ the applied kinds of were including ” as “the fertilized includes pan class="Chemical">urea, ordinary ”. Some sentences about the soil sampn>le is not clear. For exampn>le, “plant roots were removed by passing the sampn>les through a 2-mm mesh sieve”, actually, the size of mesh sieve just can remove large stone or large plant roots in the soil.

3. Still, the statistical analysis process is not clear. PCA is a method and is not a kind of parameters or varialbes.

4. In the discussion, some sentences are not easy to understand. Such as Page 11 lines 9-10 “ provide a reasonable hypothesis that for increased ”; Page 11 line 22 “ has been reported favorable ”; also, more words like “the reason maybe…”will be boring to authors.

5. It is suggested that invite native English speaker or academic researcher to polish the language throughout the whole manuscript.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

16 Apr 2020

<PLOS ONE >

<pan class="Chemical">PONE-D-20-02372>

Dear Editor,

Thank you very much for your useful comments and suggestions on our manuscript. Please convey our gratitude to the reviewers who have made useful and detailed suggestions for improvement of the manuscript. As their suggestions we have revised the language and the content with red color in the manuscript. The details of these changes were listed below in point form:

Reviewer #2:

1．P 2 Line 9：there is no explanation of what are CT, RT, NT, and RTO in the abstract.

√ In the “Abstract” section, the information about CT, RT, NT, and RTO were added, according to reviewer suggestion.

2．P 2 Line 8-11. ‘the average well color development (AWCD), richness and evenness of the functional diversity, the substrate reaction.’ This description gives too many details of quantifying functional diversity of rhizosphere soil microbial communities. Please only show the most important one.

√ In the “Abstract” section, this sentence were revised, according to reviewer suggestion.

3．P 2 A general scientific question is still missing here in the first paragraph in the Introduction.

√ In the first paragraph of “Abstract” section, a general scientific question were added, according to reviewer suggestion.

4．P 4-5. It would be better to present the four different treatments by a figure or a table, which can make it more easily understood by readers.

√ In the “Field experiment” section, it is difficult to layout a table to present the different information about four different treatments when I try to present the four different treatments by a table, and the relative information about four different treatments were well described in the present status. Therefore, I think that there is need not to present the four different treatments by a a table.

5．P7. Line 2-6. More details of how the data analysis was performed should be provided.

√In P7. Line 2-6, more information about data analysis were added and revised, according to reviewer suggestion.

6．P7. Line 9-17. P value of the differences should be provided.

√In P7. Line 9-17, P value of the differences were added, according to reviewer suggestion.

7．P 11, Line 15-17. Revise this sentence: ‘The results provided a reasonable hypothesis that enhanced soil conditions for increased rhizosphere soil microbial community function contribute due to the application of crop residue practices [22]’.

√In P 11, Line 15-17, this sentence were revised, according to reviewer suggestion.

8．P 11, Line 20-22. Revise this sentence: ‘The reason maybe that crop residue retention with tillage treatments benefits the soil by increasing the soil environment, moderating moisture and temperature variation across seasons and has been reported favorable to microbial growth in rhizosphere soil [1, 13].’

√In P 11, Line 20-22, this sentence were revised, according to reviewer suggestion.

9．P 11, Line 26-30. Revise this sentence: ‘suggesting that structure of the rhizosphere soil microbial communities may be altered by these two managements, the reason maybe that concentrations of SOC were significantly higher in the crop residue application treatments [14, 23], and soil ecological environment were also increased [8], which provided pan class="Chemical">carbon nutrient and environment for soil microbial multiply [1]’.

√In P 11, Line 26-30, these sentences were revised, according to reviewer suggestion.

Reviewer #4:

1. Some sentences in the introduction are not clear. Such as Page 3 line 1 rewritten the sentence “ to increased”. Page 3 lines 9-10 “ Frasier et al. results showed that….” is recommended to be replaced by “Frasier et al. found that ”. also Page 3 lines 11-12 “Yang et al. results showed that….”, of which what is the meaning of “most pan class="Chemical">carbon sources of microbial ”. some other similar errors such as “ results showed that or results indicated that….” should be reorganized.

√In P 3, Line 1, the “ to increased” were replaced by “increased”, according to reviewer suggestion.

√In P 3 lines 9-10, the “results showed that” were replaced by “found that”, according to reviewer suggestion.

√In P 3 lines 11-12, the “indicated” were replaced by “found”, and this sentence were revised, according to reviewer suggestion.

√Other similar errors were also revised, according to reviewer suggestion.

2. In the M & M part, some sentences about the description of study site is somehow tedious and not clear. Such as “this study was carried out on private land , the owner were gave permission to ”. By the way, gave references about the climate of the study site. Rewritten the sentences such as page 5 line 29 “ the applied kinds of were including ” as “the fertilized includes pan class="Chemical">urea, ordinary ”. Some sentences about the soil sampn>le is not clear. For exampn>le, “plant roots were removed by passing the sampn>les through a 2-mm mesh sieve”, actually, the size of mesh sieve just can remove large stone or large plant roots in the soil.

√In the M & M part, P4 lines 11-12, this sentence were revised, according to reviewer suggestion.

√In P4 lines 13-15, the reference about the climate of this study site were added, according to reviewer suggestion.

√In P5 line 29, this sentence were revised, according to reviewer suggestion.

√In P6 line 17, this sentence were revised, according to reviewer suggestion.

3. Still, the statistical analysis process is not clear. PCA is a method and is not a kind of parameters or varialbes.

√In the “Statistical analysis” section, this sentence were revised, according to reviewer suggestion.

4. In the discussion, some sentences are not easy to understand. Such as Page 11 lines 9-10 “ provide a reasonable hypothesis that for increased ”; Page 11 line 22 “ has been reported favorable ”; also, more words like “the reason maybe…”will be boring to authors.

√In P11 lines 9-10, this sentence were revised, according to reviewer suggestion.

√In P11 lines 22, these sentences were also revised, according to reviewer suggestion.

5. It is suggested that invite native English speaker or academic researcher to polish the language throughout the whole manuscript.

√ The language of this manuscript were revised by academic researcher, according to reviewer suggestion. Such as the “managements” were replaced by “management”, the “and” were replaced by “with”, and the “the” in some places were deleted, and so on.

The revised manuscript has been submitted to your journal. Once again, thank you for your help and support during the process of the improvement of the manuscript and we look forward to your positive response.

Yours sincerely,

Hai-ming Tang

Submitted filename: Response to Reviewers.doc

Click here for additional data file.

7 May 2020

pan class="Chemical">PONE-D-20-02372R2

Functional diversity of rhizosphere soil microbial communities in response to different tillage and crop residue retention in a double-cropping pan class="Species">rice field

PLOS ONE

Dear Dr. Tang,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

ACADEMIC EDITOR: The revised version has been improved.  But the manuscript still have some problems as suggested by the reviewer.

==============================

We would appreciate receiving your revised manuscript by Jun 21 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

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Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Jian Liu

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #4: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

Reviewer #4: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #4: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. pan class="Species">participant privacy or use of data from a third party—those must be spn>ecified.

Reviewer #2: Yes

Reviewer #4: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

Reviewer #4: No

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: (No Response)

Reviewer #4: This revision has been improved greatly. While, there are still some works needed to be done:

1. In the abstract section, some sentences are not clear, such as Page 2 lines 8~13 remerge the two sentences about the description about the treatment code (CT, RT, NT, RTO). Page 2 13~14 suggested to rewritten the sentence as “…the values of AWCD with application of crop residue were higher that of without crop residues.”

2. In page 3 lines 13-21, the references style in main text is not right, it is suggested to delete the year. Line 20, delete the word “some”. Also, replace “some studies” by “other…”. In lines 27-28, it will be easy to understand to rewritten the sentence as “the manipulation of returning the Chinese milk….to the paddy soil….because it provides….”.

3. In the M& M section, somehow it will be better to rewritten the sentences in page 4 lines 15-18 as “In 2015, the experiment was conducted in Ning Xiang County….China, where is the main area of double-cropping pan class="Species">rice and there were no endangered or protected spn>ecies involved”. Still, there are other mistakes about the sentences, read through this section and polish the language.

4. Although there are improvements about the description of statistical analysis, the description about the PCA is not clear, such as by which software it is performed. It is suggested to reorganized the sentences page 10 lines 10~12.

5. Still, it is suggested the author to read through the whole manuscript and reduce the grammar or language mistakes.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

Reviewer #4: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

8 May 2020

<PLOS ONE >

<pan class="Chemical">PONE-D-20-02372>

Dear Editor,

Thank you very much for your useful comments and suggestions on our manuscript. Please convey our gratitude to the reviewers who have made useful and detailed suggestions for improvement of the manuscript. As their suggestions we have revised the language and the content with red color in the manuscript. The details of these changes were listed below in point form:

Reviewer #4:

1. In the abstract section, some sentences are not clear, such as Page 2 lines 8~13 remerge the two sentences about the description about the treatment code (CT, RT, NT, RTO). Page 2 13~14 suggested to rewritten the sentence as “…the values of AWCD with application of crop residue were higher that of without crop residues.”

√ In Page 2 lines 8~13, these sentences were revised, according to reviewer suggestion.

√ In Page 2 lines 13~14, this sentences were revised, according to reviewer suggestion.

2. In page 3 lines 13-21, the references style in main text is not right, it is suggested to delete the year. Line 21, delete the word “some”. Also, replace “some studies” by “other…”. In lines 27-28, it will be easy to understand to rewritten the sentence as “the manipulation of returning the Chinese milk….to the paddy soil….because it provides….”.

√ In Page 3 lines 13~21, the year of the references were deleted, according to reviewer suggestion.

√ In Page 3 lines 21, the “some studies ” were replaced by “other studies”, according to reviewer suggestion.

√ In Page 3 lines 27-28, this sentences were revised, according to reviewer suggestion.

3. In the M& M section, somehow it will be better to rewritten the sentences in page 4 lines 15-18 as “In 2015, the experiment was conducted in Ning Xiang County….China, where is the main area of double-cropping pan class="Species">rice and there were no endangered or protected spn>ecies involved”. Still, there are other mistakes about the sentences, read through this section and polish the language.

√ In the M& M section, in page 4 lines 15-18, these sentences were revised, according to reviewer suggestion. And other places were also revised, according to reviewer suggestion.

4. Although there are improvements about the description of statistical analysis, the description about the PCA is not clear, such as by which software it is performed. It is suggested to reorganized the sentences page 7 lines 10~12.

√ In Page 7 lines 10-12, these sentences were revised, according to reviewer suggestion.

5. Still, it is suggested the author to read through the whole manuscript and reduce the grammar or language mistakes.

√ The grammar or language mistakes of this manuscript were revised, according to reviewer suggestion.

The revised manuscript has been submitted to your journal. Once again, thank you for your help and support during the process of the improvement of the manuscript and we look forward to your positive response.

Yours sincerely,

Hai-ming Tang

Submitted filename: Response to Reviewers.doc

Click here for additional data file.

11 May 2020

Functional diversity of rhizosphere soil microbial communities in response to different tillage and crop residue retention in a double-cropping pan class="Species">rice field

pan class="Chemical">PONE-D-20-02372R3

Dear Dr. Tang,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Jian Liu

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Page 12,line 16: "China China" . Please delete one word.

Reviewers' comments:

13 May 2020

pan class="Chemical">PONE-D-20-02372R3

Functional diversity of rhizosphere soil microbial communities in response to different tillage and crop residue retention in a double-cropping pan class="Species">rice field

Dear Dr. Tang:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Jian Liu

Academic Editor

PLOS ONE