Reducing residues of tetracycline and its resistance genes in soil-maize system and improving plant growth:Selecting the best remediation substance

Junmei QINJianli SONGFenwu LIUJian ZHANGHuaye XIONGWenlong BI and Yue NI

1 College of Resources and Environment,National Experimental Teaching Demonstration Center for Agricultural Resources and Environment,Shanxi Agricultural University,Taigu 030801(China)

2 Key Laborator y of Soil Environment and Nutrient Resources in Shanxi Province,Taiyuan 030000(China)

3 College of Resources and Environment,Southwest University,Chongqing 400715(China)

ABSTRACT Tetracycline(TC)and tetracycline resistance genes(TRGs)in plant edible tissues pose a potential risk to the environment and then to human health.This study used a pot experiment to investigate the effects of different remediation substances(worm castings,fungal chaff,microbial inoculum,and biochar)on the physiological characteristics of maize and the residues of TC and TRGs in the soil-maize system under TC stress.The results showed that TC significantly inhibited growth,disrupted the antioxidant defense system balance,and increased proline and malondialdehyde contents of maize plants.Tetracycline residue contents were significantly higher in root than in shoot,and followed the order root＞stem-leaf＞grain,which was consistent with the distribution of bioconcentration factors in the different organs of maize plants.The TC residue content in the soil under different treatments was 0.013—1.341 mg kg-1.The relative abundances of different antibiotic resistance genes in the soil-maize system varied greatly,and in maize plants followed the order intI1＞tetW＞tetG＞tetB＞tetM＞tetX＞tetO.In the soil,tetX had the highest relative abundance,followed by tetG and tetW.A redundancy analysis(RDA)showed that TC was positively correlated with TRGs.The addition of different remediation substances alleviated the toxicity of TC on maize physiological characteristics and reduced the TC and TRG residues in the soil-maize system,with biochar being the best remediation substance.These results provide new insights into the effect of biochar on the migration of TC and TRGs from soil to plants.

Key Words: antibiotics,biochar,fungal chaff,microbial inoculum,plant physiological characteristics,worm castings

INTRODUCTION

Antibiotics have been widely used in the livestock and poultry industries worldwide because of their great therapeutic effects and ability to promote animal growth(Awadet al.,2015;Arenas and Melo,2018).However,the environmental pollution caused by the extensive use of antibiotics is becoming increasingly serious(Phillipset al.,2004;Riazet al.,2017).Antibiotics are rarely absorbed when they enter the animal body,and approximately 85%of antibiotics are excreted in the form of their parent compounds or metabolitesviaurine and feces(Kumaret al.,2005;Luoet al.,2011;Yueet al.,2019).The overuse of antibiotics will exert selective pressure on bacteria,which will induce antibiotic resistance and increase antibiotic resistance genes(ARGs)(Prudenet al.,2006;Zhang and Zhang,2011;Zhanget al.,2018).Additionally,horizontal gene transfer,as the main mode of genetic material exchange between microorganisms,is the main way for bacteria to acquire ARGs(Ochmanet al.,2000;Huang M Het al.,2015).Some studies have confirmed a close relationship between the existence of ARGs and the use of antibiotics(Peaket al.,2007;Wuet al.,2010;Xuet al.,2015).Tetracycline antibiotics,especially tetracycline(TC)itself,are the most widely used antibiotics in the world(Qiaoet al.,2012).Due to the widespread use of TC,tetracycline resistance genes(TRGs)are prevalent in sediments,wastewater treatment plants,drinking water,and soils around the world(Huang S Let al.,2015;Keenet al.,2018).Moreover,many livestock and poultry manures are used as organic fertilizers on farmland without harmless treatment(Bagueret al.,2000).When livestock and poultry manures are used in the soil-plant system,antibiotics may accumulate in the soil and can be taken up by crops(Pan and Chu,2016).According to the European Commission for Drug Evaluation,research on the environmental safety of veterinary drugs is necessary if the drug or its metabolites can diffuse from feces and urine into soil and result in soil concentrations exceeding 0.1 mg kg-1(FEDESA,2001).After entering the environment,antibiotics and ARGs can spread and pose a potential risk to human health and the ecological environment(Mullenet al.,2019;Zhanget al.,2020).

Therefore,it is important to understand the accumulation and transport mechanisms of TC in the soil-plant system and its physiological toxicity to plants.Antibiotic residues have been detected in ginger,lettuce,spinach,carrot,tomato,potato,cucumber,rape,cabbage,corn,wheat,and other plants and in the soil in which they are grown(Ahmedet al.,2015;Riazet al.,2017;Liuet al.,2018a;Wanget al.,2019;Zhanget al.,2019).Antibiotics can be absorbed by plants,where they are transported to and accumulate in plant tissues(Huet al.,2010).Many studies have explored the accumulation and transportation of antibiotics in plants by measuring bioconcentration factors(BCF)and translocation factors(TF),but the mechanisms of accumulation and transport vary with different antibiotics and plant types(Huet al.,2010;Yuet al.,2019;Zhanget al.,2019).The accumulation of TC can produce toxic effects on plant growth,negatively affecting growth parameters,photosynthesis,fluorescence parameters,antioxidants,and genetic indices(Xieet al.,2011;Pan and Chu,2016;Liuet al.,2018a;Wanget al.,2019).The activities of superoxide dismutase(SOD),catalase(CAT),and peroxidase(POD),as common detection indicators for studying the toxic effects of antibiotics on plants,have attracted extensive attention from researchers(Xieet al.,2011;Vilvertet al.,2017).Additionally,the residual abundance of TRGs in the soil-plant system is also a research hotspot,and many studies have reported that TRGs are detected in soil and plant tissues(Wanget al.,2015;Cuiet al.,2016;Zhouet al.,2017;Liuet al.,2018a;Xiaet al.,2019).

Reducing the transfer of TC and TRGs to plants has attracted extensive attention from researchers in China and other countries.Currently,many approaches are available to repair the contamination of antibiotics and ARGs.Traditional physical and chemical methods have the advantages of a short-cycle period and quick effect(Chen and Zhang,2013),such as microwave treatment(Lvet al.,2017),membrane bioreactors(Suet al.,2017;Yanget al.,2019),ionizing radiation(Shenet al.,2019),anaerobic biodegradation(Maet al.,2018),photocatalytic degradation(Nassehet al.,2018),ultraviolet radiation,chlorination,and ozone disinfection(Zhenget al.,2017).However,these technologies consume high amounts of energy,are costly,and easily cause secondary pollution(Chen and Zhang,2013).Hence,we should actively explore ways to effectively reduce the effects of TC on plants and reduce TC and TRG residues.Studies have shown that the addition of graphene oxide(Gaoet al.,2012a;Zhanget al.,2017),biochar(Duanet al.,2017;Yueet al.,2019;Ngigiet al.,2019),and eggshells(Jiaoet al.,2018)and the establishment of vertical flow-constructed wetlands(Huang X,2015)can also reduce the residues of antibiotics and ARGs.Biochar is a potential effective absorbent of antibiotics and can effectively remove ARGs(Duanet al.,2017).The dissemination of ARGs from animal waste to the environment can be effectively mitigated by converting manure into biochar(Zhouet al.,2019).The application of biochar opens up a new way to reduce the residues of antibiotics and ARGs.Furthermore,earthworms,fungal chaff,and microbial inoculum have been investigated in many studies on the adsorption of pollutants(Yuet al.,2016;Qinet al.,2019),but their effects on reducing TC and TRG residues when they are applied alone or in combination have not yet been fully elucidated.

Many studies have evaluated the effect of TC pollution on plant physiology worldwide(Wanget al.,2015;Ahmedet al.,2015;Pan and Chu,2016;Liuet al.,2018a;Wanget al.,2019),but they have mainly focused on the effects of short-cycle vegetables and less on the effects of long-cycle crops.Additionally,many studies have focused on the use of a single substance to reduce the effects of TC on plants and the amount of TC and TRG residues in the soil-plant system(Gaoet al.,2012a;Duanet al.,2017).However,few studies have reported on the simultaneous use of multiple substances to screen the most effective substances.In this study,veterinary TC was exogenously added to explore the effects of different remediation substances(worm castings,fungal chaff,microbial inoculum,and biochar)on maize growth parameters,antioxidant system,and resistance factors.Additionally,the residues of TC and ARGs(two efflux pump genes,tetBandtetG;three ribosomal protection protein genes,tetM,tetO,andtetW;one enzymatic modification gene,tetX;and the class 1 integron-integrase gene,intI1)in the soil-maize system were analyzed.The main objectives of this study were as follows:i)to explore the physiological toxicity of TC towards maize,the regularity of TC and TRG residues in the soil-maize system,and the relationship between TC and TRGs and ii)to screen remediation substances and application methods that can effectively reduce the effects of TC and TRG residues on plants.

MATERIALS AND METHODS

Experimental material

Soil was collected from a depth of 0 to 20 cm in farmland in Taigu County,Jinzhong City,Shanxi Province,China.The soil type is classified as an Ustalf,a typical soil in Shanxi Province of China,which is widely distributed in farmland.The soil texture type is loamy soil,and its specific soil mechanical composition has been mentioned in our previous study(Songet al.,2019).The soil was air dried and passed through a 2-mm sieve and then used in a pot experiment with maize.

Maize(Zea maysL.),a common field crop in northern China,was selected as the experimental crop.The maize variety“Woyu 963”was purchased from the Shanxi Provincial Academy of Agricultural Sciences.Seeds were rinsed and soaked in deionized water for 2—3 h before planting;10 full and uniform seeds were selected for sowing.

The fertilizer used in the pot culture was a humic acid compound fertilizer(the ratio of N:P2O5:K2O was 18:18:18,the total nutrient content was more than 54%,and the application amount was 0.75 g kg-1).Veterinary TC was purchased from Ling’an Biotechnology Veterinary Drug Direct Sales Store(Guangdong,China)with a purity of more than 98%.Microbial inoculum was purchased from Shanxi Taigu Fertilizer Co.,Ltd.(Shanxi,China).The major functional bacteria in the inoculum wereBacillus megigantosandBacillus mucilaginosus,and the viable bacterial count was≥2×108cells g-1.Worm castings were purchased from Shandong Wali Biotechnology Co.,Ltd.(Shandong,China),with total nitrogen,phosphorus,and potassium contents of 23.34,25.93,and 21.52 g kg-1,respectively.Fungal chaffwas purchased from Jurongshun Agricultural Technology Co.,Ltd.(Shanxi,China),and the total nitrogen,phosphorus,and potassium contents were 12.79,5.58,and 15.66 g kg-1,respectively.Maize straw biochar was purchased from Shanxi Pingyao Shenghong Biomass Energy Development Co.,Ltd.(Shanxi,China),with a pH of 8.53,C content of 698 g kg-1,CEC content of 23.6 cmol kg-1,and total nitrogen,phosphorus,and potassium contents of 10.15,11.86,and 23.65 g kg-1,respectively.

Experimental design

A maize pot experiment was conducted at the experimental station of the College of Resources and Environment,Shanxi Agricultural University,on April 18,2018.Veterinary TC was added to soil at a concentration of 50 mg kg-1.This concentration is considered a moderate concentration and was selected after referencing a large number of relevant studies(Huet al.,2010;Chiet al.,2018;Yueet al.,2019).As shown in Table I,nine treatments were set up,and each was repeated four times.The content of the microbial inoculum was 50 mg kg-1,and that of worm castings,fungal chaff,and biochar was 15 g kg-1.The pot experiment was conducted in plastic pots(bottom inner diameter,14 cm;upper inner diameter,19 cm;height,25 cm)with 12.5 kg of soil per pot.The fertilizer,TC,and corresponding remediation substances for each treatment were fully mixed with the soil,and pots were randomly arranged in the greenhouse.This experiment adopted a new drip irrigation method(Ma and Li,2012)to prevent the uneven distribution of nutrients and antibiotics in the soil due to irrigation leaching.The bottom of each pot was covered with glass beads and a roving cloth.APVC tube was placed near the inner edge of each pot,and the bottom of the tube was extended to contact the glass beads.Water flowed into each pot through the PVC tube from a suspended container.Ten maize seeds were planted in each pot on April 18,2018;6 maize plants were harvested from each pot at the maize seedling stage on May 14,2018;3 maize plants were harvested from each pot at the jointing stage on June 23,2018;and the final maize plant was harvested from each pot at the mature stage on August 16,2018.No pesticides were added during the growth period of maize,but weeding was carried out irregularly.The soil moisture of each pot was controlled and maintained at approximately 80%using the weighing method.The air humidity in the greenhouse varied from 60%to 85%.The air temperature was measured at 9:00 a.m.each day during the experiment and varied from 12 to 34°C.The greenhouse was naturally lit;the illumination condition was not adjusted artificially.The maize roots,stems,leaves,and grains were collected at the seedling,jointing,and mature stages,carefully washed in running water to remove attached debris and soil,packed separately,and stored at-80°C to analyze and determine the relevant indicators.

TABLE I Design of treatments in the pot experiment

Determination of plant physiological characteristics

On the 121 st day of maize growth,the maize samples at the mature stage were taken to measure plant height and root length with a ruler.Maize plant fresh weight,root fresh weight,and grain weight were measured gravimetrically.

The antioxidant system and resistance factors were measured within one week of sampling.Superoxide dismutase activity was measured using the nitro-blue tetrazolium(NBT)photoreduction method(Guptaet al.,1993;Choudharyet al.,2007).Superoxide dismutase can scavenge superoxide anion radicals;when SOD is present in the reaction system,it can inhibit the reduction of NBT.The higher the enzyme activity,the stronger the inhibition effect,and the lighter the blue color of the reaction solution.Catalase activity was measured using the iodometric titration method(Lunaet al.,2005).Catalase can catalyze the decomposition of H2O2into H2O and O2,and its activity is represented by the amount of hydrogen peroxide decomposed in a certain period of time.Under the catalysis of ammonium molybdate,H2O2reacts with KI to release free iodine.The iodine was titrated with sodium thiosulfate to calculate the amount of H2O2catalyzed by the enzyme.In addition,in the presence of H2O2,POD can act on guaiacol to generate tetra-o-methoxyphenol.The product has a special absorption peak at 470 nm,and the color depth is proportional to the product concentration within a certain range,so the POD activity can be indirectly measured by the guaiacol colorimetric method(Prabhukarthikeyanet al.,2018).

The proline(Pro)content was determined by the acid ninhydrin colorimetry method(Kuanget al.,2017).Only the proline in plants can react with ninhydrin acid to produce a stable red product.The product has a maximum absorption peak at 515 nm,and its absorption value has a linear relationship with the Pro content.The malondialdehyde(MDA)content was determined by the thiobarbituric acid method(Hagegeet al.,1990).The MDA can react with 2-thiobarbituric acid under a high-temperature and acidic environment to produce a reddish-brown product.The product has an absorption peak at 532 nm and a small light absorption at 660 nm.Aldehydes and soluble sugars interfere with this reaction,and there is an absorption peak at 450 nm,which can be eliminated by two-component spectrophotometry.

Quantitative determination of TC

To determine TC content,an appropriate sample amount was placed in a 50-mL centrifuge tube and then extracted by ultrasound three times with 0.1 mol L-1Na2EDTAMcllvaine buffer in an ice water bath.Next,the supernatants were combined and diluted in a 10-mL volumetric flask.The extract was passed through an HLB(hydrophilic-lipophilic balance)cartridge at a flow rate of 1 mL min-1using a solid-phase extraction apparatus.The column was then eluted using 5 mL of a solution containing 0.01 mol L-1oxalic acid and methanol,and all eluents were collected.The eluate was dried at 40°C with a nitrogen blower and then diluted with the hybrid moving phase(oxalic acid:acetonitrile:methanol,76:16:8,volume:volume:volume)to 1 mL and passed through a 0.22-μm filter,followed by transfer to an autosampler vial for analyses by high-performance liquid chromatography-tandem(HPLC)mass spectrometry(Agilent 1290-6460 Triple Quad LC/MS,Agilent Technologies,USA).

The samples were separated using an Agilent Eclipse Plus C18 column(5μm,4.6 mm×250 mm).The HPLC conditions were set to a column temperature of 40°C,the mobile phase was solvent A(0.1%aqueous formic acid)and solvent B(acetonitrile)at a flow rate of 0.4 mL min-1,and the injection volume was 5μL.The gradient elution procedure was as follows:0 min,8%B;0—12 min,B increased from 8%to 55%;12—13 min,B decreased from 55%to 8%;and 13—16 min,B remained at 8%.The mass spectrometry conditions were as follows:the ionization method used electrospray ionization(ESI+);the quantitative measurement used the multiple reaction monitoring(MRM)mode,monitoring ion transitions at mass-to-charge ratio from 445(precursor ion)to 427(quantification ions)for TC;the retention time was 80 ms;the capillary voltage was 10 kV;and the fragment electric voltage was 120 V.Based on the relative response factors(RRFs),tetracycline-d6 was selected as the internal standard,and the concentration of tetracycline was determined by the internal standard method.The limit of detection(LOD)was defined as a signal-to-noise ratio(S/N)greater than 3.The recovery of the selected target compound was determined by the standard addition method.The recovery of TC was between 83%and 102%,and the LOD was 0.12μg kg-1.

Quantification of TRGs,intI1 and 16S-rRNA

To extract DNA,maize root,stem-leaf,grain,and soil samples were each placed in a 50-mL centrifuge tube.Next,sterile ddH2O was added,and the samples were shaken by table oscillation for 30 min.All eluents were filtered by suction to obtain the corresponding membrane.The DNA of homogenized maize tissue and soil samples were extracted using a TIANAMP DNA kit(TIANGEN Biotech,China)according to the manufacturer’s instructions.The sample concentration was determined using an ultraviolet spectrophotometer(Quawell Q3000,Quawell Technologie,Inc.,USA).

Two efflux pump genes(tetBandtetG),three ribosomal protection protein genes(tetM,tetO,andtetW),one enzymatic modification gene(tetX),and the class 1 integronintegrase gene(intI1)were selected for quantitative polymerase chain reaction(qPCR)detection using the StepOne-Plus real-time PCR system(Thermo,Applied Biosystems Inc.,Foster City,USA).The ARGs selected have a wide range of hosts,high abundance levels,and are common in the environment(Wuet al.,2010;Cuiet al.,2016;Duanet al.,2017;Fanet al.,2019).The total volume of the qPCR was 10μL,including 5μL of TB Green Premix Ex Taq II(Tli RNaseH Plus,Takara Biotech,Co.,Ltd.,Dalian,China)(2×),0.4μL of primer F,0.4μL of primer R,0.2μL of R OX Reference Dye(50×),1μL of the DNA sample,and 3μL of ddH2O.The qPCR conditions were as follows:an initial predenaturation step at 95°C for 30 s followed by 40 cycles of denaturation at 95°Cfor 5 s and annealing extension at 60°C for 30 s.The cycle threshold(Ct)detection limit was set at 40 cycles.The solubility curve was used to analyze whether the primers were amplified.The sequence information of qPCR primers is listed in Table SI(See Supplementary Material for Table SI),and primer synthesis was provided by WC Gene Biotechnology Co.,Ltd.(Shanghai,China).The amplified fragments of each resistance gene were inserted into PUC57 plasmids by molecular cloning,and standard plasmids were prepared.According to 6 gradients diluted 10 times,standard curves were made,and the absolute abundances of ARGs were calculated according to the standard curves of the resistance gene standards.To minimize the differences caused by different DNA manipulation efficiencies and background bacterial abundance,the 16S rRNA gene was used as an internal control for data normalization.The relative abundances of ARGs were calculated using the 2-ΔCtmethod(Linet al.,2016),whereΔCt=Ctfor target gene,-Ctfor 16S rRNA gene.

Statistical analysis

All the data were analyzed by single-factor variance analysis and least significant difference(LSD)multiple comparison using SPSS 20 statistical software(IBMCorp.,Armonk,USA).The significance of each index was tested,and differences were considered significant atP＜0.05.A heat map was generated using OmicShare tools,a free online platform for data analysis(http://www.omicshare.com/tools).A redundancy analysis(RDA)of the correlations between ARGs and environmental factors was performed using Canoco 5 software.All the data were analyzed using Microsoft Excel 2010 software(Microsoft Corporation,Redmond,USA).

TABLE II Effect of tetracycline(TC)on maize growth at the mature stage

RESULTS

Selected properties of the soil used

The basic physical and chemical properties of the original soil were as follows:pH,8.63;total nitrogen,0.51 g kg-1,total phosphorus,0.26 g kg-1,total potassium,1.34 g kg-1;available nitrogen,37.75 mg kg-1,available phosphorus,22.39 mg kg-1,and available potassium,143.42 mg kg-1;organic matter content,24.99 g kg-1;cation exchange capacity,8.81 cmol kg-1;total salt content,0.39 g kg-1;and TC concentration,0.015 mg kg-1.The relative abundances ofintI1,tetG,tetM,andtetOwere 2.21×10-5,7.18×10-6,1.51×10-6,and 1.03×10-6,respectively;tetB,tetW,andtetXwere not detected in the soil.

Growth parameters of maize

Tetracycline significantly inhibited the growth of maize,with inhibition of belowground biomass being greater than that of aboveground biomass.Compared with under the NT treatment,the plant height,root length,plant fresh weight,root fresh weight,and grain weight under the TC treatment decreased by 8.92%,15.46%,13.91%,17.61%,and 15.22%,respectively(Table II).The maize growth parameters under remediation substance treatments were higher than those under the TC treatment.Specifically,the growth parameters of maize plants under the TC+MI+WC,TC+MI+B,TC+WC,and TC+B treatments were significantly higher than that under the TC treatment(P＜0.05),with growth parameters following the order TC+MI+WC＞TC+MI+B＞TC+WC＞TC+B,indicating that the combined applications of worm castings or biochar with microbial inoculum were better than single applications.The roots of maize plants under the TC+MI+B,TC+MI+WC,TC+MI+FC,and TC+B treatments were significantly longer than those under the TC treatment(P＜0.05),with increases of 43.46%,39.68%,33.36%,and 19.98%,respectively.Moreover,combined substance applications resulted in significantly longer roots compared with single applications.The fresh weights of maize plants under the remediation substance treatments were significantly greater than that under the TC treatment(P＜0.05).The fresh weights under the different treatments followed the order TC+MI+FC＞TC+FC＞TC+MI+B＞TC+B＞TC+MI+WC＞TC+WC＞TC+MI,indicating that the combined applications of different remediation substances with microbial inoculum were better than single applications.The root weights of the maize plants under the TC+MI+FC,TC+B,TC+MI+B,TC+MI,and TC+MI+WC treatments were significantly higher than that under the TC treatment(P＜0.05),with increases of 46.18%,36.65%,36.09%,31.96%,and 25.89%,respectively.The maize grain weights under the TC+FC,TC+MI+FC,TC+MI+WC,TC+MI,and TC+WC treatments were significantly higher than that under the TC treatment(P＜0.05).The TC+FC treatment resulted in the greatest grain weight,60.68%greater than that under the TC treatment,followed by the TC+MI+FC treatment,which was 40.27%greater than that under the TC treatment.In general,the combined applications were better than single applications at reducing the inhibition of TC on maize growth.

Antioxidant system of maize

Superoxide dismutase is the first line of defense against oxidative stress and can scavenge superoxide anion free radicals in plants and catalyze their disproportionation(Alscheret al.,2002).At the seedling stage,SOD activity under the TC treatment was significantly higher than that under the NT treatment(P＜0.05),with an increase of 3.34%(Fig.1a).In contrast,at the jointing and mature stages,SOD activities decreased by 16.47%and 0.99%,respectively.At the maize seedling stage,SOD activities under the TC+WC and TC+MI treatments were significantly lower than that under the TC treatment(P＜0.05),with decreases of 2.48%and 6.78%,respectively.At the jointing stage,there were no differences in SOD activity between remediation substance treatments and the TC treatment.At the mature stage,SOD activities under the TC+MI and TC+MI+B treatments were significantly lower than that under the TC treatment(P＜0.05),with decreases of 3.17%and 5.68%,respectively.Overall,the SOD activity in the maize growth period followed the order seedling stage＞mature stage＞jointing stage.

Catalase can eliminate the production of H2O2in plant metabolism to avoid excessive accumulation of H2O2,causing oxidative damage to cells(Fornazieret al.,2002).At the seedling and jointing stages,the CAT activity under the TC treatment was lower than that under the NT treatment,with decreases of 10.98%and 8.09%,respectively.Conversely,CAT activity was significantly higher under the TC treatment than that under the NT treatment at the mature stage(P＜0.05),with an increase of 181.18%(Fig.1b).At the seedling stage,CAT activities under the remediation substance treatments were significantly higher than that under the TC treatment(P＜0.05),except for the TC+FC and TC+MI+FC treatments;the TC+MI+WC treatment showed the most significant increase(129.45%).At the jointing stage,the CAT activities under the TC+MI,TC+MI+B,TC+MI+WC,and TC+FC treatments were significantly higher than the activity under the TC treatment(P＜0.05),with increases of 83.45%,70.07%,35.92%,and 21.48%,respectively.At the mature stage,CAT activities under the remediation substance treatments were not significantly higher than the activity under the TC treatment.Thus,CAT activity first increased and then decreased throughout the maize growth period.

Peroxidase is a protective enzyme of the enzymatic defense system in plants that converts H2O2to H2O and O2(Fornazieret al.,2002).At the maize seedling stage,POD activity under the TC treatment was significantly lower than that under the NT treatment(P＜0.05),with a decrease of 46.68%(Fig.1c).At the jointing stage,POD activity under the TC treatment was significantly higher than that under the NT treatment(P＜0.05),with an increase of 14.94%.However,there was no difference in POD activity between the TC and NT treatments at the mature stage.At the maize seedling stage,TC toxicity could be alleviated by adding different remediation substances.Among them,the TC+MI+FC,TC+WC,TC+MI+WC,TC+MI,and TC+FC treatments showed significantly higher POD activities than the TC treatment(P＜0.05),and the TC+MI+FC treatment showed the most significant effect,with an increase of 162.56%.At the jointing stage,the TC+WC treatment exhibited higher POD activity than the TC treatment,with an increase of 1.31%.At the mature stage,POD activities under the TC+MI+WC,TC+FC,TC+MI+FC,TC+MI,TC+WC,and TC+B treatments were significantly higher than the activity under the TC treatment;the TC+MI+WC treatment showed the most significant effect,with an increase of 82.46%.Overall,POD activity in maize throughout the growth period followed the order mature stage＞jointing stage＞seedling stage.

Fig.1 Superoxide dismutase(SOD)(a),catalase(CAT)(b),and peroxidase(POD)(c)activities in maize plants during the growth period under different treatments.Error bars represent standard deviations of means(n=4).The different letters above each column indicate significant differences within each stage at P＜0.05.See Table I for the description of different treatments.FW=fresh weight;U=units.

Resistance factors of maize

Proline plays an important role in osmotic regulation in plants(Surekhaet al.,2014).Under normal growth conditions,the free Pro content in plants is low.However,under stress conditions,the Pro content will increase significantly,reflecting the degree of plant stress and its use as a suitable indicator of plant stress resistance.During the three periods of maize growth(Fig.2a),the contents of Pro in maize under the different treatments gradually decreased with time,and the Pro contents under the TC treatment were higher than that under the NT treatment,with increases of 46.92%,5.15%,and 5.85%,respectively.At the maize seedling stage,the Pro contents under the remediation substance treatments were lower than that under the NT treatment.The TC+FC,TC+MI+FC,TC+B,TC+WC,and TC+MI treatments showed significantly lower Pro contents than did the TC treatment(P＜0.05),with decreases of 29.09%,20.34%,19.40%,14.19%,and 13.48%,respectively.At the jointing stage,the Pro contents under the TC+MI+B,TC+MI+FC,TC+FC,TC+WC,and TC+B treatments were significantly lower than that under the TC treatment(P＜0.05),with decreases of 24.48%,23.14%,17.36%,14.23%,and 10.23%,respectively.At the mature stage,the Pro contents under the TC+MI+WC,TC+FC,TC+WC,and TC+MI+FC treatments were significantly lower than that under the TC treatment(P＜0.05),with decreases of 20.22%,19.33%,18.40%,and 16.55%,respectively.Overall,the Pro contents under the TC+MI+FC,TC+FC,and TC+WC treatments were lower at all stages of maize growth.

Malondialdehyde is an important product of cell membrane lipid peroxidation,and its content can reflect the degree of stress and cell membrane lipid peroxidation in plants(Kocaet al.,2007).Throughout the growth period of maize(Fig.2b),the MDA contents under the TC treatment were higher than those under the NT treatment,increasing by 11.22%,1.69%,and 1.44%,respectively,at each stage.At the maize seedling stage,except under the TC+B and TC+MI+B treatments,the MDA contents of maize under the remediation substance treatments were significantly lower than that under the TC treatment(P＜0.05);the TC+MI+FC treatment showed the lowest MDA content,with a decrease of 50.88%.At the jointing stage,the MDA contents under the TC+MI+FC,TC+MI+WC,TC+B,and TC+MI+B treatments were significantly lower than that under the TC treatment(P＜0.05),with decreases of 20.27%,17.61%,16.28%,and 14.29%,respectively.At the mature stage,except under the TC+FC treatment,the MDA contents under the remediation substance treatments were significantly lower than that under the TC treatment(P＜0.05).The TC+MI+WC treatment showed the lowest MDA content,with a decrease of 42.55%,followed by the TC+MI+FC treatment,with a decrease of 23.05%.Overall,the MDA content throughout the maize growth period followed the order jointing stage＞mature stage＞seedling stage.

Fig.2 Contents of proline(Pro,a)and malondialdehyde(MDA,b)in maize plants during the growth period under different treatments.Error bars represent standard deviations of means(n=4).Different letters above each column indicate significant differences within each stage at P＜0.05.See Table I for the description of different treatments.FW=fresh weight.

Residues and transport of TC in the soil-maize system

After the TC in soil is absorbed by maize root,it is transferred to the stem-leaf and grain through the Casparian strip in the root system(Liuet al.,2018a),and residual TC can be found in various organs of the plant.The total residual amount of TC in plants differed depending on the treatment(Fig.3).The total concentration of TC in the plants under the TC treatment was 12.39 mg kg-1higher than that under the NT treatment.The total concentrations of TC in plants under the remediation substance treatments were lower than that under the TC treatment,and followed the order TC+WC＞TC+MI+WC＞TC+MI＞TC+MI+B＞TC+FC＞TC+B＞TC+MI+FC,with removal rates ranging from 3.48%—68.84%.Compared with under the TC treatment,the concentration of TC in the maize root under the TC+MI+FC treatment was the lowest(2.36 mg kg-1),followed by the TC+B treatment(2.72 mg kg-1),with removal rates of 76.06%and 72.38%,respectively.In maize stem-leaf,the concentrations of TC under the TC+MI+B,TC+MI,TC+FC,TC+MI+FC,and TC+B treatments were significantly lower than that under the TC treatment,and the removal rates ranged from 18.08%to 57.59%.In maize grain,the concentrations of TC under the TC+MI+FC,TC+MI+WC,TC+FC,and TC+B treatments were significantly lower than that under the TC treatment,and the removal rates ranged from 22.12%—29.14%.Thus,the residual concentrations of TC in the different plant organs followed the order root＞stem-leaf＞grain.Additionally,the removal rates of different organs under the TC+MI+FC treatment were highest,and the total residual concentrations were lowest,followed by those under the TC+B treatment.

Fig.3 Concentrations of tetracycline(TC)in the soil-maize system under different treatments.See Table I for the description of different treatments.

The residue of TC in the soil under the different treatments ranged from 0.013 to 1.341 mg kg-1(Fig.3).The residue of TC in the soil under the TC treatment was significantly higher than that under the NT treatment,while the residue of TC in soil under the remediation substance treatments was different from that in plants.The TC+MI+Btreatment had the lowest TCresidue in the soil,followed by TC+MI+WC,TC+MI,TC+WC,TC+FC,TC+MI+FC,and TC+B,with removal rates of 21.90%—50.61%.

To compare the effects of different remediation substances on the accumulation and transport of TC in maize,we calculated the BCF and TF(Table SII,See Supplementary Material for Table SII).The BCF is the ratio of the concentration of antibiotics in different parts(root,stem-leaf,and grain)of the plant to the concentration of antibiotics in the soil environment(Gao and Zhu,2004;Linet al.,2007),and is expressed as the root concentration factor(RCF),stem-leaf concentration factor(SLCF),and grain concentration factor(GCF),respectively.Tetracycline refers to the ratio of the antibiotic concentration in aboveground plant biomass to that in belowground plant biomass(Zhanget al.,2019).Table SII shows that the BCF and TF values under the NT treatment were greater than those under the TCtreatment.Of the remediation substance treatments,the RCF,SLCF,and GCF values under the TC+B treatment were the lowest.The BCF values under each treatment differed depending on the organ,but BCF generally followed the order RCF＞SLCF＞GCF.In addition,the TF values under the remediation substance treatments were higher than that under the TC treatment,with the TF value under the TC+MI+FC treatment being the greatest.

Relative abundances of intI1 and TRGs in the soil-maize system

The relative abundances of ARGs in maize plants varied greatly(Fig.4).The relative abundance ofintI1(1.67×10-3)was significantly higher than those of the TRGs.Of the TRGs,the relative abundance oftetWwas the highest(1.24×10-3),and the others followed the ordertetG＞tetB＞tetM＞tetX＞tetO.The residues of different genes in different organs of maize varied.Specifically,the abundances oftetM,tetO,andtetWfollowed the order stem-leaf＞grain＞root.The abundances ofintI1,tetB,andtetXwere the highest in maize grain,and that oftetGwas the highest in maize root.In maize root under the remediation substance treatments,the relative abundance of total TRGs under the TC+MI treatment was the lowest,followed by the TC+B and TC+MI+B treatments,with these treatments having 82.07%,40.29%,and 35.10%lower abundances than the TC treatment,respectively.In maize stems-leaves,the relative abundance of TRGs under the TC treatment was significantly higher than that under the NT treatment,with an increase of 87.50%.Additionally,compared with under the TC treatment,the lowest relative abundance of TRGs occurred under the TC+B treatment,with a decrease of 90.17%,followed by that under the TC+MI+B treatment,with a decrease of 58.73%.The relative abundance of TRGs in grain under the TC treatment was 2 orders of magnitude higher than that under the NT treatment.The relative abundances of TRGs in grain under the remediation substance treatments followed the order TC+B＜TC+FC＜TC+MI+WC＜TC+MI+B＜TC+MI+FC＜TC+MI＜TC+WC.The relative abundances of genes in soil were different from those in maize plants,with relative abundances in soil following the ordertetX＞tetG＞intI1＞tetW＞tetB＞tetM＞tetO.The relative abundance of TRGs in soil under the TC treatment was higher than that under the NT treatment.The relative abundances of TRGs in soil under the remediation substance treatments were less than that under the TC treatment,and the relative abundance of TRGs under the TC+B treatment was the lowest,followed by the TC+MI+B treatment.In summary,the TC+B treatment resulted in the lowest relative abundance of TRGs in soil,maize plants,and all organs of maize.Thus,the addition of biochar can effectively reduce the residues of TRGs in the soil-maize system.

Fig.4 Relative abundances of intI1 and tetracycline resistance genes(TRGs)in the soil-maize system under different treatments.See Table I for the description of different treatments.

Relationships between TRGs,TC and other environmental factors

According to the RDA(Fig.5),the 1st axis explained 70.26%of the variation,and the 1st and 2nd axes together explained 86.20%of the variation.The amount of variation in the genes explained by each environmental factor followed the order TC＞grain weight＞Pro＞CAT＞intI1＞plant fresh weight＞root fresh weight＞root length,indicating that the main factor affecting the relative abundance of TRGs was the residue of TC.The RDA showed that TRGs were positively correlated with TC,grain weight,CAT,andintI1,and negatively correlated with Pro,plant fresh weight,root fresh weight,and root length,Additionally,intI1was positively correlated with TRGs,except fortetX.The analysis of the relationship between remediation substance treatments and TC and TRGs found that the residues of TC and TRGs in different treatments followed the order TC+MI＞TC+FC＞TC+MI+WC＞TC+WC＞TC+MI+B＞TC+MI+FC＞TC+B,indicating that the application of biochar alone or in combination with the microbial inoculum had a good effect on the reduction of TC and TRGs.

Fig.5 Redundancy analysis(RDA)of the relationship between tetracycline resistance genes(TRGs)and environmental factors(tetracycline(TC),intI1,the antioxidant system,resistance factors,and growth parameters)under different treatments.Blue arrows represent genes,red arrows represent environmental factors,and open circles represent different treatments.See Table I for the description of different treatments.CAT=catalase;Pro=proline;Rfw=root fresh weight;Rl=root length;Pfw=plant fresh weight;Gw=grain weight.

DISCUSSION

Antibiotics kill bacteria by inhibiting essential cellular processes and by activating cellular response pathways that contribute to cell death(Kohanskiet al.,2010).Specifically,tetracycline antibiotics inhibit the binding of amino acidtRNA with the 30S ribosomal subunit and the synthesis of the peptide chain,thus inhibiting the synthesis of cell proteins(Kohanskiet al.,2010).In this study,the exogenous addition of 50 mg kg-1TCsignificantly inhibited the growth of maize.As shown in Table II,the inhibitory effect of TC on the belowground biomass of maize was distinctly greater than that on the aboveground biomass,which is consistent with the results of previous studies(Jiaoet al.,2018;Liuet al.,2018a).When plants grow in antibiotic-contaminated soil,damage to the root system is the main mechanism by which TC inhibits plant growth,so root growth can be used as a sensitive index of the ecological toxicity of antibiotics to vegetables(Liuet al.,2018a).Remediation substances improved the growth of maize in TC-contaminated soil(Table II),with the TC+MI+WC,TC+MI+B,TC+B,and TC+MI+FC treatments being the most effective,indicating that various remediation substances can effectively alleviate the toxic effects of TC on plants.

The antioxidant enzyme system is the protection system for plants under adversity.It can maintain the balance of active oxygen metabolismin vivo,thereby affecting the expression of defense-related genes and protecting the membrane structure so that plants can resist environmental stress to a certain extent(Whanet al.,2008;Liet al.,2013).Under normal growth conditions,the active oxygen species and antioxidant enzyme systems in plants are in a dynamic balance(Zhanget al.,2015).When plants are under stress,many reactive oxygen species are producedin vivo.If the reactive oxygen species cannot be removed in time,the growth and development of plants is inhibited(Prabhukarthikeyanet al.,2018).Therefore,to maintain normal growth,plants will resist external stress through the antioxidant enzyme system.SOD can convert active oxygen radicals into H2O2and O2,and PODand CAT can decompose H2O2into harmless H2O.In this study,under TC stress,the balance of the antioxidant system in maize was disrupted,the defense system was activated,antioxidant enzyme(SOD)activity and MDA and Pro contents increased,and related proteins and genes were induced and expressed.However,the addition of different remediation substances enhanced the reaction of the antioxidant enzyme system,the expression of defense-related genes,and the ability of antioxidant enzymes to scavenge active oxygen.Furthermore,the peroxidation of membrane lipids,damage to membrane structure and function,and damage to the plant cell membrane were further reduced,which decreased the MDA and Pro contents in maize.In addition,different remediation substances have different effects on defense gene expression,including expression speed,intensity,and spatial distribution,which need to be further explored in future research.In this study,TC stress induced an increase in SOD activity and inhibited the activities of CAT and POD at the seedling stage,whereas this was reversed in the jointing and mature stages(Fig.1).When plants are subjected to external environmental stress,they activate the enzyme defense system to enhance the activity of SOD,which is the first line of defense(Alscheret al.,2002).When the stress reaches a certain level,plants produce many peroxides,which exceed the scavenging capacity of SOD enzymes and cause damage to plant somatic cells,resulting in a decrease in SOD activity.Thus,maize can respond positively to stress,but the response mechanism was not consistent with that of Vilvertet al.(2017),likely because of the difference in plant species.After adding different remediation substances,the cells jointly resisted TC pollution stress through the synergistic action of antioxidant enzymes.The TC+WC,TC+MI+WC,and TC+MI treatments effectively promoted the balance of the antioxidant enzyme system,but the mechanism of worm casting application alone and its combination with microbial inoculum on the antioxidant system remains unclear,and further research is needed.Additionally,membrane lipid peroxidation caused by excessive reactive oxygen species exceeding the scavenging capacity of plant antioxidant systems is an important mechanism of plant damage(Kocaet al.,2007).When plants were exposed to TC stress,lipid peroxidation of the plant cell membrane promoted the production of Pro and MDA in maize.The content of Pro in maize gradually decreased with time,indicating that the toxicity of TC gradually weakened.Malondialdehyde is an important product of lipid peroxidation in cell membranes(Kocaet al.,2007),and its content first increased and then decreased with time.The decrease in MDA content at the mature stage may be related to the decrease in the toxicity of TC residues.The contents of Pro and MDA in maize treated with different remediation substances were significantly decreased(Fig.2),and the TC+MI+FC treatment had a significant effect,probably because the beneficial microorganisms in the microbial inoculum combined with fungal chaffproduced a synergistic effect to resist TC stress.

When wastewater or animal manure is used in the soilplant system,antibiotics may accumulate in soil and can be taken up by,accumulated in,and transported by crops(Pan and Chu,2016).In this study,the TC residue concentration in maize was higher belowground than aboveground,which is consistent with TC residues in ginger,lettuce,and other plants(Duanet al.,2017;Liuet al.,2018a).The BCFs of TC in the soil-maize system followed the order RCF＞SLCF＞GCF(Table SII),which was consistent with the residual concentration of TC in different organs(root＞stem-leaf＞grain)(Fig.3).The abilities of plants to transport antibiotics from belowground to aboveground biomass varies due to the different sensitivities of different plants to antibiotics.Additionally,differences in rhizosphere enzymatic activities and microbiological characteristics between different plants also affect the dissipation and bioavailability of pollutants(Yueet al.,2019).The accumulation of antibiotics in plant roots may be related to the active and physicochemical absorption of antibiotics by plant roots.In this study,the TF of TC in the soil-plant system was calculated,and the TF value was less than 1,which indicates that the ability of maize to transport TC from belowground to aboveground was weak.The TF is mainly affected by the crop transpiration rate,which can promote the absorption of antibiotics by plants from soil(Huet al.,2010).In addition,the physical and chemical properties of different components in plant tissues and the properties(e.g.,molecular weight,hydrophobicity,and electrical properties)of TC may affect the distribution of pollutants in plants(Dettenmaieret al.,2009;Yuet al.,2019).Pan and Chu(2016)studied the relationship between the physical and chemical properties of veterinary antibiotics and plant toxicity and found that hydrophobicity was the most important factor in controlling the toxicity of antibiotics to plants,while other physicochemical parameters(e.g.,electrical properties)of veterinary antibiotics showed poor correlations with their phytotoxicity.Other previous studies have also found hydrophobicity to be a crucial factor influencing the phytotoxicity of organic compounds(Hulzeboset al.,1993;Gramatica,2007).Antibiotic resistance genes migrate through stomata in plant tissues,exist in roots,and reach stems,leaves,and grains during plant growth.Furthermore,the induction and mutation of antibiotics in the soil-plant system also promotes ARG transmission.We found that TC was positively correlated with TRGs(Fig.5),which is consistent with previous studies(Wuet al.,2010;Yanet al.,2018;Fanet al.,2020).In contrast,some studies have found no significant correlation between antibiotics and ARGs(Gaoet al.,2012b;Wanget al.2015),which may be because different antibiotics and gene types were selected.IntI1is generally considered an indicator for mobile gene components(Duanet al.,2017),which are highly abundant in soil-plant systems and can promote the accumulation and spread of ARGs through horizontal gene transfer.In this study,intI1was positively correlated with TRGs,indicating thatintI1played an important role in the horizontal transfer of ARGs.The correlation betweenintI1and ARGs may be due to their location on the same host bacteria or gene elements,such as binding plasmids,insertion sequences,etc.(Duanet al.,2017).

The addition of different remediation substances can help plants resist TC stress due to synergistic action between the antioxidant enzyme system and other resistance factors,reducing the damage of excessive reactive oxygen species to cell membrane lipids,maintaining the integrity of plant somatic cells,promoting the normal growth and metabolism of plants,degrading TC to a certain extent,and reducing the abundance of ARGs.In the soil-maize system,the residues of TC and relative abundances of TRGs under the TC+MI+FC and TC+B treatments were lower(Figs.3 and 4).This may be because the combination of fungal chaffand microbial inoculum,and the addition of biochar alone increased the adsorption capacity of soil to TC and the activity of soil microbial biomass and metabolism,accelerating the biodegradation and non-biological degradation of TC in soil,reducing the selective pressure on resistant bacteria,and reducing the residues of TC and TRGs in the soil-maize system.The residues of TC and relative abundance of TRGs under the TC+WC treatment were higher.This may be because the residue of TC and TRGs in livestock and poultry excrement entered the earthworm intestine during earthworm composting,but could not be metabolized by earthworms and remained in worm castings(Liuet al.,2018b).This process requires further study.Overall,the combined applications of either worm castings or fungal chaffwith microbial inoculum(TC+MI+WC and TC+MI+FC)were found to be superior to the single applications of worm castings or fungal chaff(TC+WC and TC+FC).This may be because the beneficial microorganisms in the microbial inoculum could cooperate with the castings and fungal chaffto reduce the TC residue concentration and promote plant growth.However,the single application of biochar(TC+B)was superior to the combined application of biochar with microbial inoculum(TC+MI+B),a finding that may be related to the nature of the biochar itself.In this study,the BCF values under the TC+MI+FC,TC+MI+B,and TC+B treatments were smaller(Table SII),which indicated that the addition of these remediation substances could hinder the transport of TC from soil to plant tissue.The TF values of these treatments were higher(Table SII),which indicated that they had a strong transport ability from belowground to aboveground plant biomass,which was also related to the degree of plant root damage.From the growth parameters under these treatments(Table II),it can be seen that the root growth of maize was less affected by TC stress,which shows that adding these remediation substances can effectively alleviate TC stress and promote plant growth.In general,biochar was found to be the best remediation substance,effectively alleviating the physiological toxicity of TC stress on plants and reducing the residues of TC and TRGs in the soil-maize system.This may be because biochar can increase the adsorption of TC in soil throughπ-πbonds and metal bridging,which can transform more free TC into bound TC(Liet al.,2017),reduce its mobility and bioavailability in soil,alleviate selective pressure on microorganisms,and ultimately reduce the TC content and TRG abundance in plants.In addition,many studies(Duanet al.,2017;Jiaoet al.,2018)have shown that biochar can significantly reduce the content of effective antibiotics in soil,can effectively control the transfer of antibiotics and ARGs from the soil to the edible parts of plants,and is an effective soil amendment.

CONCLUSIONS

Tetracycline significantly inhibited maize growth,disrupted the antioxidant defense system balance,and increased the Pro and MDA contents in maize plants.The distribution of TC residues in different organs of maize plants followed the order root＞stem-leaf＞grain.The relative abundances of resistance genes in maize plants followed the orderintI1＞

tetW＞tetG＞tetB＞tetM＞tetX＞tetO,while in soil,the relative abundance oftetXwas the highest,followed by those oftetGandtetW.An RDA showed that TC was positively correlated with TRGs.Antibiotic residues pose a great threat to food safety and human health.This study found that adding different remediation substances can alleviate the inhibition effect of TC on maize growth and reduce the residue contents of TC and TRGs in the soil-maize system.The application of biochar,alone or in combination with microbial inoculum,had the most obvious reducing effect on TC and TRG residues,with broad application prospects.However,the specific molecular mechanisms of the interaction between biochar and TC and the relationships between antibiotics,ARGs,and microbial communities need further exploration.

CONTRIBUTION OF AUTHORS

Junmei QIN and Jianli SONG contributed equally to this work.

ACKNOWLEDGEMENTS

We greatly appreciate the financial support of the Key R&D Program in Shanxi Province,China(Nos.201903D 221015 and 201803D221002-2),the Project 1331 in Shanxi Province,China(No.20211331-15),and the Open Fund Project of Shanxi Key Laboratory of Soil,Environment and Nutrient Resources,China(No.2019004).We thank WC Gene Biotechnology Co.Ltd.,Shanghai,China for providing technical support for gene testing.We thank American Journal Experts(AJE)for their professional English language editing services.

SUPPLEMENTARY MATERIAL

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