Responses of soil microbial communities to manure and biochar in wheat cultivation of a rice-wheat rotation agroecosystem in East China

Jun MENG,Wenjin LI,Yingbo QIU,Zhangtao LI,Linze LI,Yu LUO,Haipeng GUO,Yijun YU,Shengdao SHAN and Huaihai CHEN,

1Zhejiang Province Key Laboratory of Recycling and Ecological Treatment of Waste Biomass,School of Environmental and Natural Resources,Zhejiang University of Science and Technology,Hangzhou 310023(China)

2State Key Laboratory of Biocontrol,School of Ecology,Shenzhen Campus of Sun Yat-sen University,Shenzhen 518107(China)

3Zhejiang Provincial Key Laboratory of Agricultural Resources and Environment, College of Environmental & Natural Resource Sciences, Zhejiang University,Hangzhou 310058(China)

4State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products,Ningbo University,Ningbo 315211(China)5Arable Soil Quality and Fertilizer Administration Bureau of Zhejiang Province,Hangzhou 310020(China)

ABSTRACT Soil contamination in agroecosystems remains a global environmental problem.Biochar has been suggested as an organic amendment to alleviate soil pollution,sequester carbon(C),and improve soil fertility.However,information on how bacterial and fungal communities in acidic bulk and rhizosphere soils respond to swine manure and its biochar is still lacking.In this study,biochar and swine manure were applied at two rates of 1.5 and 3 t ha-1 in a rice-wheat rotation field to assess how soil characteristics,especially pH and chemical element availability,correlate to compositional variations of bacteria and fungi in bulk and rhizosphere soils.Our results showed that high rates of biochar and manure promoted the bacterial richness in bulk and rhizosphere soils by increasing soil pH and reducing soil arsenic(As)and copper(Cu)availability.Compared with soil As and Cu availability,soil pH had opposite effects on beta diversity of both the bacterial and fungal communities.Specifically,biochar and swine manure applications stimulated the bacterial classes Gemmatimonadetes,Deltaproteobacteria,and Gammaproteobacteria by increasing soil pH and decreasing soil available chemical elements.Opposite trends were observed in fungal communities responding to biochar and manure.For example,biochar restrained the fungal class Eurotiomycetes by decreasing soil As and Cu availability,but manure inhibited Leotiomycetes mainly because of an increase in soil pH and a decrease in soil dissolved organic C.These suggest that both bacterial and fungal communities respond significantly to biochar and manure amendments in both bulk and rhizosphere soils,possibly because of their sensitive adaptation to variations in soil environmental factors,such as pH level and chemical element availability.

Key Words: bacteria,chemical element,element availability,fungi,rhizosphere,soil dissolved organic C,soil pH

INTRODUCTION

Soil contamination in agroecosystems has drawn increasing attention with the rapid development of the agricultural industry(Akramet al.,2018;O’Connoret al.,2018).Recently,anthropogenic practices,such as the application of biosolids,composts,commercial fertilizers,pesticides,and sewage wastewater,and atmospheric deposition have polluted soils (Akramet al., 2018; Butaet al., 2021).The adverse effects of contaminants threaten crop yields and human health(Breviket al.,2020).Wheat(Triticum aestivumL.) has been shown to effectively take up and translocate chemical pollutants,resulting in food security issues worldwide(Abtahiet al.,2017;Wanget al.,2017).During wheat growth,the availability of chemical elements,including both nutrients and pollutants,can directly control the transfer of ions from soils to crop roots,and therefore,it is the principal cause of chemical toxicity in wheat(Aliet al.,2020).The transfer processes of chemical elements primarily rely on the critical zones of the surface between soils and crops,which highlights the importance of environmental studies on rhizosphere soil(Seshadriet al.,2015).The complexity of bulk(non-rhizosphere)and rhizosphere soils could regulate the bioavailability and toxicity of chemical elements;thus,the bioremediation of contaminated land greatly depends on both bulk and rhizosphere soil characteristics,such as pH and organic carbon(C)levels(Lwinet al.,2018).Therefore,understanding the bioremediation of chemical elements in wheat and rice rotation systems,especially with the inclusion of their assessment in crop rhizosphere soils,is a global task to promote soil health and crop quality.

Soil organic amendments,such as animal manure,have been shown to immobilize chemical elements in fields(Zhenet al., 2020), but since chemical elements such as heavy metals can be excreted from manure, contributing to soil pollution(Liet al.,2020),animal manure requires proper treatment(e.g.,preparation of biochar)before application to agricultural soils(Baiet al.,2017).Biochar is a renewable product mainly made from agricultural biomass wastes(Kwoczynski and ?melík,2021),and it is recalcitrant C pyrolyzed from organic materials at relatively low temperatures and limited O2(Panahiet al.,2020).It has been estimated that biochar can reduce global C emissions from human activity by 12% every year after returning to soil (Woolfet al.,2010).In comparison to traditional C substrates such as animal manure, biochar is more resistant to microbial decomposition with a longer mean residence time(Lehmann,2007;Wang L Wet al.,2020),and thus the transition from traditional C substrate to biochar would have greater C use efficiency,resulting in an approximately 100%increase in C sequestration(Lehmannet al.,2006).In recent decades,owing to its capability of long-term C sequestration, biochar has been applied to agricultural lands as an organic amendment to promote soil health(Lehmannet al.,2006;Chanet al., 2007) and to regulate soil chemistry through physicochemical processes(Castaldiet al.,2011;Oliveiraet al., 2017).Through physical adsorption and chemical reactions such as ion exchange, electrostatic interactions,and complexation,biochar can adsorb and immobilize chemical ions to minimize their mobility and bioavailability,leading to a reduction in environmental risks in soil-crop systems(Wanget al.,2022).Therefore,understanding the effects of biochar and manure addition to regulate chemical elements in the soil and the subsequent changes to microbial communities is of great importance to better elucidate the environmental benefits of biochar or other organic materials to agroecosystems.

Soil microorganisms play an important role in mediating biochemical reactions in ecosystem processes(Naziret al.,2010;Nielsenet al.,2011).Thus,the interaction between microbes and the soil environment has a vital influence on the availability of soil elements in the soil(Van Der Heijdenet al.,2008).In addition to increasing soil organic C levels,the addition of biochar and animal manure can also affect soil chemical properties,such as pH and dissolved organic C(DOC)(Yanget al.,2016).Thus,biochar and animal manure amendments can shift soil habitats,favoring certain microbial groups that can quickly become accustomed to the new soil environment(Lehmannet al.,2011).Consequential shifts in soil bacterial and fungal communities induced by organic addition can in turn affect soil C sequestration, nutrient cycles, and crop productivity (Biederman and Harpole,2013).However,our understanding of how soil properties regulate bacterial and fungal communities due to organic amendments of biochar and manure,especially in both bulk and rhizosphere soils, remains unclear.As a result, it is necessary to assess the microbial habitats affected by biochar and its raw manure feedstock, as well as consequent soil environmental changes,such as soil pH and the availability of soil chemical elements.

With the progressive development of molecular biology and bioinformatics (Singeret al., 2016), high-throughput sequencing technology offers an opportunity to assess microbial composition variation in soil efficiently and economically(Zhouet al.,2015).Compared to traditional microbial community analyses,16S and ITS rRNA amplicon sequencing can provide deeper insights into even rare groups of bacterial and fungal communities (Onget al., 2013; Singeret al.,2016)and thus greatly improve the phylogenetic resolution for microbial community analysis (Bartramet al., 2011;Reuteret al.,2015).However,there is no consensus on the specific microbial species in bulk and rhizosphere soils that are responsive to biochar compared to its raw feedstock swine manure.Here,using a high-throughput sequencing approach,we established a field study to assess how soil bacterial and fungal communities in both bulk and rhizosphere soils react to manure amendment in comparison to its biochar in the lower middle reaches of the Yangtze River basin,Jinhua City,Zhejiang Province,East China.We hypothesized that biochar addition could affect the soil pH and chemical element availability,leading to environmental selection for certain soil microorganisms.Specifically, we aimed to determine i) whether biochar and manure amendments can choose favorable bacterial and fungal groups in both bulk and rhizosphere soils and ii)how soil properties,especially soil pH and chemical element availability,can explain the dynamics of bacterial and fungal compositions induced by biochar and manure amendments.

MATERIALS AND METHODS

Preparation of organic materials

The biochar was pyrolyzed at 550—600?C from swine manure acquired from Jiangsu Benenv Environmental Technologies Co.,Ltd.(Yixing,China)(Liuet al.,2020).Swine manure was collected from an intensive swine production unit in Hangzhou,Zhejiang Province,East China.The airdried swine manure and biochar samples were ground to pass through a 0.25-mm sieve.Swine manure (pH 7.70)contained 247.4 g C kg-1,2.83 mg kg-1As,0.44 mg kg-1Cd,319.75 mg kg-1Cu,12.40 mg kg-1Ni,1.62 mg kg-1Pb,764.16 mg kg-1Zn,and 5.28 g kg-1Fe.Biochar(pH 10.75)contained 310.9 g C kg-1,7.59 mg kg-1As,0.74 mg kg-1Cd,680.10 mg kg-1Cu,24.40 mg kg-1Ni,2.82 mg kg-1Pb,1 668.72 mg kg-1Zn,and 11.62 g kg-1Fe.

Study site and experimental design

The test field was located in Lanxi County,Jinhua City,Zhejiang Province, China (29?10′21′′N, 119?24′08′′E).The mean annual temperature and precipitation were 17.7?C and 1 439 mm, respectively.Soil type was identified as sandy loam according to United States Department of Agriculture(USDA)Soil Taxonomy(59.24%sand,28.70%silt,and 12.06%clay).Soil pH and organic C content were 5.35±0.04 and 20.00±0.38 g kg-1, respectively.The total concentrations of As, Cu, and Fe in the soil were 9.94±0.79,14.54±0.79,and 23 258±1 689 mg kg-1,respectively.Our previous survey results showed that the As concentrations in wheat grains were above the maximum allowable limit for As(0.5 mg kg-1)in cereal used in China(GB 2762—2017).Because Cu and Fe are two common heavy metals and As is a typical metalloid often found in agricultural top soils,As,Cu,and Fe were chosen as typical chemical elements tested in this study.Considering that Fe is an essential element for microbes and enzymes,the response of Fe availability may be distinct from that of As and Cu.

The study field was divided into 15 plots of area(5 m× 6 m) using a completely randomized design with five treatments and three replicates.A 0.6-m inter-plot “alley way” was set up to separate the plots between treatments and replicates.Biochar was applied at two rates of 1.5 and 3 t ha-1,and swine manure was applied at the same rates for comparison.Therefore, five treatments were set up as follows:i)control soil without organic amendments(CK),ii)soil amendment with biochar at a low rate of 1.5 t ha-1(BL),iii)soil amendment with biochar at a high rate of 3 t ha-1(BH),iv)soil amendment with manure at a low rate of 1.5 t ha-1(ML),and v)soil amendment with manure at a high rate of 3 t ha-1(MH).Biochar and swine manure were evenly applied with a base fertilizer to a depth of 15 cm in November 2018.Detailed information on field crop rotation management has been reported in previous studies(Liuet al.,2020;Tanget al.,2020).

Soil sampling and analysis

During the harvest period,wheat plants from each plot were sampled with roots, and the rhizosphere soil was carefully removed from the roots and placed in sterile 50-mL tubes.Bulk soil(0—15 cm)from each plot was sampled based on a five-point sampling approach using a 2-cm diameter soil auger before mixing for a composite sample.The samples were kept on ice before being transported to the laboratory.Both bulk and rhizosphere soils were sieved through a 2-mm mesh screen before soil property analysis and DNA extraction.

Except for DNA extraction, all soil properties were analyzed in dry soil samples.The soil pH was measured using a pH meter(S470,SevenExcellence?,Mettler-Toledo GmbH, Gie?en, Germany) at a soil/water ratio of 1:2.5(weight/volume).Soil DOC was extracted with deionized water at a soil/water ratio of 1:5(weight/volume)by centrifugation at 200 r min-1for 1 h,followed by centrifugation at 4 000×gfor 20 min and filtering through 0.45-μm membrane filters.The DOC concentration was determined using a TOC analyzer(Multi N/C 3100,Analytik Jena AG,Jena,Germany).Soil available As concentration was measured using a double-channel atomic fluorescence spectrometer(AFS 9130, Beijing Titan Instruments Co.Ltd., Beijing,China)after extraction in 0.5 mol L-1NaH2PO4solution at a 1:15 ratio(weight/volume)(Khanet al.,2010).Soil available Cu and Fe concentrations were determined by inductively coupled plasma mass spectrometry(ICP-MS;PerkinElmer NexION 300X,Waltham,USA)after extraction in a 0.1 mol L-1CaCl2solution at a ratio of 1:10(weight/volume).

DNA extraction, amplification, sequencing, and bioinformatics

Soil DNA was extracted from 0.5 g soil using a FastDNA spin kit for soil (MP Bio, Solon, USA) according to the manufacturer’s instructions before being column-purified using a OneStep PCR inhibitor removal kit(Zymo Research,Irvine,USA).Soil DNA concentrations were measured,and purity was verified by the ratio of absorbance at 260 to 280 nm(about 1.8)using a NanoDrop spectrophotometer(Thermo Scientific,Wilmington,USA)(Chenet al.,2019).

Bacterial 16S and fungal ITS rRNA genes were PCRamplified using primer sets targeting V3—V4 and ITS1—ITS2,respectively (Tojuet al., 2012; Klindworthet al., 2013).The PCR was conducted using 2× KAPA HiFi HotStart ReadyMix (KAPA Biosystems, Wilmington, USA) in a C1000 Touch?thermal cycler(Bio-Rad,Hercules,USA).The PCR products were purified using AMPure XP beads(Beckman Coulter Genomics,Danvers,USA).Barcode sequences were then added using a Nextera XT index kit(Illumina,San Diego,USA)and paired-end sequencing was conducted on the Illumina MiSeq platform (300× 2 bp paired-end,v3 chemistry,Illumina)(Chenet al.,2018).

Barcodes and primers were removed from the sequences using Cutadapt(Martin,2011).Paired-end sequences were processed using QIIME (Caporasoet al., 2010) with a threshold quality score Phred> 19 to ensure sequence accuracy> 99%, as recommended by the default setting.Sequence chimeras were eliminated using the USEARCH method(Edgar,2010)in the QIIME software.Operational taxonomic units(OTUs)with 97%identity were selected,and taxonomy was assigned using the Ribosomal Database Project (RDP) taxonomy-assignment method for 16S and ITS rRNA sequencing data(Wanget al.,2007).

Statistical analyses

Two-way analysis of variance(ANOVA)(SAS 9.3,SAS Institute Inc.,Cary,USA)was used to evaluate significant differences in alpha diversity and dominant classes of soil bacterial and fungal communities among the five organic amendment treatments(Factor 1)in bulkvs.rhizosphere soil(Factor 2).A two-way permutational multivariate analysis of variance (PEMANOVA) with 9999 permutations was conducted to assess significant differences in beta diversity of bacterial and fungal communities among the five organic amendment treatments(Factor 1)in bulkvs.rhizosphere soil(Factor 2)in Plymouth Routines in Multivariate Ecological Research(PRIMER)statistical software(v7.0.13,PRIMERE Ltd.,Plymouth,UK)(Clarke and Gorley,2015).Distancebased redundancy analysis(dbRDA),distance-based linear models(DistLM),and RELATE test in PRIMER were used to show the associations between soil properties and bacterial and fungal community structures (Anderson, 2004).Pearson’s correlation coefficients were determined to further assess the significant correlation between soil properties and the relative abundance of the dominant classes of soil bacterial and fungal community compositions.

RESULTS

Microbial alpha diversity

In the bacterial community,two-way ANOVA showed no significant interaction between the two tested factors,organic amendment treatment and rhizosphere, for both observed OTUs and Chao 1; therefore,direct comparison was conducted among organic amendment treatments across bulk and rhizosphere soils(Fig.1).Compared to CK,both bacterial OTUs and Chao 1 were greater in the soils with application of biochar and manure,especially at the higher rates (BH and MH) (P< 0.05), indicating that both biochar and manure addition could promote bacterial richness in bulk and rhizosphere soils.However, the Shannon and Simpson indices were not significantly different among the organic amendment treatments(Table SI,see Supplementary Material for Table SI).Fungal alpha diversity showed no significant differences in bulk and rhizosphere soils among organic amendment treatments(Table SII,see Supplementary Material for Table SII),suggesting that fungal community richness and evenness did not respond significantly to organic amendments.

Soil pH,DOC,and available metals(As, Cu, and Fe)were further evaluated to determine the relationships between soil properties and bacterial and fungal communities in bulk and rhizosphere soils.Generally, soil properties were significantly affected by organic amendment treatments(P<0.001)(Table I).Specifically,BL,BH and MH significantly increased the pH of bulk and rhizosphere soils(P<0.05).Generally, DOC in the rhizosphere soil was higher than that in the bulk soil(P<0.05).In the bulk soil,DOC was not significantly affected by organic amendment treatments.However,in the rhizosphere soil,DOC significantly decreased in BL,BH,and ML(P<0.05).In the bulk soil,both biochar and manure treatments reduced soil available As(P<0.05).Only ML decreased the soil available Cu(P<0.05).Soil available Fe was lower in ML of the bulk soil and in both biochar and manure treatments of the rhizosphere soil(P<0.05).Both the observed OTUs and the Chao 1 were positively associated with soil pH (r= 0.547,P<0.001)(Fig.1).However,available As and Cu concentrations were negatively correlated with bacterial richness indices(r=-0.80 to-0.75,P<0.001).

Microbial beta diversity

The main test of two-way PERMANOVA showed that bacterial (16S) and fungal (ITS) communities differed markedly among organic amendment treatments and between rhizosphere and bulk soils (P< 0.01), and there was no significant interaction between two factors(Table SIII,see Supplementary Material for Table SIII).These two factors,organic amendment treatment and rhizosphere, explained 7.9%and 9.5%of the bacterial community variation,respectively.Generally,DistLM analysis showed that all tested soil properties were significantly associated with bacterial community structure (Table SIV, see Supplementary Material for Table SIV),together explaining 31.9%of the community variation(Table SV,see Supplementary Material Table SV).The dbRDA analysis showed that soil pH affected the bacterial community in both bulk and rhizosphere soils with increasing application rates of biochar and manure(Fig.2).Compared with soil pH,available As and Cu had opposite effects on the bacterial community.The rhizosphere bacterial community was generally separated from that in the bulk soil,which was associated with the soil DOC and available Fe.

For soil fungi,the contributions of two factors,that is,organic amendment treatment and rhizosphere,to community variation were 17.3%and 14.2%,respectively(Table SIII).The DistLM analysis revealed that all tested soil properties could be related to fungal community distribution (Table SIV),together explaining 28.8%of the community variation(Table SV).However,the contribution of chemical elements was less significant to the community variation of fungi(6.0%—7.4%)than bacteria(7.0%—13.6%),suggesting that bacteria may be more sensitive to the availability of chemical elements than fungi.The effects of soil properties on the fungal community were similar to those observed in the bacterial community(Fig.2).Soil pH and Cu availability had opposite effects to shift fungal community between biochar and manure treatments, whereas DOC and Fe availability drove community variation between bulk and rhizosphere soils(Fig.2).The RELATE test showed a significant correlation between the pairwise similarity of bacterial and fungal communities(Spearman’s rank correlation coefficient Rho=0.26,P<0.05).In addition, both bacterial and fungal diversities gradually decayed with increasing Euclidean distance between soil properties(r=-0.16 and-0.20,P<0.001)(Fig.S1,see Supplementary Material for Fig.S1).

Fig.1 Alpha diversity indices of bacterial(16S)community in bulk and rhizosphere soils treated without(control,CK)or with biochar(B)and manure(M)at application rates of 1.5(L)and 3(H)t ha-1 and their Pearson’s correlations with soil properties(pH,As,and Cu).For assessing the effects of different organic amendment treatments,the data of bulk and rhizosphere soils were combined.Bars with different letters indicate significant differences at P <0.05.The rarefied sequence depths were 9 584 for all samples.The asterisks*and**indicate significant differences at P <0.05 and P <0.01,respectively.OTUs=operational taxonomic units;NS=not significant.

TABLE I Properties of bulk and rhizosphere soils treated without(control,CK)or with biochar(B)and manure(M)at application rates of 1.5(L)and 3(H)t ha-1

Fig.2 Distance-based redundancy analysis(dbRDA)of bacterial(16S)and fungal(ITS)community with soil properties as explanatory variables in bulk and rhizosphere soils treated without(control,CK)or with biochar(B)and manure(M)at application rates of 1.5(L)and 3(H)t ha-1.Fitted and total variations explained by each dimension are given in percentages.

Microbial composition

The relative abundances of major bacterial or fungal phyla showed no significant differences between the biochar and manure treatments.Thus,microbial composition was mainly discussed at the class level.The dominant bacterial classes in all treatments were Acidobacteriia from the phylum Acidobacteria (34%—47%), Alphaproteobacteria(13%—17%),Bacteroidia from the phylum Bacteroidetes(3%—11%), and Anaerolineae from the phylum Chloroflexi(5%—7%)(Fig.2S,see Supplementary Material for Fig.2S).Two-way ANOVA showed that Gemmatimonadetes,Deltaproteobacteria,and Gammaproteobacteria were significantly affected by organic amendment treatments without a significant interaction between the two factors;the data of bulk and rhizosphere soils were thus combined to assess the effects of different organic amendment treatments(Fig.3).Compared with CK,BL,BH,and MH stimulated Gemmatimonadetes by on average 91%,Deltaproteobacteria by 37%, and Gammaproteobacteria by 52% (P< 0.05).Pearson’s correlations showed that the relative abundances of Gemmatimonadetes,Deltaproteobacteria,and Gammaproteobacteria had a significantly positive correlation with soil pH but a negative association with soil available As and Cu(Fig.4).These results suggest that biochar and manure amendments could stimulate Gemmatimonadetes,Deltaproteobacteria,and Gammaproteobacteria by increasing soil pH but reducing the availability of As and Cu.

Fig.3 Relative abundances of dominant classes (Gemmatimonadetes,Deltaproteobacteria, and Gammaproteobacteria) in soil bacterial (16S)community(average>1%)in bulk and rhizosphere soils treated without(CK)or with biochar(B)and manure(M)at application rates of 1.5(L)and 3(H)t ha-1.For assessing the effects of different organic amendment treatments,the data of bulk and rhizosphere soils were combined.Different letters above the bars indicate significant differences at P < 0.05.The asterisks*,**,and***indicate significant differences at P <0.05,P <0.01,and P <0.001,respectively.NS=not significant.

Fig.4 Pearson’s correlations between soil properties and relative abundances of dominant classes (Gemmatimonadetes, Deltaproteobacteria, and Gammaproteobacteria)in soil bacterial(16S)community(average>1%)in bulk and rhizosphere soils treated without(control,CK)or with biochar(B)and manure(M)at application rates of 1.5(L)and 3(H)t ha-1.

The dominant classes of soil fungi were Sordariomycetes from the phylum Ascomycota,accounting for 34%—47%of the total fungal sequences, followed by Agaricomycetes from the phylum Basidiomycota (9%—20%), Mortierellomycotina from the phylum Zygomycota(4%—23%),and Dothideomycetes from the phylum Ascomycota(3%—10%)(Fig.3S, see Supplementary Material Fig.3S).Only Eurotiomycetes and Leotiomycetes, both from the phylum Ascomycota,showed significant differences among organic amendment treatments(P<0.05)(Fig.5).Since there was no interaction between the two factors,organic amendment treatment and rhizosphere,the bulk and rhizosphere soil data were combined to directly compare the effects of organic amendment treatments across bulk and rhizosphere soils.Compared with CK, ML and MH significantly repressed Eurotiomycetes by on average 55%, which was positively associated with available As and Cu(r=0.479 and 0.387,P<0.05)(Fig.5).However,BL and BH significantly decreased the relative abundance of Leotiomycetes by 67%,which was negatively correlated with soil pH(r=-0.465,P<0.01)(Fig.5)but positively associated with soil DOC(r=0.669,P<0.001).These results suggest that the effects of biochar and manure amendments on the fungal community have different mechanisms,because manure amendment restrained Eurotiomycetes by reducing the availability of As and Cu, whereas biochar addition reduced Leotiomycetes due to soil pH increase and DOC reduction.

DISCUSSION

Fig.5 Relative abundances of dominant classes(Eurotiomycetes and Leotiomycetes)in soil fungal(ITS)community(average>1%)in bulk and rhizosphere soils treated without(CK)or with biochar(B)and manure(M)at application rates of 1.5(L)and 3(H)t ha-1 and their Pearson’s correlations with soil properties.For assessing the effects of different organic amendment treatments,the data of bulk and rhizosphere soils were combined.Different letters above bars indicate significant differences at P <0.05.The asterisks*and***indicate significant difference at P <0.05 and P <0.001,respectively.NS=not significant.

The high-throughput sequencing approach implemented in this study has provided new insights into how biochar and manure addition can favor certain bacterial and fungal groups in both bulk and rhizosphere soils,and how soil chemical properties,especially soil pH and chemical element availability,can explain the dynamics of bacterial and fungal composition induced by biochar and manure amendments.Previous studies have mainly focused on the effects of biochar on soil physical properties,thus improving soil structure and drainage(Herathet al.,2013;Blanco-Canqui,2017),such as soil porosity,bulk density,saturated hydraulic conductivity,water holding capacity,and aggregate stability.It has been assumed that biochar and manure amendments can also affect soil pH,DOC,and availability of chemical elements,thus creating soil habitats that favor general microbial activity and abundance(Wang W Pet al.,2020;Yeet al.,2021;Liet al.,2022;Xuet al.,2022).We showed that bacterial community richness was significantly promoted by the application of biochar or manure in both bulk and rhizosphere soils(Fig.2),introducing the question of what specific microbial composition was responsible for this increase in richness as well as their regulating factors.Thus,we further examined the variation in the community composition of the soil bacteria.

Our study strongly supported that organic amendments can not only stimulate overall bacterial richness but also select certain bacterial or fungal groups that were better adapted to the new niches of both bulk and rhizosphere soils.Soil properties could be used as potential predictors for these community variations in soil microbes impacted by biochar and manure amendments and could partially explain the significant stimulating effects of biochar and manure on bacterial richness indices.In the bacterial community,our results revealed that in both bulk and rhizosphere soils,biochar and manure amendments typically increased the relative abundances of Gemmatimonadetes,Deltaproteobacteria,and Gammaproteobacteria(Fig.3).These observations are concomitant with other studies,suggesting that biochar drives bacterial dynamics.For example,a field study on the effects of biochar on the diversity of soil bacterial communities revealed that Gemmatimonadetes were present only in topsoil amended with the highest amount of biochar at 10%(Wonget al., 2019).A field experiment exploring the effects of biochar on microbial abundance and activity in temperate vineyard topsoil showed that biochar increased the abundance of Gemmatimonadetes(Mackieet al.,2015).However,other studies have revealed that the relative abundance of Gemmatimonadetes decreased with biochar addition in layered soil columns(Xuet al.2016).Our results showed that,compared to CK, adding biochar and manure stimulated Gemmatimonadetes by on average 91%(Fig.3).In addition,as observed in our study, Gemmatimonadetes were more abundant in the biochar treatments than in the manure treatments,similar to the trends observed for Deltaproteobacteria and Gammaproteobacteria(Fig.3).Previous studies have demonstrated that Gemmatimonadetes are slow-growing oligotrophs adapted to poor substrate environments(Mackieet al.,2015; Ibrahimet al.,2020).However,other studies have shown that the relative abundances of Deltaproteobacteria and Gammaproteobacteria are reduced by the addition of biochar to manure compost (Abujabhahet al., 2016;Azeemet al., 2020; Luet al., 2022).In response to these contradictory conclusions,additional studies incorporating more detailed information on both microbial biomass and composition of taxa are needed to elucidate the interactions between biochar and bacterial community dynamics, and how they can be associated with changes in soil properties in agroecosystems.

Enrichment of Deltaproteobacteria and Gammaproteobacteria was often found in biochar-amended bulk soil.For instance,a 3-year application of biochar at three levels(2%,4%,and 8%)significantly increased the relative abundance of Deltaproteobacteria in an annual cropping rotation system(Yaoet al.,2017).In addition,it has been shown that 1%biochar application in a laboratory experiment facilitated the colonization of Deltaproteobacteria and Gammaproteobacteria in the rhizosphere of rice grains grown on contaminated soils (Wang W Pet al., 2020).Similarly, in a temperate apple orchard site,the abundance of Deltaproteobacteria and Gammaproteobacteria increased by 10% in biochar treatments at 5 kg biochar per tree space compared to the control(Abujabhahet al.,2016).Therefore,consistent with these studies(Ibrahimet al.,2020;Zhanget al.,2022),our results further indicate taxa-specific changes in Proteobacteria at the class level,not only in bulk soil but also in rhizosphere soil.These variations in the bacterial community composition also highlight the importance of environmental selection for certain bacterial groups induced by biochar or other organic amendments.

Both bacterial composition and fungal groups could be affected by the organic additions of biochar and manure.For example, 10 months after the incorporation of biochar, a progressive reduction in Leotiomycetes abundance occurred in the biochar-amended soil samples,suggesting that biochar loading could reduce the relative abundance of certain fungi at the class level.In contrast,the abundance of Leotiomycetes in the soil was significantly higher under 6-year continuous straw-derived biochar amendments (Baiet al., 2019).In addition, Helotiales, an order of Leotiomycetes, was less abundant in biochar-amended acidic tea soil(Zhenget al.,2019).For the fungal community at the class level, our study showed that Leotiomycetes,belonging to Ascomycota,significantly decreased in biochar-amended soils (Fig.5).Interestingly,in manure-amended soils,the relative abundance of Eurotiomycetes was significantly repressed by on average 55%(Fig.5),suggesting that the effects of manure compost on the fungal community may involve different mechanisms than those of biochar.A 32-year fertilization field experiment in the North China Plain revealed that Eurotiomycetes were more abundant in the organic manure treatments than the control,as their abundance was correlated with N concentration(Wenet al.,2020).Additionally,in a 50-year N input experiment of black soil in Northeast China,N inputs at 150 and 300 kg N ha-1year-1increased the abundance of Eurotiomycetes,suggesting that higher N in soil may favor Eurotiomycetes(Zhouet al.,2016).Our study suggested that Eurotiomycetes were more abundant in the biochar treatments than in the manure treatments(Fig.5).However,other studies have shown that Eurotiomycetes were significantly higher under pig manure treatment than under other treatments (Abujabhahet al., 2019; Hanet al.,2021).Therefore,these contradictory findings indicate that the reaction of fungal groups to organic additions of biochar and manure compost may be more complex than that of their bacterial counterparts.However, our results clearly showed that the effects of manure on Eurotiomycetes could be associated with variations in available chemical pollutants,whereas biochar addition mainly influenced Leotiomycetes by altering soil pH and DOC(Fig.5).

Due to changes in soil pH and availability of chemical elements, amendment of organic biochar could influence the microbial community in several ways(Gulet al.,2015).First, because of its alkaline pH and negatively charged surface groups,biochar can bind soil H+ions to alleviate acidity in low-pH environments(Chintalaet al.,2014).As one of the best predictors of microbial community composition(Rousket al.,2010),an increase in soil pH can drive both bacterial and fungal distributions(Lauberet al.,2008).We further showed that the increase in bacterial groups of Gemmatimonadetes,Deltaproteobacteria,and Gammaproteobacteria could be significantly associated with the pH increase due to biochar and manure amendments.In addition,biochar can also affect the levels of low-molecular-weight DOC in the soil(Stewartet al.,2013),which is a favorable C substrate for soil microbes that are capable of rapidly utilizing these easily decomposable C sources(De Boeret al.,2005).Our findings showed that soil DOC mainly drove the community variations of soil bacteria and fungi between bulk and rhizosphere soils;however,due to the changes in DOC levels,biochar addition could significantly decrease the relative abundance of the fungal class Leotiomycetes.

Moreover, because biochar has numerous pores capable of the sorption and desorption of toxic elements and compounds (Ahmadet al., 2014) such as trace elements and heavy metals, biochar-soil surfaces can further affect the microbial groups sensitive to environmentally hazardous substances (Zhuet al., 2017).Biochar affects the accumulation and translocation of chemical elements,and thus impacts microbial activity, mainly due to an increase in soil enzymes and microbial activity (Nieet al., 2018).In this study, we found that biochar and manure amendments led to a decrease in available As and Cu in both bulk and rhizosphere soils,promoting bacterial community richness and driving bacterial community changes, such as an increase in Gemmatimonadetes,Deltaproteobacteria,and Gammaproteobacteria.Organic additions of biochar and manure compost have been suggested as convenient and costeffective ways to reduce chemical element bioavailability and uptake by plants,and their significant effects on soil bacterial communities highlight the importance of investigating how microbial communities regulate biogeochemical cycling(Muhammadet al., 2016; Xuet al., 2022).Interestingly,biochar and manure had similar intrinsic pH values and thus comparable pH effects on soil conditions and bacterial composition.For the fungal community, manure significantly decreased Eurotiomycetes due to the reduction in As and Cu availability, which, as potential pollutants in the soil,may have negative effects on most fungal groups but not on Eurotiomycetes.However,biochar reduced Leotiomycetes,which could be associated with pH and DOC, indicating that Leotiomycetes may react more significantly to nutrient availability regulated by soil pH and DOC compared to other fungal groups.Therefore,our results showed that soil pH and the availability of As and Cu in bulk and rhizosphere soils may have divergent influences on the fungal community.Thus,future biochar studies should focus on the evaluation of fungal communities in response to individual edaphic factors to better elucidate the effects of biochar and manure amendments.

CONCLUSIONS

With the help of a high-throughput sequencing approach,we conducted a field study to connect soil pH and chemical element availability with the effects of biochar and swine manure amendments on bacterial and fungal compositions in both bulk and rhizosphere soils during the growth of wheat in an acidic soil of East China.By increasing soil pH and reducing As and Cu availability, biochar and manure amendments promoted bacterial richness and favored the bacterial classes Gemmatimonadetes,Deltaproteobacteria,and Gammaproteobacteria in both bulk and rhizosphere soils.Although there was no effect on fungal alpha diversity,manure restrained the fungal class Eurotiomycetes because of decreased As and Cu availability,whereas biochar significantly inhibited Leotiomycetes because of increased soil pH and decreased soil DOC.Our study identified specific microbial taxa selected by biochar and manure amendments,thus offering an opportunity to potentially link certain microbes to their habitats and,consequently,their ecological significance.Future work should focus on an in-depth understanding of microbial functional traits based on shotgun metagenomics to identify specific microbial processes affected by biochar and manure amendments in agroecosystems.

ACKNOWLEDGEMENTS

This work was financially funded by the National Natural Science Foundation of China (Nos.42277282 and 41601334), the Public Welfare Technology Application Research Project of Zhejiang Province, China (No.LGF21D010002),the Key Research and Development Program of Zhejiang Province,China(No.2020C01017),the State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products,Ningbo University, China (No.KF20190106), the Basic and Applied Basic Research Foundation of Guangdong Province,China(No.2022A1515010861),the Shenzhen Science and Technology Program(No.JCYJ20220530150201003),and the Young Teachers Team Project of Fundamental Research Funds for the Central Universities,Sun Yat-sen University,China(No.22qntd2702).

SUPPLEMENTARY MATERIAL

The supplementary material for this article can be found in the online version.