Fertilization is an important management option to increase soil fertility and crop yield,but the effects of fertilization regimes are soil specific.Microbial community composition and enzyme activity are sensitive indicators of ongoing changes in soil,which cannot be easily achieved with other parameters at early stages(B¨orjesson et al.,2012).Long-term fertilization has been shown to strongly in fluence soil microbial composition and increase the expression of enzyme-encoding genes(Su et al.,2015).Compared with the application of mineral fertilizers,combined mineral and organic fertilization accelerates microbial growth and soil enzyme activities(Liu et al.,2010;Lazcano et al.,2013;Ai et al.,2015).Microbial coummunity and enzyme activities are sensitive to changes in soil moisture(Gordon et al.,2008;Unger et al.,2009;Kechavarzi et al.,2010),but it is not clear how the microbial community and enzyme activities respond to changes in soil moisture depending on fertilization regimes.The effects of moisture on microbial properties can be modi-fied by soil fertility.Biological properties(i.e.,microbial biomass carbon,basal respiration rate,enzyme activities)responding to drying-rewetting were found to be negatively correlated with the content of soil organic carbon(SOC)(Zhao et al.,2010).The SOC is the dominant factor controlling the size of microbial communities in paddy soils(Pan et al.,2016).Soil moisture was identified as the critical factor modifying microbial community structure;however,nutrients were not prominent co-limiting factors(Ng et al.,2015).Microorganisms can maintain their primary functions in the face of environmental disturbance depending on physiological acclimation and adaptations mechanisms(Schimel et al.,2007).Because of these inherent constraints and lack of adaptation to distinct environmental conditions,wetting pulses may have specific effects on biochemical processes in various soil types(Borken and Matzner,2009;Manzoni et al.,2012).Fertilization and drying-rewetting affected biochemical properties in a sandy loam soil,whereas soils with higher fertility were able to maintain their original biological functions in response to those cycles(Zhao et al.,2010).However,higher fertility grasslands exhibited greater decreases in microbial biomass nitrogen(N)and soil nutrients in response to drying-rewetting(Gordon et al.,2008).Available data on the self-regulation of soil properties in response to changes in moisture along fertility levels are inconsistent.

桑干河发源于宁武县管涔山庙儿沟，属海河流域永定河水系，控制流域面积17 744 km2。桑干河山阴县河头村生态蓄水工程，位于山阴县合盛堡乡河头村附近的桑干河干流上，控制流域面积4 892 km2。

[36]吴毅、贺雪峰、罗兴佐等：《村治研究的路径与主体——兼答应星先生》，《开放时代》2005年第4期。

总之，教师需要将互联网运用在高中德育管理中，指导家长开展针对学生的德育实践，突破时空界线实现教育资源的共享，从而增强德育教学的实效性，提高高中生的道德修养，塑造其健全的人格。在互联网的环境条件下，搭建家庭与学校之间全程、全员及全方位育人的立交桥，使交流更为畅通高效，使家庭与学校的关系更加和谐，是促进家校共创与教育健康发展的新颖平台。

Paddy soil derived from uncultivated barren land is generally deficient in nutrients,and fertilization can improve soil fertility in crop production(Li et al.,2000).Application of mineral fertilizers increases SOC to a limited extent,and organic manure increases microbial biomass carbon(C)and its functional diversity(Zhong and Cai,2007).This study investigated the responses of soil microbial community and enzyme activities to changes in moisture along a gradient of soil fertility formed within a long-term(24 years) field experiment.We hypothesized that after long-term fertilization,the gradients of soil fertility would have formed,which would lead to different responses of microbial properties to changes in soil moisture.To test this hypothesis,three moisture regimes(submergence,low moisture,and submerging-draining cycles)were considered.

MATERIALS AND METHODS

Experimental site and soil sampling

The long-term field experiment was conducted at the Yingtan Red Soil Ecological Experiment Station(28°15′30′′N,116°55′30′′E),Chinese Academy of Sciences in Yujiang County,Jiangxi Province of China.This site has a typical subtropical monsoon climate with a mean annual temperature of 17.6°C and an annual precipitation of 1795 mm.The experiment field was established in 1990 on an uncultivated barren area with soil derived from Quaternary red clay.The initial chemical properties of the soil were as follows∶pH 4.5,organic C 5.7 g kg−1,total N 0.43 g kg−1,total phosphorus(P)0.65 g kg−1,total potassium(K)13.4 g kg−1,alkali-hydrolyzable N 90.2 mg kg−1,available P 5.6 mg kg−1,available K 105.9 mg kg−1,and 38%clay(＜ 1 µm).The cropping system was double-cropped rice(Oryza sativa L.)including early and late season rice.Planting dates were early April to the end of October,and the rest of the year was fallow.Four fertilizer treatments were established∶unfertilized control(CK),organic manure(M),N,P,and K fertilizers(NPK),and NPK plus M(NPK+M).Each treatment was replicated in three plots(each 30 m2)in a completely randomized design.Manure was applied as follows∶in each plot of M treatment,all rice straw(9.9 g N kg−1,1.1 g P kg−1,31.0 g K kg−1,and 387 g C kg−1)from the plot was returned,and 833 kg ha−1(dry weight)of pig manure(21.2 g N kg−1,27.6 g P kg−1,18.1 g K kg−1,and 267 g C kg−1)was added.Mineral fertilizers N,P,and K were applied as urea(115 kg N ha−1),calcium-magnesium phosphate(68 kg P2O5ha−1),and potassium chloride(42 kg K2O ha−1),respectively,in each growing season.N fertilizer was added as a basal dressing and a top dressing at a ratio of 8∶7;P and K fertilizers were applied as a basal dressing.

where βG, βX,NAG,and AP are the activities of β-glucosidase,β-xylosidase,N-acetyl-glucosaminidase,and acid phosphatase,respectively(Garc´ıa-Ruiz et al.,2008).

Laboratory incubation at three soil moisture regimes

A 150 g(dry weight)soil sample from each fertilizer treatment was placed in a glass jar(300 mL),and subjected to one of three soil moisture regimes∶i)submergence(continuously submerged)at a water∶soil ratio of 1.2∶1(volume∶weight),ii) five submerging-draining cycles(S-D cycles)with 7-d waterlogging followed by 7-d draining,and iii)constant soil moisture content at 40%water-holding capacity(low moisture).All soil samples were incubated for 70 d at 25°C in darkness.Deionized water was added every 5 d to replenish water loss under low moisture.At the end,soil water was drained offfor the submergence treatment.All soils were kept at room temperature for 1 d,and then passed through a 2-mm sieve.Each soil sample was divided into two parts∶one was freeze-dried at−80 °C for phospholipid fatty acid(PLFA)analysis,and the other was stored at 4°C for enzyme activity assays.

Measurement of soil chemical properties

Soil pH and nutrient contents were assayed prior to incubation.Soil pH was determined using a pH meter(FE30,Mettler-Toledo,Switzerland)with a 1∶2.5(weight∶volume)soil∶water suspension.The SOC was measured using the dichromate method(Nelson and Sommers,1982).Total and available N were measured using the semimicro-Kjeldahl method(Lu,1999);total P by the molybdenum blue method after HF-HClO4 digestion(Jackson,1958);available P by the molybdenum blue method after sodium bicarbonate extraction;total K by HF-HClO4digestion(Jackson,1958)and a flame photometer;and available K by ammonium acetate extraction and a flame photometer(Lu,1999).

Phospholipid fatty acid analysis

The PLFA analysis was used to discriminate microbial community composition among fertilizer treatments with changes in soil moisture.A modified Bligh-Dyer method(Bossio et al.,1998)was used to extract PLFAs.Briefly,3 g of freeze-dried soil sample were added to a Teflon centrifuge tube with 15.2 mL buffer(chloroform∶methanol∶phosphate at 1∶2∶0.8 volume ratio)to extract the PLFAs.Polar lipids were separated from the extracted fatty acids in chloroform by using a silica-bonded phase column(SPE-Si,Supelco,UK)with chloroform,acetone,and methanol in that order.Alkaline methanolysis trans-esterified the polar lipids to fatty acid methyl esters(FAMES).The FAMES were quantified using nonadecanoic acid methyl ester(19∶0)as the internal standard and determined by a gas chromatograph mass spectrometer(Agilent 7890,USA)and the MIDI Sherlock Microbial Identification System(MIDI,USA).The content of PLFAs was expressed as nmol g−1.

The CVs of biochemical indexes were calculated for each fertilizer treatment to evaluate the stability of soil microbial properties in response to changes in soil moisture.The RDA analysis showed that the CK soil was clearly separated from the fertilizer-treated soils along the RDA 1 axis,which explained the major difference(54.5%).The CV values of biochemical indexes were negatively correlated with the SOC,total P,and available N(Fig.3).These results indicated that paddy soils with higher fertility were better able to maintain their original biological functions in response to changes in moisture.

Assays of soil enzyme activity

10月24日下午，习近平总书记在广东考察时，来到广州明珞汽车装备有限公司。他指出：“民营企业对我国经济发展贡献很大，前途不可限量。”

Long-term fertilization in fluenced SOC and soil nutrient contents and availability∶compared with the CK treatment,the fertilization treatments increased SOC,total N,available N,total P,and available P by 30.1%–36.3%,27.3%–38.4%,35.9%–56.4%,28.6%–102.9%,and 61.4%–440.9%,respectively(Table I).Total and available P in the fertilization treatments differed,following the pattern of NPK+M＞NPK＞M.Enzyme activities depending on soil moisture regimes

After the late season rice harvest in November 2014, five soil cores(5 cm×10 cm×18 cm)were collected on a W-shaped transect across each plot and then pooled to form one composite sample.After removing visible root debris,the soils were ground to pass a 2-mm sieve and thoroughly mixed before use for chemical analyses and incubation.

Data analysis

第一个环节“佳篇有约”进行中，临时增加了“作者答问”。课前我的预设是：作者先朗读自己的作品，而后介绍写作背景，同学们聆听，听后讨论、点评。但第一个作者就没有准备好。不能冷场，于是临时调整为“思后问”，即作者先朗读完自己的作品，同学们静静思考2分钟，然后向作者提问。

The effects of fertilization on soil pH and nutrient contents were analyzed using one-way analysis of variance(ANOVA)with SPSS version 16.0 for Windows(SPSS Inc.,USA).Duncan’s test was used to test for significant effects of fertilization(P ＜ 0.05).Twoway ANOVA was used to determine whether the differences among moisture regimes and fertilization were significant.Nonmetric multidimensional scaling(NMDS)analysis was used to identify the pro files based on the data of individual PLFAs.Redundancy analysis(RDA)was conducted to investigate the relationship between soil nutrient contents and coefficients of variation(CVs)from seven biochemical indexes.The biochemical indexes included GMea,total PLFAs,ac-tinomycete PLFAs,fungal PLFAs,bacterial PLFAs,Gram-positive bacterial PLFAs/Gram-negative bacterial PLFAs,and bacterial PLFAs/fungal PLFAs.The analyses of NMDS and RDA were performed using the‘vegan’package in R software version 3.2.5.

RESULTS

Changes in soil nutrient after long-term fertilization

由于黏膜出现肿胀问题，导致胆管阻塞，胆汁无法顺利进入肠道，导致细菌在大量繁殖的同时，产生大量毒液，这些毒液进入到血液中会致使患病机体中毒。此外，患病羊脱水现象明显，致使血液粘稠度不断增加，外周循环阻力大幅度提升，机体心脏压力不断增大。在心脏的代偿作用减弱以后，会导致患病体的心力衰竭，外周循环也不断趋于衰竭化，进而导致患病体出现休克现象。慢性胃肠炎具体是由于结缔组织增生而导致的。

The ranges of βG,βX,NAG,and AP in the tested soils were 32.78–109.80,7.48–17.66,15.14–46.83,and 177.80–575.37 nmol h−1g−1,respectively(Table II).Under different soil moisture conditions,the ranges of βG, βX,NAG,and AP in the CK soil were 32.78–61.79,7.48–10.70,15.14–28.18,and 177.80–461.58 nmol h−1g−1,respectively(Table II).In the fertilizer-treated soil,the corresponding enzyme activities were 42.59–109.80,8.80–17.66,25.19–46.83,and 189.23–575.37 nmol h−1g−1(Table II).The ANOVA analysis showed that both soil moisture and fertilizerapplication markedly in fluenced the enzyme activities(Table II).In the CK soil,the βG, βX,NAG,and AP values under S-D cycles were lower than those under low moisture by 46.1%,27.7%,46.3%,and 61.5%,respectively.In the fertilization treatments,the corresponding decreases were 42.5%–55.7%,1.7%–22.6%,20.5%–33.8%,and 40.7%–54.4%,respectively.Soil fertility conditions buffered the effects of soil moisture regimes on enzymatic activities differently depending on the type of enzyme.

TABLE I Effects of fertilization on soil pH,soil organic carbon(SOC),and nutrient contents

a)CK=unfertilized control;M=organic manure;NPK=N,P,and K fertilizers;NPK+M=N,P,and K fertilizers plus organic manure. b)Means±standard errors(n=3). c)Means followed by the same letter(s)within each column are not significantly different at P ＜ 0.05 according to Duncan’s test.

Fertilizera) pH SOC Total Available N P K N P K g kg−1mg kg−1 CK 5.8±0.1b)ac) 8.4±0.5b 1.0±0.01b 0.35±0.02d 13.9±0.5a 47.8±2.1b 4.4±0.6d 118.0±9.8a M 5.7±0.1a 10.9±0.8a 1.3±0.04a 0.45±0.02c 13.6±0.3a 64.9±2.5ab 7.1±0.9c 65.0±10b NPK 5.6±0.07a 11.0±0.6a 1.4±0.10a 0.58±0.03b 13.9±0.6a 73.5±9.3a 13.7±0.9b 65.0±1.4b NPK+M 5.6±0.1a 11.5±0.7a 1.4±0.07a 0.71±0.02a 13.3±0.5a 74.7±6.5a 23.8±0.8a 98.8±3.8a

TABLE II Effects of soil moisture regimes and fertilization on the activities of β-glucosidase(βG),β-xylosidase(βX),N-acetyl-glucosaminidase(NAG),and acid phosphatase(AP)and analysis of variance(ANOVA)for soil moisture,fertilization,and their interaction

a)See Table I for the detailed descriptions of the abbreviations for fertilizers.b)Submerging-draining. c)Means±standard errors(n=3).

Enzyme Fertilizera) Soil moisture regime Moisture Fertilization Interaction activity Submergence S-Db)cycles Low moisture F value P value F value P value F value P value nmol h−1g−1 βG CK 32.78±2.58c) 33.30±3.61 61.79±7.80 28.4 0.00 8.2 0.001 1.2 0.32 M 43.23±2.80 45.98±3.14 103.77±6.28 NPK 42.59±4.28 51.56±3.68 109.80±16.44 NPK+M 66.21±12.93 56.80±5.17 98.84±7.47 βX CK 7.48±1.42 7.74±0.34 10.70±1.66 3.3 0.05 14.1 0.00 0.8 0.6 M 16.32±1.17 14.62±0.64 17.66±1.57 NPK 8.80±0.45 11.77±0.92 11.97±1.78 NPK+M 13.98±0.62 12.58±0.83 16.25±2.37 NAG CK 17.64±1.23 15.14±2.20 28.18±7.59 6.8 0.005 8.6 0.00 0.6 0.7 M 27.03±4.18 26.84±1.02 33.75±2.62 NPK 25.19±2.01 27.48±1.81 40.92±6.57 NPK+M 43.51±11.56 31.02±2.56 46.83±2.47 AP CK 195.86±26.47 177.80±8.04 461.58±45.24 70.1 0.00 3.6 0.03 0.8 0.6 M 274.17±57.12 262.45±18.00 575.37±47.82 NPK 189.23±6.12 261.94±24.14 485.46±39.39 NPK+M 258.49±33.79 285.02±36.15 480.30±49.87

Response of microbial community composition to soil moisture regimes

Soil microbial community composition,discriminated by PLFA analysis,was sensitive to changes in soil moisture.The NMDS analysis placed the soil samples under low moisture regime in the positive terminal of the NMDS 1 coordinate axis,and those under S-D cycles and submergence were located close together in the negative terminal(Fig.1).Substantial overlap of the PLFA data for the soil samples under S-D cycles and submergence on the NMDS 1 coordinate axis suggested that submerging stress was a major contributor to the shift in community structure for the soils under S-D cycles.Under S-D cycles,the CK soil was clearly separated from the three fertilizer-treated soils along the NMDS 2 coordinate axis.

Total PLFAs in the tested soils ranged from 18.74 to 38.27 nmol g−1.Under three soil moisture regimes,total PLFAs in the CK soil ranged from 18.74 to 28.93 nmol g−1and in the fertilized soils from 29.16 to 38.27 nmol g−1(Table III).Under all soil moisture regimes,the contents of PLFA markers for actinomycete,fungal,and bacterial communities in the CK soil were lower than those in the fertilizer-treated soils.Application of fertilizer increased the microbial richness.The ANOVA analysis showed that both soil moisture and fertilization significantly in fluenced microbial community composition(Table III).

The β-glucosidase,β-xylosidase,N-acetyl-glucosaminidase,and acid phosphatase activities were measured using fluorogenically labeled substrates∶4-methylumbelliferone(MUB)-β-D-glucoside,4-MUB-β-D-xylopyranoside,4-MUB-N-acetyl-β-D-glucosaminide,and 4-MUB-phosphate,respectively(Darrah and Harris,1986;Saiya-Cork et al.,2002).This method was described by DeForest(2009).Briefly,1 g of fresh soil was added to 125 mL of 50 mmol L−1acetate buffer(pH=5.5)and homogenized for 2 min with an OMNI mixer(OMNI,USA)to obtain soil suspensions.We used 96-well,300-µL black polystyrene microplates(Corning,USA).After running the blank,negative control,quench standard,and the sample,the microplates were incubated in darkness at 20°C for 2 h.Enzymatic activities were determined fluorometrically using a microplate fluorometer(SpectraMax i3x,Molecular Devices,USA)with 365 nm excitation and 450 nm emission filters.Absolute enzymatic activities were calculated by correcting for homogenate control,substrate control,and quenching,which were expressed in unit of nmol g−1soil h−1.The geometric mean of the four soil enzyme activities(GMea)in each sample was calculated using Eq.1.

Fertilization strongly in fluenced the effects of soil moisture on the content of total PLFAs(Fig.2).In the CK soil,the content of total PLFAs under S-D cycles was 54.4%higher than that under low moisture(Fig.2);however,in the fertilization treatments,total PLFAs were 12.4%–29.4%higher under S-D cycles than under low moisture.Compared with low moisture regime,the degree of changes in total,actinomycete,fungal,and bacterial PLFAs under S-D cycles was mediated by fertilization.The greatest changes occurred in the CK soils and the lowest in the manure-treated soils(Fig.2).Fertilizer application maintained the stability of microbial composition in response to fluctuation in soil moisture.

Fig.1 Nonmetric multidimensional scaling(NMDS)analysis of individual phospholipid fatty acid data in soils under long-term fertilizer treatments and different soil moisture regimes.Soil moisture regimes are discriminated by colors:black stands for submerging-draining cycles,grey for low moisture,and white for submergence.See Table I for the detailed descriptions of the abbreviations for fertilizers(CK,M,NPK,and NPK+M).

Fig.2 Changes of phospholipid fatty acids(PLFAs)in the soils after long-term fertilizer treatments under soil moisture regime of five submerging-draining cycles compared with those under low moisture regime.See Table I for the detailed descriptions of the abbreviations for fertilizers(CK,M,NPK,and NPK+M).

Responses of soil properties to soil moisture regimes

The PLFAs were separated into various taxonomic groups according to PLFA biomarker data(Frosteg˚ard et al.,1993;Bossio et al.,1998;Green and Scow,2000).The polyunsaturated PLFAs 18∶2ω6c and 18∶1ω9c were chosen as fungal biomarkers(Kaiser et al.,2010);the fatty acids 10Me16∶0,10Me17∶0,and 10Me18∶0 were chosen as biomarkers of actinomycetes(Vestal and White,1989);and the PLFAs 15∶0,16∶0,17∶0,and 18∶0 were chosen as biomarkers for general bacteria(Zelles et al.,1997).We used i15∶0,a15∶0,i16∶0,i17∶0,and a17∶0 as Gram-positive bacterial biomarkers and 16∶1ω7c,18∶1ω7c,cy17∶0ω7c,and cy19∶0ω7c as Gram-negative bacterial biomarkers(Green and Scow,2000).

DISCUSSION

After 24 years of fertilization,the SOC content increased in the fertilized soils compared with the unfertilized soil(Table I).Long-term manure input increased both the content and availability of soil organic matter(SOM)(Carter,2002;Da Silva Oliveira et al.,2017).In the CK and NPK treatments,crop roots and rhizodeposits were the sole sources of organic input into the soil,since aboveground biomass was completely removed from the field.Rice plants allocated about 30%–60%of net photosynthesized C to roots,and 40%–90%of this fraction was translocated into the soil(Lynch and Whipps,1990).In the present study,the rice yield under the NPK treatment(4722±375 kg ha−1)was much higher than that without fertilizer application(1566± 157 kg ha−1).This indirectly promoted SOM accumulation via the corresponding increase of roots and rhizodeposits.The increased SOM stimulated microbial growth and thus enzyme release(Kuzyakov et al.,2000).

（4）按“以废治废”，覆盖50cm厚度的底泥计，每平方米可消耗0.5m3的底泥废弃物，明显减轻了矿企的底泥处理压力，节约了大量处理费用。并且，由于底泥的使用减少了原有改良材料的添加，相比降低了约6%的修复成本。

Enzyme activities under low moisture were higher than those under S-D cycles or submergence(Table II).Decreased enzyme activities in flooded soil are associa-ted with∶i)shifts in microbial community(Rinklebe and Langer,2006),ii)decreased enzyme production(Kang et al.,1998),or iii)increased inhibitors such as free metal ions(Freeman et al.,1996).The effective life span of enzymes depends on both the intrinsic properties of the enzymes and the capacity of soil matrix to bond and stabilize enzymes through associations with mineral surfaces(Tietjen and Wetzel,2003;Allison,2006).Application of manure and/or NPK increased the content of SOM,which buffered the effect of moisture on enzyme activities.

TABLE III Effects of soil moisture regimes and fertilization on microbial community composition,discriminated by phospholipid fatty acid(PLFA)analysis,and analysis of variance(ANOVA)for soil moisture,fertilization,and their interaction

a)See Table I for the detailed descriptions of the abbreviations for fertilizers.b)Submerging-draining. c)Means±standard errors(n=3).

PLFAs Fertilizera)Soil moisture regime Moisture Fertilization Interaction Submergence S-Db)cycles Low moisture F value P value F value P value F value P value nmol g−1 Total CK 23.49±0.59c)28.93±5.41 18.74±2.17 8.0 0.002 13.8 0.00 0.8 0.56 M 38.27±2.38 34.46±0.68 30.67±2.35 NPK 34.55±2.08 36.61±0.85 32.45±2.28 NPK+M 36.79±1.55 37.74±1.52 29.16±3.91 Actino- CK 2.77±0.48 2.81±0.01 2.37±0.33 4.3 0.03 13.3 0.00 1.1 0.39 mycete M 4.27±0.45 3.80±0.07 3.76±0.12 NPK 3.71±0.32 4.04±0.10 3.74±0.09 NPK+M 3.92±0.17 4.16±0.32 3.38±0.30 Fungal CK 1.47±0.13 1.25±0.03 1.29±0.14 2.5 0.10 8.1 0.001 0.32 0.92 M 2.43±0.23 2.19±0.07 2.09±0.22 NPK 2.40±0.12 2.45±0.25 2.17±0.12 NPK+M 2.40±0.31 2.92±0.08 1.92±0.30 Bacterial CK 19.73±0.62 20.10±0.15 15.54±1.84 8.3 0.002 13.8 0.00 0.87 0.53 M 32.59±1.99 29.34±0.67 25.79±2.06 NPK 29.30±1.92 31.07±0.97 27.51±2.10 NPK+M 31.37±1.15 31.94±1.03 24.75±3.47 Gram-negative CK 4.50±0.28 4.48±0.19 3.76±0.32 12.3 0.00 13.8 0.00 0.62 0.71 bacterial(G−) M 7.55±0.62 7.55±0.97 6.05±0.42 NPK 7.45±0.46 9.42±0.48 6.46±0.62 NPK+M 7.15±0.44 9.19±0.24 6.06±1.10 Gram-positive CK 8.61±0.41 9.34±0.21 6.47±1.03 7.4 0.003 9.4 0.00 1.8 0.14 bacterial(G+) M 14.38±1.02 12.41±0.55 10.88±0.95 NPK 12.02±0.98 11.99±0.55 11.77±0.71 NPK+M 13.88±0.57 12.84±0.93 10.28±1.12 G+/G− CK 1.93±0.16 2.09±0.14 1.70±0.19 0.7 0.51 0.4 0.76 0.6 0.70 M 1.92±0.15 1.71±0.26 1.79±0.03 NPK 1.63±0.16 1.28±0.10 1.84±0.07 NPK+M 1.95±0.11 1.40±0.14 1.74±0.12 Bacterial/fungal CK 13.62±1.36 16.09±0.26 12.02±0.75 1.1 0.37 0.4 0.73 0.6 0.74 M 13.46±0.40 13.41±0.42 12.41±0.46 NPK 12.26±0.92 14.29±0.53 12.66±0.30 NPK+M 13.39±1.30 11.12±0.22 13.04±0.91

Fig.3 Redundancy analysis(RDA)of soil nutrients and coeffi-cients of variation(CVs)of seven biochemical indexes under four fertilizer treatments and three soil moisture regimes.The explanatory variables are indicated by different arrows.Soil nutrients included soil organic carbon(SOC),total phosphorus(TP),available nitrogen(AN),and available potassium(AK)and biochemical indexes included the geometric mean of four soil enzyme activities(GMea),total phospholipid fatty acids(PLFAs)(T),actinomycete PLFAs(A),fungal PLFAs(F),bacterial PLFAs(B),Gram-positive bacterial PLFAs/Gram-negative bacterial PLFAs(G+/G−),and bacterial PLFAs/fungal PLFAs(B/F).The CV value was calculated from 100 times the ratio of standard deviation to mean of soils subjected to different soil moisture regimes.See Table I for the detailed descriptions of the abbreviations for fertilizers(CK,M,NPK,and NPK+M).

Soil microbial community composition was sensitive to changes in soil moisture.Compared with the soils under low moisture,those under submergence and S-D cycles had a higher PLFA content(Table III).Microbial biomass in subsurface soils increased by eight times after drying/rewetting compared with that under constant moisture(Xiang et al.,2008).Soil moisture regimes in fluenced microbial community structure both directly and indirectly through effects on nutrient availability and oxygen concentrations(Drenovsky et al.,2004).Waterlogging with lower oxygen levels leads to the selection of facultative and obligate anaerobic microorganisms.Flooding is a part of paddy cultivation,and most microorganisms are adapted to flooding.When paddy soil is exposed to fluctuations in moisture,the submergence was more beneficial for the microbial communities compared with the aerobic condition.

Application of manure or mineral fertilizers increased the stability of microbial community response to changes in moisture.Application of organic fertilizers promoted soil resilience to disturbance(Griffiths and Philippot,2013).Shifts in microbial community structure induced by drought stress were more pronounced in soils without manure application than in those amended with manure(Hueso et al.,2012).Microorganisms need more nutrients and energy to complete colonization and increase their activity to survive under severe disturbance(Ohsowski et al.,2012).In addition to nutrients and energy,manure application may also improve soil structure,water-holding capacity,and cation exchange capacity,all of which may benefit the resistance of microorganisms to disturbance(Ryals et al.,2014;Ng et al.,2015).Increased soil fertility can mitigate the effects of moisture regimes on microbial community structure.

Variations in soil biochemical indexes were higher in the CK soil than in the fertilized soils,suggesting that fertilization increased the stability of microbial properties and their response to moisture changes.The CVs of these biochemical indexes were negatively correlated with SOC,total P,and available N.Zhao et al.(2010)also found a significant negative relationship between the variations in soil biological properties caused by drying-rewetting cycles and SOC determined prior to drying-rewetting cycles.The soil after dryingrewetting cycles was deficient in nutrients,especially N and P.Long-term fertilization increased soil nutrients,which were available for soil microorganisms and thus increased enzyme secretion(Su et al.,2015;Xu et al.,2015).It is advantageous for microorganisms responding to drying-rewetting cycles to accumulate more enzymes in soil because this induces a greater accumulation of depolymerized products during dry periods,which can be used upon rewetting(Manzoni et al.,2012).High soil fertility reduces the fluctuations in microbial community and activity in response to moisture changes.

CONCLUSIONS

Soil submergence or S-D cycles decreased enzyme activities,but increased the contents of PLFAs compared with low moisture.This indicates the lower specific activity of microorganisms by oxygen limitation.Microbial properties(enzyme activities and PLFA contents)were modified by moisture to a greater extent in the unfertilized soil compared to the soils with long-term fertilization.The SOC dominated the response of microbial properties to soil moisture.It can be concluded that long-term(24 years)organic and mineral fertilization increased soil fertility,which in turn,buffered the negative effects of over flooding on microbial community composition and enzyme activities.

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