Effects of Biochar,Arbuscular Mycorrhizal Fungi and Nitrogen Application on Crop (Cichorium intybus L.) Growth and Soil Properties in Cadmium Contaminated Soil

Su Li-fei ,Li Yan-cong ,Wang Sui ,Sun Xiao-he ,Liu Bo-wen ,and Sun Yan-kun*

1 College of Resources and Environment,Northeast Agricultural University,Harbin 150030,China

2 College of Agriculture,Northeast Agricultural University,Harbin 150030,China

Abstract:The effects of biochar (BC),arbuscular mycorrhizal fungi (AM),nitrogen (N) and their composite treatments (BC+N,AM+N,BC+AM and BC+AM+N) application on Cichorium intybus L.(C.intybus L.) nutrient uptake,soil properties and cadmium(Cd) accumulation were investigated in Cd contaminated soil (0.11 mg·kg-1).The results showed that the addition of BC increased the rate of mycorrhizal infection.However,the addition of N slightly inhibited mycorrhizal colonization,and the shoot and root bioaccumulation of chicory was positively influenced by BC and N when inoculated with AM fungi.Compared with the single component treatment (AM,BC or N) or two-component treatment (BC+N,AM+N or BC+AM),the three-component composite treatment (BC+AM+N) had the highest shoot bioaccumulation,whereas BC+AM treatment was considered the best for root biomass bioaccumulation.Compared with the control treatment,the single component treatment (AM,BC or N) and the composite treatment resulted in an overall improvement of the chicory shoot,root related nutrient uptake (N,P,K,Mg,Ca,Mn and Fe) and some soil physicochemical properties;in addition,these treatments showed better results than BC+AM+N and BC+AM treatments.Among the Cd-related indexes,Cd concentrations in the shoot,root and soil of C.intybus L.were reduced through treatment with AM and BC.However,a lower bioconcentration coefficient (BCF) and a higher transfer coefficient (TF) were observed in both treatments,and the most desirable effect was observed following the combination treatment (BC+AM).Compared with other single management,the shoot and root Cd concentrations of C.intybus L.after the management of N alone were higher,and the value of BCF (2.63%)was higher,but the value of TF (1.05%) was lower.Indexes related to Cd improved concurrently following the application of N in combination with BC or AM.Therefore,in Cd contaminated soils,single or combined application of BC,AM and N could promote chicory growth and nutrient uptake and improve some soil physicochemical properties.However,N should not be applied alone and needed to be combined with AM and BC;furthermore,it was evident that the treatment with the three composites (BC+AM+N) was optimal from an application point of view.

Key words:biochar,arbuscular mycorrhizal fungi,nitrogen,nutrient absorption,cadmium accumulation,soil nutrient

Introduction

Different agro-industrial practices,such as metal smelting,mining and sewage sludge applications,and high geological background (HGB) area can lead to an excessive amount of heavy metal in the soil,and heavy metal pollution has become an environmental concern in developing and developed countries alike (Sunet al.,2021).Cadmium (Cd) is a widespread heavy metal that is particularly found in agricultural soils.Reportedly,Cd is highly toxic to plants,reduces photosynthesis and mineral nutrient uptake,inhibits plant growth,and may even directly lead to plant death (Liuet al.,2019).Owing to the fact that Cd is highly mobile,highly toxic and non-biodegradable,once it enters into the animal body,it will accumulate and cannot be excreted (Liet al.,2020b).Therefore,agricultural ecosystems and human health can be adversely affected through the transfer of Cd in the food chain.As a result,mitigating the harmful effects of heavy metals on crops and ensuring their safe production are pressing issues in agricultural production.

Cichorium intybusL.is a commonly farmed vegetable crop with significant nutritional and economic values,as well as a crop with a high Cd accumulation capacity(Zhanet al.,2013).Reducing cadmium pollution withCichorium intybusL.has become a research hotspot in the fields of soil science and environmental science(Zhanet al.,2013).Studies on the influence of soil conditioners,such as biochar and nitrogen fertilizer,on the absorption and transfer of soil cadmium ofCichorium intybusL.to reduce Cd pollution on agricultural land have garnered a lot of interest in recent years (Wuet al.,2022).Biochar (BC) is a carbon-rich product that can be obtained through the thermal cracking of biomass materials,such as agricultural and forestry wastes,livestock manure and sludge,at low-to-medium temperatures and in oxygenlimited or anaerobic conditions (Huet al.,2020).The structure,physicochemical properties and structurefunction relationship of BC and the effect of BC on soil properties have been studied extensively (Houet al., 2021).The high cation exchange capacity,pH,porous structure,large specific surface area and active functional groups of BC enable it to effectively adsorb and immobilize heavy metal ions,owing to which it has been applied to remediate heavy (class) metal ions,such as nickel (Ni),arsenic (As) and chromium (Cr) in soils (Maet al.,2017;Liet al.,2020a).Furthermore,in Cd contaminated soils,BC reportedly reduces the soil bioavailability of Cd,improves plant yield,and reduces plant uptake of Cd (Chenet al.,2020).

Arbuscular mycorrhizal (AM) fungi belong to the subphylumGlomeromycotanand are widespread in soils,where they can establish reciprocal symbiosis with ＞80% of plant species in terrestrial ecosystems to improve plant mineral nutrient uptake,promote plant growth,and mitigate environmental stresses (Caoet al.,2021b).AM fungi are rich in the free radical mycelium and the numerous mycelia are known to form communal mycelial networks (CMNs) in the soil,which can subsequently improve plant adaptation to environmental stresses (Caoet al.,2021a).Studies have reported that CMNs can secrete glomalin in order to ensure the complexation of heavy metals and reduce their content in the soil;in addition,they can intracellularly adsorb and immobilize heavy metals in the mycelium (Suet al.,2020).Alternatively,AM fungi are important functional microorganisms in the soil and can influence the interroot microdomain environment and microbial community to affect the uptake of heavy metals by plants in various ways.

Nitrogen (N) is an important component of genetic materials and metabolic compounds,such as proteins and nucleic acids in plants.The uptake and transport processes of N by plants are similar to those of Cd,and the uptake and utilization of N by plants are affected by the types of crops,types of N,application rates and application time(Liet al.,2016),which can minimize the accumulation of Cd in crops.NH4+-N and NO3--N are the main types of N taken up by plants,and studies have shown that the addition of N fertilizers that are based on NH4+-N will directly lead to soil nitrification and indirectly release H+,thereby accelerating soil acidification (Sajjadet al.,2020).The acidic soil environment subsequently increases Cd bioavailability and solubility (Yeet al.,2019).The positive correlation between N and Cd concentrations in plants has been reported previously;however,a dilution effect is typically observed,i.e.,and the concentration of Cd is reduced in the plant body with the increase in plant biomass (Yanget al.,2019).

Previous studies have shown that BC can improve the ability of AM fungi to colonize plant roots and enhance the acquisition of soil nutrients (Rafiqueet al.,2019).The application of AM fungi in combination with BC can promote plant nutrient uptake,reduce the soil availability of Cd,and limit the upward transport of Cd.This is attributable to the synergistic effect of AM fungi and BC on the acceleration of Cd fixation and reduction of Cd plant availability (Linget al.,2018).Alternatively,the interaction between BC and N elements can enhance N residues in the soil,thereby improving nutrient use efficiency and crop yield (Ibrahimet al.,2020).However,when they were used in combination,BC and N fertilizers can enhance the stability of microbial communities and positively affect biological activity in the soil (Songet al.,2020).Additionally,the adsorption of BC can effectively reduce the loss of N fertilizer through nitrification,and it can effectively reduce the available concentration of Cd in acidic soil (Sajjadet al.,2020).However,the effects of the combined application of AM fungi,BC and N on plant growth and development and its effectiveness in Cd contaminated soils remain unclear.Cichorium intybusL.,a perennial herb in theAsteraceae Bercht.&J.Preslfamily,is used as both medicine and food.The shoots and roots of the plant can be used for medicinal purposes,the leaves can be prepared as lettuce,the roots contain inulin and aromatic substances,and can tolerate fluctuating climatic and soil conditions (Zhanget al.,2021).However,researches on the related physical and biological measures of the growth and development ofC.intybusL.require further elucidation.Therefore,in the present study,the research aim was to examine the effects of the combined application of AM fungi,BC and N on plant nutrient accumulation,Cd uptake and its related soil properties in chicory plants.

Materials and Methods

Test site and materials

The experiment was performed in the artificial climate chamber from May to September,2020 in Northeast Agricultural University,Harbin,China.And the cultivation conditions were the same as Chenet al(2019).AM fungal inoculum consisted ofGlomus magnicaule,Funneliformis mosseae,Claroideoglomus etunicatumandRhizophagus claruswas mixed according to a mass ratio of 1:1:1:1;and the inoculum consisted of spores and culture soil.The test N element was ammonium chloride (NH4Cl) (N,24.0%).The test plant wasC.intybusL.

BC,pH 10.24;the total C,430.14 g · kg-1;the total N,8.66 g · kg-1;the total P,2.38 g · kg-1;the total Ka,37.43 g · kg-1;cation exchange capacity,23.03 cmol · kg-1;and specific surface area,217.25 m2· g-1.

The test soil was collected from the abandoned farmland soil around the Wanshan mine with the longitude and latitude for 109.24° and 28.67°,respectively.The physicochemical properties were as follows: pH,5.83;Cd,0.11 mg · kg-1;soil organic matter content,22.09 g · kg-1;the total N,1.16 g · kg-1;the total P,1.06 g · kg-1;available N,74.26 mg · kg-1;available P,25.33 mg · kg-1;and available K,122.51 mg · kg-1.According to the national standard of the People's Republic of China GB15618-2018,when the soil pH was 5.5-6.5,the critical value of Cd risk of agricultural soil pollution was 0.3 mg · kg-1,and the test soil was considered to have a moderate degree of Cd pollution.

Experimental design

A completely randomized design was used with three factors including a single application of N fertilizer,a single application of BC and single inoculation of AM fungi,and the corresponding composite treatments were as follows: BC+N,AM+N,BC+AM and BC+AM+N;in addition,no application of BC+no inoculation of AM fungi+no N fertilizer was used as the control (CK).A total of eight treatments were set up in the experiment and each treatment was replicated three times.

The round pots with a bottom diameter of 10 cm and a height of 11 cm were used in this experiment.Each pot was filled with 1 kg of soil and 10,50 and 0.5 g of BC,AM fungal treatment and N fertilizer,respectively.The corresponding treatments were mixed with the soil at the abovementioned application rates.Three seeds were sown in each pot and thin to one plant after 15 days (training time from May 28 to September 1,2020).Distilled water was added from time to time during planting to maintain 60% of soil water-holding capacity.Other management measures were the same as the crop cultivation method,and the experimental cultivation period was 97 days.

Across the grassy6 field, the stone monument stood erect3 and proud against the French sky. A Canadian flag rippled7 softly in the calm breeze. The passengers, each deep in their own thoughts, stared silently out the windows. Lost in the moment, I could visualize8 the sights and sounds of war. Suddenly an unexpected wave of emotion swept over me. I felt immense sadness for those men who never returned home to Canadian soil, but at the same time my heart swelled9 against my chest with an enormous sense of pride. Tears swelled up in my eyes. I was embarrassed by my uncontrolled reaction. As I turned around I realized that each of my friends had experienced the same feelings, their eyes also wet with tears. We smiled knowingly at each other not speaking a word, our eyes rich with understanding.

Sample collection and measurement

Biomass and infestation rate of chicory plants

After cultivation,the potted apparatus was dissected and carefully washed with water to obtain intact roots,and the roots were cut into 1 cm segments and stained with trypan blue using the GridSearchCV under a light microscope to calculate the rate of mycorrhizal infestation (Kormaniket al.,1980).The shoot and root systems ofC.intybusL.were separated and then placed in an oven at 105℃ for 30 min for enzyme inactivation,and dried at 65℃ until a constant weight was achieved.

Nutrient content and Cd content of chicory plants

The dried chicory shoot and root samples were added to 6 mL HNO3:HClO4(2:1,V:V) for microwave digestion;the concentrations of P,K,calcium (Ca),magnesium (Mg),manganese (Mn),iron (Fe) and Cd were then determined through inductively coupled plasma optical emission spectroscopy.

Cd content in different parts of chicory plants and Cd content of soil

Soil Cd,Mg,Mn,Fe and copper (Cu) concentrations were extracted using the ammonium bicarbonate(NH4HCO3)-diethylenetriaminepentaacetic acid(AB-DTPA) universal leaching method.The rest of the soil indicators were assayed according to the method reported by Bao (2000).Analysis of soil pH (water:soil=5:1) was performed using a pH meter,and the concentration levels of NO3-and NH4+were determined through double-wavelength spectrophotometry and indophenol blue method,respectively,and available phosphorous (AP) and available potassium (AK) levels were determined using the phosphomolybdenum blue method and flame photometric method,respectively.

Data processing and statistical analysis

Microsoft Excel 2013 was used for data organization,and SPSS 23.0 and Origin 9 was used for the multifactor analysis of variance (ANOVA) (α=0.05)and correlation graphing,respectively.

Results

Effects of application of BC,AM fungi and N fertilizer on chicory biomass accumulation

Fig.1a showed that the infestation rate of each AM fungal inoculation treatment was 34.63%-77.56%,and the infestation rate of each treatment was in the order of increasing from AM to AM+N to BC+AM to BC+AM+N.The highest infestation rate was observed in BC+AM+N treatment,followed by BC+AM treatment;however,although both treatments were not considerably different from each other,they showed significantly higher infestation rates than those in AM and AM+N treatments.Therefore,the infestation rates increased and decreased significantly with the addition of BC and N,respectively,under identical inoculation conditions.

Fig.1b showed that in terms of shoot biomass,the content of each treatment was in the order of increasing from CK to N to BC to AM to BC+N to BC+AM to AM+N to BC+AM+N,where the CK treatment was significantly lower than the other treatments,and the other treatments were significantly higher than CK treatment by 260.98%-758.54%;in addition,AM+N,BC+AM and BC+AM+N treatments were significantly higher than the remaining treatments.As shown in Fig.1c,the root biomass was the highest after BC+AM treatment and significantly lower than the other treatments by 26.56%-91.10%.The lowest value was observed in the CK treatment,followed by N and BC+N accumulation;the three treatments showed no significant differences;however,they were significantly lower than the remaining treatments.

Fig.1 Effects of biochar,AM fungi and N application on chicory biomass accumulation and mycorrhizal infection in Cd contaminated soils

Effects of application of BC,AM fungi and N on shoot nutrient accumulation in chicory

According to Table 1,in terms of the shoot nutrient uptake ofC.intybusL.,N,P,K,Mg,Ca,Mn and Fe were lower in all the treatments than those in the CK treatment,and BC,AM fungi and N treatments were significantly higher than the CK treatment in the above seven indicators by 281.27%-810.44%,162.50%-1 120.31%,156.68%-822.39%,216.00%-1782.67%,225.04%-883.07%,125.71%-1217.14% and 77.27%-877.27%,respectively.Multifactor ANOVA showed that AM,BC and N significantly affected the uptake of major,medium and trace elements in the above indexes;in the two-factor analysis,AM+BC and BC+N interacted with N,P,K and Ca;similarly,AM+BC+N also interacted with N,P,K,Mg and Ca.

Effects of BC,AM fungi and N application on nutrient accumulation in chicory root system

According to Table 2,in terms of the nutrient uptake of chicory roots,N,P,Mg,Ca,Mn and Fe (except K) were lower after treatment with the CK,among which N,P,K and Fe contents were the highest in BC+AM,Ca and Mn contents were the highest in AM+N treatment,and Mg content was the highest in AM treatment.Generally,the inoculation with AM fungi significantly increased root N,P,K,Mg,Ca,Mn and Fe contents,the addition of BC significantly increased N,K and P contents,and the application of N fertilizer significantly increased N,P,K and Fe contents.In the absence of AM fungi,BC significantly increased K,Mn and P contents of the roots.Notably,after inoculation with AM fungi,the application of N fertilizer decreased P and K contents of the roots with or without BC application.After adding BC,AM fungi significantly increased Ca content in the roots,whereas the application of N fertilizer significantly decreased Fe content in the roots.The three-way ANOVA showed that there was no significant interaction among N,P,K,Mg,Ca,Mn and Fe in the roots.

Table 1 Effects of biochar,AM fungi and N application on nutrient uptake in chicory aerial parts in Cd contaminated soils

Table 2 Effects of biochar,AM fungi and N application on nutrient uptake in chicory root system in Cd contaminated soils

Effects of BC,AM fungi and N application on Cd concentration,bioconcentration and transport

Fig.2 showed that Cd concentrations in both shoot and root systems were reduced more in AM fungal inoculation treatment than those in the treatment without AM fungal inoculation.Overall,the concentrations of Cd in the shoot (28.90%,57.68%,52.48% and 5.32%) and root (52.61%,49.18%,88.31% and 61.83%) were reduced in AM,AM+N,BC+AM and BC+AM+N treatments,respectively,compared to the CK treatment in the corresponding plant site.In the absence of AM fungi inoculation and regardless of the presence or absence of BC application,N application increased the shoot and root Cd concentrations,whereas BC application significantly reduced root Cd concentrations following AM fungi inoculation (Fig.2b).Among the soil Cd concentrations,the application of BC,AM fungi and N alone or in combination significantly reduced the soil Cd concentrations,and they were all significantly lower than the CK treatment by 12.61%-21.51% (Fig.2c).

The bioconcentration factor (BCF) was an evaluation index of the magnitude of heavy metal uptake capacity of plants,which could be used to identify the difficulty of heavy metal migration from soil to plant body in the soil-plant system.In terms of BCF,BCF value of each treatment was in the order of increasing from BC+AM to BC to AM+N to AM to CK to BC+AM+N to N to BC+N;BC+N and N treatments were significantly higher than the other treatments (Fig.2d).In terms of the transfer coefficient (TF),TF value of each treatment was in the order of increasing from AM+N to CK to N to AM to BC+N to BC+AM+N to BC to BC+AM.Therefore,it was evident that the application of N fertilizer decreased TF value under the premise of the multicomponent application containing AM fungi and/or added BC (Fig.2e).

Fig.2 Effects of biochar,AM fungi and N application on Cd accumulation,bioconcentration and translocation in chicory grown in Cd contaminated soils

Effects of BC,AM fungi and N application on soil properties

According to Table 3,the lowest pH of 5.11 was obtained by N treatment,which was significantly lower than the CK treatment by 14.69%.BC+AM treatment had the highest pH,and the remaining treatments were lower than the same by 5.52%-19.40%,which were significantly higher than the other treatments,except for the absence of significant differences with BC and the CK treatments.All the N treatments had higher NH4+-N concentrations in the soil compared to the treatments without N application,and NH4+-N concentrations were significantly lower in the treatments inoculated with AM fungi than those in the treatments without AM fungi.Application of N significantly increased NO3--N concentration in the absence of AM fungi inoculation and AM fungi treatment significantly decreased NO3--N concentration when N was applied regardless of the presence or absence of BC.In AP,AK,available magnesium (AMg) and available manganese (AMn),the maximum value was for BC+N treatment;furthermore,in terms of the factors,AP concentration was significantly reduced when a single AM fungus was inoculated.However,when BC was added and no N fertilizer was applied,AP showed a significant increase following AM treatment.In the absence of BC application and the presence or absence of N application,inoculation with AM fungi reduced AK concentration;however,when AM fungi were inoculated and BC was added,N application significantly reduced AK concentration.In addition,BC significantly increased AMg and AMn concentrations,whereas N addition significantly increased AMn levels.In terms of available iron(AFe),the content of each treatment was in the order of increasing from BC+N to BC+AM+N to N to AM+N to CK to BC to BC+AM to AM;herein,N,BC+N,AM+N and BC+AM+N treatments significantly reduced AFe levels by 50.84%,58.43%,45.57% and 54.82%,respectively,compared to AM treatment.According to ANOVA,there were highly significant interactions of AM+BC in AMn,AM+N in AMg,BC+N in AP and AMn,and AM+BC+N in AK and AMn.

Table 3 Effects of biochar,AM fungi and N application on soil properties

Correlation analysis of chicory indicators with soil indicators under BC,AM fungi and N application

According to Table 4,the correlation analysis between the growth indicators of chicory and soil indicators under the conditions of BC,AM fungi,and N application in Cd contaminated soil revealed the presence of a significant positive association(P＜0.05) between soil pH and TF;NH4+-N showed significant and highly significant (P＜0.01) positive associations with shoot Cd and root Cd concentrations,respectively;AMg showed highly significant positive and significant positive associations with shoot K content and TF,respectively;AMn showed highly significant positive associations with shoot K and Fe contents.Soil pH and root Cd concentration,NH4+-N and chicory indicators (root biomass,shoot P content,shoot Ca content,root N content,root P content,root K content,root Mn content and root Fe content);AK and root Mg and Ca contents,and AFe and chicory indicators (shoot K content,shoot Mn content,shoot Cd concentration,root Cd concentration,and TF);and soil Cd concentration showed a significant negative or a highly significant negative correlation with root N and Mg contents.

Table 4 Correlation analysis among biochar,AM fungi and indexes of chicory plants under N application in Cd contaminated soil and soil indexes

Discussion

Excessive soil Cd concentrations caused by human behavior and the HGB area have become a constraint to the safe development of agriculture (Xuet al.,2022).Applications of pertinent physical amendments and functional microbial agents were effective strategies to reduce soil concentrations of Cd and the accumulation of Cd in plants (Xuet al.,2022;Wuet al.,2022).Furthermore,there was a certain synergistic effect among the associated measures(Xuet al.,2022).The results of the present study demonstrated that single AM,BC and N treatments increased bioaccumulation in both shoot and root systems of chicory.AM fungi were an important functional microorganism widely existing in the soil,and they reportedly could improve the plant root uptake area and uptake efficiency,which consequently promoted the growth and development of the host plant (Caoet al.,2021b).N was the major mineral nutrient with the highest uptake by plants and was the main contributor to ensuring a suitable harvesting yield;therefore,the application of N was beneficial to the accumulation of the plant biomass.The addition of BC was necessary for the process of plant growth and development,which might be attributable to the fact that the addition of BC could improve soil fertility and nutrient effectiveness,thereby increasing N sorption and consequently increasing plant yield (Shenet al.,2022).Furthermore,the addition of BC stabilized soil pH through liming effect,which facilitated the accumulation of plant biomass (Ahmadet al.,2014).

For the biomass accumulation ofC.intybusL.: this study showed that among the combined treatments of BC,AM and N,the shoot biomass of the twocomponent treatments (BC+AM,BC+N and AM+N)was greater than that of the single-component and the CK treatment;however,in terms of root growth,the root biomass after N treatment was lower than that following the application of the corresponding non-N treatments,BC+N treatment in particular.BC was a charcoal-rich product with multiple pores and a large specific surface area;thus,adding BC could reduce N loss by adsorbing excessive N and attenuating root growth in the early stages of growth (Shenet al.,2022).So,in this study,the shoot bioaccumulation was higher which indicated that BC promoted shoot growth.Furthermore,the results of the present study showed that the addition of BC significantly increased the colonization rate of AM fungi in all the treatments compared with those without BC application.This might be attributable to the better pore structure and nutrient substrate effectiveness of BC,which consequently provided a suitable environment for AM fungal growth,and this result was consistent with those reported by prior investigations (Lianget al.,2019;Yuet al.,2019).

For nutrient uptake ofC.intybusL.: results of this study showed that both single and multicomponent treatments increased the overall nutrient uptake(N,P,K,Mg,Ca,Mn and Fe) in the shoot and root systems ofC.intybusL.compared with the CK treatment.The addition of BC significantly increased P contents in the shoot and root systems following AM fungal inoculation.AM fungi infesting plants reportedly formed mycorrhizal symbiotic structures that could secrete extracellular phosphatase and solubilize insoluble P,which consequently increased host P levels (Gul and Whalen,2016).Furthermore,inoculation with AM fungi significantly increased the amount of N,K,Ca,Mg,Mn and Fe contents overall.However,it was observed that BC application also increased P content of the shoot and root system,which might be attributable to the fact that BC promoted organophosphorus mineralization (Zainulet al.,2020).BC also improved the accumulation of N,K,Mg and Mn inC.intybusL.to different degrees,indicating that AM fungi and BC had positive effects on nutrient uptake inC.intybusL.

For Cd concentration: in this study,inoculation with AM fungi significantly reduced Cd concentrations in the shoot and root system.Previous studies had shown that AM fungi could affect the uptake of Cd in plants uptake by mediating the expression levels of the plant Cd transporter protein and the abundance of the soil bacterial community (Chenet al.,2019).In Cd contaminated soil,both AM fungal hyphae and glomalin proteins could absorb or sequester soil Cd to reduce the transfer of Cd to the host (Suet al.,2020).In the present study,inoculation of AM fungi increased shoot and root biomass.Therefore,AM fungi might reduce Cd concentration inC.intybusL.through direct action or by indirectly increasing biomass.The addition of BC significantly reduced the concentration of Cd in the roots following the inoculation of AM fungi.Previous studies had shown that BC had excellent cation exchange properties and surface functional groups which could effectively precipitate Cd-P compounds (Parket al.,2019).In soils contaminated with heavy metals,BC could increase the soil pH;thus,the higher pH caused by BC could explain the reduction of Cd concentration in plants.Studies showed that BC could be combined with plant roots to promote the reduction of pollutants in the soil,thereby effectively controlling their migration and accumulation to crops (Wuet al.,2022).In addition,some researches indicated that dissolved organic matter might regulate the physiological process of plant uptake of cations,thereby reducing plant Cd concentration (Xuet al.,2022;Wuet al.,2022).The results were consistent with previous studies.

However,N application increased soil NH4+-N and NO3--N concentrations as well as Cd concentrations in shoot and root systems of uninoculated AM fungi.N significantly increased BCF of uninoculated AM fungi,with the highest BCF levels in BC+N treatment.Previous studies had shown that N application enhanced the bioaccumulation of Cd from soil to plants,such as wheat,rice and tobacco when the available amount of Cd in the soil was relatively high (Yanget al.,2019).However,BC reduced the available Cd concentration in the soil through sorption to reduce the uptake of Cd by plants (Zhanget al.,2020).AM fungi could reduce soil Cd concentration through mycelial action and secretions (Caoet al.,2021a;Huet al.,2014;Suet al.,2020).Therefore,it could be explained that BCF was significantly reduced in AM inoculation treatment,whereas BC+N treatments resulted in the highest amount of BCF.In this study,through the application of N fertilizer in the form of NH4Cl,NH4+could reduce pH of the soil and compete with Cd2+for soil sorption sites,which resulted in the presence of more Cd2+in the soil solution.This subsequently led to the enrichment of Cd in the soil and plants (Zhanget al.,2019);therefore,soil NH4+-N was positively correlated with BCF.

In addition,N application reduced soil pH,which had a significantly negative correlation with Cd concentration and content in the roots.This might be attributable to the fact that NH4+could release H+,thereby enhancing nitrification and lowering soil pH(Mitchell,2000).Soil pH was an important factor for plant uptake of Cd.When the soil pH was low,the available state of soil Cd increased sharply,therefore,plant uptake consequently increased (Yanget al.,2021).The sorption of Cd by plants increased with decreasing pH (Sajjadet al.,2020).The present study further demonstrated that N application reduced TF values and soil NH4+-N was negatively correlated with TF,indicating that NH4+-N inhibited the transfer of Cd from the root system to the shoot.This was consistent with the results of related studies in the past (Chenget al.,2018).This might be attributable to the fact that NH4+-N could reduce the expression levels ofOsHMA2 andOsHMA3,which encoded protein transporter genes responsible for the transfer of Cd ions from the root to the shoot.In the case of AM fungi inoculation and BC application,N fertilization reduced shoot and root Cd concentrations but increased shoot biomass.After N application,Cd content decreased with the plant biomass increasing;therefore,the decrease in Cd concentration and content could be attributed to the dilution effect of increasing biomass (Yanget al.,2019).

Conclusions

In conclusion,a single or multicomponent treatment of BC,AM and N could improveC.intybusL.growth and nutrient uptake and some soil properties;however,application of N alone could mediate soil acidification,thereby leading to an increased accumulation of Cd inC.intybusL.Therefore,in Cd contaminated soil,it was suggested that N fertilizers combined with AM fungi and BC could reduce the risk of cadmium contamination of crops.