Wheat straw biochar amendment suppresses tomato bacterial wilt caused by Ralstonia solanacearum:Potential effects of rhizosphere organic acids and amino acids

TIAN Ji-hui,RAO Shuang,GAO Yang,LU Yang,CAI Kun-zheng

1 Guangdong Provincial Key Laboratory of Eco-Circular Agriculture,South China Agricultural University,Guangzhou 510642,P.R.China

2 College of Natural Resources and Environment,South China Agricultural University,Guangzhou 510642,P.R.China

3 Key Laboratory of Tropical Agro-Environment,Ministry of Agriculture and Rural Affairs,South China Agricultural University,Guangzhou 510642,P.R.China

Abstract Complex interactions based on host plant,rhizosphere microorganisms and soil microenvironment are presumed to be responsible for the suppressive properties of biochar against soil-borne diseases,although the underlying mechanisms are not well understood.This study is designed to evaluate the efficacy of biochar amendment for controlling tomato bacterial wilt caused by Ralstonia solanacearum,and to explore the interactions between biochar-induced changes in rhizosphere compound composition,the pathogen and tomato growth.The results showed that biochar amendment decreased disease incidence by 61–78% and simultaneously improved plant growth.The positive ‘biochar effect’ could be associated with enhanced microbial activity and alterations in the rhizosphere organic acid and amino acid composition.Specifically,elevated rhizosphere citric acid and lysine,but reduced salicylic acid,were induced by biochar which improved microbial activity and rendered the rhizosphere unsuitable for the development of R.solanacearum.In addition,nutrients which were either made more available by the stimulated microbial activity or supplied by the biochar could improve plant vigor and potentially enhance tomato resistance to diseases.Our findings highlight that biochar’s ability to control tomato bacterial wilt could be associated with the alteration of the rhizosphere organic acid and amino acid composition,however,further research is required to verify these ‘biochar effects’ in field conditions.

Keywords:rhizosphere compounds,microbial activity,citric acid,lysine,salicylic acid

1.Introduction

Bacterial wilt caused byRalstonia solanacearumis one of the most destructive soil-borne plant diseases,and it is distributed worldwide (Yuliaret al.2015).The extensive host range ofR.solanacearumincludesover 200 species representing more than 40 plant families,including economically important crops such as tomato and potato(Hayward 1991).Conventional management strategies,such as resistant cultivars,crop rotation and soil solarization do not always succeed in controllingR.solanacearumdue to its abilities to survive in adverse soil conditions and infect various hosts (Hayward 1991;Yuliaret al.2015).Furthermore,the failure of host resistance against pathogens,the development of pathogen resistance to pesticides,and increasing risks of excessive pesticide application to the surrounding environment are among the forces driving the development of alternative methods for bacterial wilt management (Yuliaret al.2015).

Biochar is a carbon (C)-rich product of the pyrolysis of organic materials under low oxygen conditions.In addition to its potential for long-term sequestration of C,biochar amendment has been shown to improve soil physicochemical properties,reduce greenhouse gas emissions and enhance plant productivity (Zhanget al.2016).Recently,biochar has been proven to be effective at suppressing a wide range of soil-borne plant diseases in 22 pathosystems caused by 11 soil-borne pathogens (Graberet al.2014).For instance,rice straw biochar application reduced the incidence and severity of tobacco bacterial wilt caused byR.solanacearum,and that reduction was due to the improvement of soil physiochemical properties and an increase in bacterial diversity and richness (Zhanget al.2017).It was also reported that biochar application significantly suppresses tomato bacterial wilt directly,by its ability to adsorb pathogen,and indirectly,viaadsorption of root exudatesin vitro(Guet al.2017).Research has suggested various ways in which biochar could influence the progress of diseases caused by soil-borne pathogens,including:(1) induction of systematic plant defenses;(2)changes in the soil physical and chemical microenvironment;(3) enhanced abundance and/or activities of beneficial microbes;(4) direct inhibition of pathogen growth by toxic organic compounds;and (5) changes in the chemistry of rhizosphere exudates (Graber and Elad 2013;Graberet al.2014).The last of these mechanisms is the subject of this study.

The rhizosphere represents a highly dynamic front,in which biological and chemical processes are subjected to the influences of various compounds (Graberet al.2014).Low molecular weight compounds such as organic acids and amino acids,mainly derived from root exudates,plant litter and microbial metabolites,account for many of the major rhizosphere compounds (Jones 1998;Baiset al.2006).As the primary nutrients and energy sources for the general rhizosphere microorganisms,as well as the pathogens in the environment,these compounds are a major driving force in the regulation of rhizosphere microorganisms (Wuet al.2017).Therefore,the interactions between indigenous microorganisms,soil-borne pathogens and their host plants are mediatedviarhizosphere compounds to a substantial extent (Baiset al.2006;Graberet al.2014).For instance,R.solanacearumwas found to be specifically attracted to the diverse amino acids and organic acids present in root exudates of tomato (Yao and Allen 2006;Liet al.2017b).In contrast,certain compounds that cannot be utilized byR.solanacearum,e.g.,lysine and serine,were reported to suppress tomato bacterial wilt (Posas and Toyota 2010).Thus,altering the composition of the main rhizosphere low molecular weight compounds appears to be an alternative method for controlling soil-borne pathogens (Guet al.2017;Wuet al.2017).

Biochar is reportedly able to change the chemistry of plant root exudates (Graber and Elad 2013;Chenget al.2018),which may consequently influence the suitability of the rhizosphere for the development of pathogens.Akhteret al.(2015) demonstrated that green waste biochar and wood biochar altered tomato exudates differentially,and root exudates of plants grown with wood biochar amendment effectively suppressed the development ofFusarium oxysporumf.sp.lycopersici.However,the chemical differences in root exudates and their potential influences on pathogen growth and development are still unknown(Akhteret al.2015).To date,no comprehensive relationship has been established between biochar-induced changes in rhizosphere compound composition and the concomitant suppression of soil-borne pathogens and enhanced plant performance.Here we tested these possibilities experimentally in laboratory assays and a greenhouse experiment.The objective of this study was to evaluate the efficacy of biochar amendment for suppressing tomato bacterial wilt caused byR.solanacearum,and to identify the underlying mechanisms.We hypothesized that biochar soil amendment could alter the rhizosphere organic acid and amino acid composition,and that such changes might contribute to the potential suppression of bacterial wilt.

2.Materials and methods

2.1.Pot trial

The soil (Eutric Cambisol,FAO) was collected from a relatively unimpacted field under continuous cropping of tomato in Zhucun Village (113°42´E,23°17´N),Zengcheng District,Guangdong Province,China.The collected soil was air-dried and mixed to homogeneity before use.The selected properties of the soil are as follows:pH 4.9,organic C 9.5 g kg–1,total N 0.9 g kg–1,total P 1.3 g kg–1,total K 2.9 g kg–1,available N 115 mg kg–1,Olsen P 151 mg kg–1,and available K 83 mg kg–1.The biochar used in this study was a commercial wheat straw biochar produced by Sanli New Energy Company,Henan,China.This biochar was chosen because wheat straw is a valuable renewable resource that is easily accessible in large quantities in China(Biet al.2009),and preliminary experiments suggested better performance of wheat biochar than peanut biochar in bacterial wilt control (Luet al.2016).Pyrolysis of the wheat straw was performed at 500°C,and the basic properties of the resulting biochar were:pH 10.0,C 47.4%,N 1.0%,ash content 47.6%,Olsen P 325 mg kg–1and available silicon(Si) 2.4 g kg–1.

The tomato (Solanum lycopersicum,cv.Taiwan Red cherry,susceptible toR.solanacearum) seeds were surface sterilized for 30 min at 50°C,germinated on moist filter paper in Petri dishes for two days,then sown to a tray filled with nursery soil and incubated in a growth chamber with a 14 h light period (light intensity 200 µmol m–2s–1) at 30°C/25°C (day/night).After five weeks,two seedlings were transplanted to each pot (19 cm diameter and 16.5 cm height) containing 2 kg soil that was previously homogenized with or without biochar (2%,chosen based on a preliminary experiment examining the effects of different biochar rates (from 1 to 4%) on tomato bacterial wilt which revealed the best control at 2% (Lu 2015)),that was either pathogen-free or inoculated withR.solanacearumas described below.Thus,four treatments were included in the current experiment:non-inoculation ofR.solanacearumwithout biochar amendment (NI-CK),non-inoculation ofR.solanacearumwith 2% biochar amendment (NI-BC),inoculation ofR.solanacearumwithout biochar amendment(I-CK),and inoculation ofR.solanacearumwith 2% biochar amendment (I-BC).Each treatment was replicated five times,and each replicate consisted of a pot with two tomato plants (ten plants per treatment).The experiment was conducted in three separate trials with similar results,so only one representative experiment is presented here.

Culture,preparation and inoculation ofR.solanacearumwere performed according to Chenet al.(2015).Briefly,a highly virulentR.solanacearumstrain (physiological race 1,biovar 3,provided by College of Horticulture,South China Agricultural University,China) was cultured on 2,3,5-triphenyltetrazolium chloride (TTC) selective medium and incubated at 30°C for 48 h (Kelman 1954).The cell density was adjusted to 3×108CFU mL–1before inoculation.The roots of tomato were lightly stabbed and inoculated withR.solanacearumby pouring 10 mL of the bacterial suspension into each pot when the sixth leaf of the tomato seedlings appeared.The roots of non-inoculated tomato were also stabbed with 10 mL deionized water added as control.The disease index was recorded daily based on a scale of 0–9:0=no wilting,1=one leaf wilted,3=two or three leaves wilted,5=all wilted except the top leaves,7=all leaves wilted,and 9=stems collapsed or plants died (Chenet al.2015;Zhanget al.2017).The investigation began when the first symptoms arose and continued until the plants were all dead (21 d after transplanting).Disease incidence was calculated as described by Weiet al.(2011),where Disease incidence (%)=∑(Number of diseased plants in this index×Disease index)/(Total number of plants×Highest disease index)×100.

2.2.Sampling and analysis

At the end of the experiment,the roots of each tomato plant were dug up carefully,the loosely adhering soil was shaken off,and the tightly adhering soil masses were collected and combined into a single rhizosphere soil sample for each plot.Each soil sample was sieved (<2 mm) and divided into two parts.One part was stored at 4°C for assessment of soil microbial and biochemical properties within two weeks,while the other was air-dried for determination of soil chemical properties.

Soil microbial biomass C (Cmic) and N (Nmic) were determined after ethanol-free chloroform fumigation and extraction with 0.5 mol L–1K2SO4(1:4,w/v) (Joergensen 1996).Soil microbial biomass P (Pmic) was analyzed after ethanol-free chloroform fumigation and extraction with 0.5 mol L–1NaHCO3(Morelet al.1996).Soil basal respiration (mg CO2-C kg–1soil h–1) was determined by static incubation.The metabolic quotient,i.e.,qCO2,was calculated as soil basal respiration per unit of Cmic.Microbial quotient (MQ) refers to the ratio between Cmicand soil organic C (Corg) (Anderson and Domsch 1993).Soil and plant C and N were analyzed using a Vario MACRO Cube Analyzer (Elementar Analysensysteme Vario MACRO cube,Germany).As no inorganic C forms are present in the soils used for this study,a total C analysis was used to determine Corg(Nelson and Sommers 1996).Soil pH was determined in a 1:2.5 soil:water suspension.The concentration of Si in soil and shoot tissues was determined colorimetrically following digestion with 1 mol L–1HCl and 2.3 mol L–1HF (1:2) (Diogo and Wydra 2007).Each of the lab measurements described above was conducted with three technical replicates,and the average was then used for statistical analysis.

The plants were separated into shoot and root,with shoot biomass determined by drying plant materials at 72°C for 3 days.The roots were washed and scanned using a root scanner (Epson Expression 1680),and the root length,surface area,diameter and volume were analyzed using WinRHIZO.The population density ofR.solanacearumin tomato stem (Rs-stem) was determined according to Diogo and Wydra (2007).Briefly,mid-stem (5 cm section) pieces were surface sterilized with 70% alcohol for 20 s,macerated in sterile water and centrifuged for 10 min at 7 000 r min–1.Then,ten-fold serial dilutions of stem suspensions were prepared and 1 mL of each dilution was plated with three replicates on 20 mL TTC selective medium (Kelman 1954).Plates were incubated for 48 h at 30°C and the number of colonies counted.

2.3.Growth of R.solanacearum in rhizosphere extractions

Subsamples of each soil sample were extracted with 0.1%H3PO4(1:5,w/v,3 min,200 r min–1),centrifuged (5 min,5 000 r min–1) and membrane filtered (Millex HV 0.45 m,Millipore),and then 10 mL of the tomato rhizosphere extractions and 1 mL aliquot of the pre-incubatedR.solanacearum(3×108CFU mL–1) was inoculated into 150 mL of medium(Jinet al.2013;Bobilleet al.2016).The culture without rhizosphere extractions (0.1% H3PO4-the extracting reagent only and deionized water only) was performed as control,and each treatment was replicated five times.Cultures were then incubated at 30°C under 130 r min–1shaking,and growth rates were determined by measuring the optical density (OD) at 600 nm every 3 h (UV spectrophotometer,TU-1901,PERSEE,Beijing) (Wanget al.2013).

2.4.ldentification of organic acids and amino acids in rhizosphere extractions

Low molecular weight compounds present in the rhizosphere extractions prepared as described above were identified.The specific organic acids and amino acids reported to be involved in various rhizosphere processes (Jones 1998;Moe 2013),including citric acid,malic acid,succinic acid,fumaric acid,tartaric acid,salicylic acid,valine,threonine,lysine,histidine,arginine,methionine,phenylalanine and alanine,were selected as the standards.These standard compounds (purchased from Sigma,USA) and the rhizosphere extractions were analyzed by UPLC-MS/MS (Zevo-TQD,Waters,USA) sequentially and run under the same conditions.The standard compounds were chromatographed alone and as a mixture.Organic acid and amino acid compounds in the rhizosphere extractions were identified by comparing their retention times and peak areas with those of the standards.Specifically,an ACQUITY UPLC BEH C18 (1.7 µm,2.1 mm×50 mm) column was used.The mobile phase used for organic acid analysis consisted of (A)0.1% formic acid,and (B) acetonitrile with 0.1% formic acid in the following gradients:0 min,100% A;2.5 min,87.5%A+12.5% B;3–4 min,50% A+50% B;5–6 min,100% A,and the flow rate was 0.3 mL min–1.The mobile phase used for amino acid and salicylic acid analysis consisted of (A) 0.1%formic acid,and (B) methanol with 0.1% formic acid in the following gradients:0 min,95% A+5% B;1 min,90% A+10%B;1.5–4.5 min,30% A+70% B;5–6 min,95% A+5% B,and the flow rate was 0.2 mL min–1.The detailed mass spectra conditions are provided in Appendix A.

2.5.Statistical analysis

The effects of biochar amendment,R.solanacearumand their interactions were evaluated by two-way analysis of variance (ANOVA) with a Duncan test at theP=0.05 level.The correlation of soil and plant parameters was based on the Pearson correlation coefficients.Clustering analysis was conducted with the Squared Euclidean distance method to assess the interrelationships among all the variables and samples,and the data were standardized using the Z-score method before analysis.All statistical analyses mentioned above were conducted using SPSS 16.0 (SPSS,Chicago,IL,USA).Non-linear fitting of the pathogen growth curve was conducted with Origin 8.0 (OriginLab,Massachusetts,USA) using the DoseResp function.Redundancy analysis(RDA) was performed using CANOCO 4.5 to gain insights into the relationships of plant growth and disease development(response variables:Rs-stem,biomass,plant C,plant N,plant Si,Cmic,Nmic,Pmic,soil respiration,qCO2,MQ) with the composition of rhizosphere organic acids and amino acids(explanatory variables:citric acid,malic acid,succinic acid,fumaric acid,tartaric acid,salicylic acid,methionine,arginine,histidine,alanine,phenylalanine,valine,threonine,lysine) under biochar amendment and pathogen inoculation.The plant and microbial variables were standardized [Log transformation Y´=log (A×Y+B),A=1,B=1] before RDA.The explanatory variables that significantly (P<0.05) contributed to the explanation of variation in the response variables were selected into the model stepwise based on Monte Carlo Permutation Tests using 499 permutations.

3.Results

3.1.Biochar amendment suppressed tomato bacterial wilt and improved plant growth

To assess the efficacy of biochar amendment for controlling tomato bacterial wilt disease,we evaluated a time-resolved development of disease symptoms inR.solanacearuminfected tomato,either with or without the application of biochar (Appendix B).Tomato leaves grown in pots without biochar (I-CK) began to wilt at least four days earlier than those amended with biochar (I-BC,Fig.1-A).In comparison to the control without biochar (I-CK),the disease incidence was reduced by 61 to 78% by biochar amendment (I-BC)(Fig.1-A).Similarly,biochar amendment significantly decreased the population density ofR.solanacearumin tomato stems (Rs-stem) by as much as 43% at 12 days postR.solanacearuminoculation (Fig.1-B).

Fig.1 The disease incidence (A) and pathogen density (B) in tomato terms with or without biochar amendment that was either pathogen-free or inoculated with Ralstonia solanacearum. NI-CK,non-inoculation of R.solanacearum without biochar amendment;NI-BC,non-inoculation of R.solanacearum with 2% biochar amendment;I-CK,inoculation of R.solanacearum without biochar amendment;I-BC,inoculation of R.solanacearum with 2% biochar amendment.Data are mean and SE (n=10).＊＊＊indicates significance level at P<0.001.

Previous studies have shown that biochar amendment could improve plant growth,which may enhance the success of plant resistance to diseases (Graber and Elad 2013).Therefore,we evaluated the responses of tomato biomass and C content toR.solanacearuminoculation with and without biochar amendment (Table 1).Inoculation ofR.solanacearumresulted in significant decreases of shoot biomass and C content (Ivs.NI),regardless of the presence or absence of biochar.In contrast,biochar amendment (NI-BC and I-BC) significantly increased both shoot biomass (except NI-BC) and C content compared with the treatments without biochar (NI-CK and I-CK) (Table 1).On the other hand,enhanced plant nutrients could improve plant vigor,and hence influence the ability of pathogens to infect the plants (Graberet al.2014),so we investigated whether biochar amendment could improve tomato N and Si concentrations and potential disease control.The results demonstrated significant increases of both plant N and Si concentrations by biochar amendment in the absence of pathogen,whereas no such differences were evident when tomato was inoculated withR.solanacearum(Table 1).In addition,the root system is an important agronomic trait for plant growth and survival because of its role in resource acquisition (Badri and Vivanco 2009).The responses of root architecture to bothR.solanacearuminoculation and biochar amendment were thus investigated.There were significant decreases of root length,surface area and volume after inoculation withR.solanacearum.In contrast,biochar amendment significantly enhanced root surface area,diameter and volume of tomato inoculated withR.solanacearum(Table 1).

Table 1 Plant biomass,nutrient and root traits for tomato with or without biochar amendment that was either pathogen-free or inoculated with Ralstonia solanacearum1)

3.2.Pathogen inoculation and biochar amendment altered rhizosphere soil chemical and microbial properties

Previous studies have demonstrated that acidic conditions favored the outbreak of bacterial wilt caused byR.solanacearum(Liet al.2017a),whereas biochar,which is commonly alkaline,has been often reported to increase soil pH (Graberet al.2014).Hence,we investigated whether biochar amendment can increase soil pH,and potentially influence the rhizosphere environment forR.solanacearumdevelopment (Table 2).We found small,but significant,changes in soil pH (6.7vs.6.6) uponR.solanacearuminfection.In contrast,biochar amendment significantly increased soil pH.On the other hand,biochar amendment significantly increased soil Corg,total N and available Si contents,regardless of inoculation withR.solanacearum(Table 2).

The rhizosphere microorganisms,which are often influenced by biochar addition (Graberet al.2014),have been linked to soil nutrient turnover,plant growth promotion and soil-borne pathogen suppression in many ways (Jaiswalet al.2017).Therefore,changes in rhizosphere microbial biomass,activity and metabolic efficiency in response toR.solanacearuminoculation and biochar amendment were also investigated (Table 2).Inoculation ofR.solanacearumresulted in significant decreases of Cmic,Nmicand Pmicin rhizosphere soils.In the presence of pathogen,biochar amendment significantly increased rhizosphere Nmicand Pmicwithout influencing Cmic,resulting in a significant decrease of the Cmic-to-Corgratio,i.e.,the microbial quotient.In addition,Nmic,Pmic,soil respiration andqCO2were significantly enhanced by biochar amendment,either in the presence or absence of the pathogen (Table 2).

3.3.Rhizosphere extractions under biochar amendment suppressed pathogen growth

Rhizosphere compounds are widely known to be involved in the interactions between plants and pathogens (Akhteret al.2015;Wuet al.2015).To test the direct effect of compounds obtained from tomato rhizosphere on pathogen development in this system,the growth ofR.solanacearumin the rhizosphere extractions from different treatments was studied for a total of 40 h.Deionized water and 0.1% H3PO4(the extracting reagent) served as controls.The growth of pathogen in each treatment displayed a similar pattern,i.e.,a typical sigmoid curve.The growth ofR.solanacearumwas very slow in the initial stage (0–12 h),but was greatly accelerated at the rapid growth stage (12–33 h) and tended to stabilize thereafter (>33 h).Furthermore,compared with the control,the rhizosphere extractions from different treatments showed varying degrees of influence on the growth ofR.solanacearum(Fig.2).

Fig.2 A time-resolved growth curve of Ralstonia solanacearum incubated into rhizosphere extractions of tomato with or without biochar amendment that was either pathogen-free or inoculated with R.solanacearum.The culture without rhizosphere extractions (0.1% H3PO4 and deionized water only) was performed as control.NI-CK,non-inoculation of R.solanacearum without biochar amendment;NI-BC,noninoculation of R.solanacearum with 2% biochar amendment;I-CK,inoculation of R.solanacearum without biochar amendment;I-BC,inoculation of R.solanacearum with 2%biochar amendment.Data are mean and SE (n=10).＊,＊＊ and＊＊＊ indicates significance levels at P<0.05,P<0.01 and P<0.001,respectively.

The pathogen growth curve was well-fitted by a nonlinear fitting using the DoseResp function,and the maximum growth rate (hill slope,p) and theoretical maximum population density (top asymptote,A2) were also calculated(Table 3).There was no significant difference in the maximum growth rate ofR.solanacearumin rhizosphere extractions from different treatments.However,rhizosphere extractions without biochar amendment (NI-CK:2.652,I-CK:2.635) clearly produced a higher maximum population density in comparison with the control (H2O:2.573,0.1%H3PO4:2.472).In contrast,rhizosphere extractions with biochar amendment (NI-BC:2.400,I-BC:2.168) produced lower maximum population density in comparison with the control (Table 3).

3.4.Biochar amendment altered rhizosphere organic acid and amino acid compositions

Since the rhizosphere extractions from biochar amended plants can effectively suppress pathogen growthin vitro(see above),we hypothesized that biochar amendment could have changed certain rhizosphere compounds which may exert specific effects on pathogen development.To test this hypothesis,six organic acids and eight amino acids reported to be involved in various rhizosphere processes were identified and quantified in this study (Table 4).Wefound an order of magnitude difference in the concentrations of different organic acids and amino acids.Among them,citric acid was the most abundant organic acid in the tomato rhizosphere,followed by succinic acid,malic acid and fumaric acid,while salicylic acid had the lowest concentration.Lysine was the most abundant amino acid,followed by threonine and valine,while histidine had the lowest concentration (Table 4).

Table 2 Rhizosphere soil and microbial attributes for tomato with or without biochar amendment that was either pathogen-free or inoculated with Ralstonia solanacearum1)

Inoculation ofR.solanacearum,biochar amendment and their interaction showed significant effects on most of the detected organic acids and amino acids (except for fumaric acid,arginine,histidine and phenylalanine) (Table 4;Appendix C).Specifically,inoculation ofR.solanacearumresulted in significant decreases of citric acid (48%),malic acid (46%),tartaric acid (11%),valine (29%),threonine(27%),lysine (23%),arginine (20%),alanine (16%),phenylalanine (7%),and a five-fold increase of salicylic acid(Table 4;Appendix C).In the absence of pathogen,biochar amendment significantly increased fumaric acid (125%),methionine (178%) and lysine (19%),while it decreased malic acid (40%),tartaric acid (67%),alanine (34%),valine(27%) and threonine (33%).In the presence of pathogen,biochar amendment resulted in significant increases of citric acid (31%),fumaric acid (54%),lysine (27%),and methionine (170%);and decreases of succinic acid (29%),tartaric acid (68%),salicylic acid (58%),threonine (37%)and alanine (29%) (Table 4;Appendix C).

Table 3 Nonlinear fitting equation of Ralstonia solanacearum growth in rhizosphere extractions of different treatments

3.5.Linking the rhizosphere compound composition to microbial dynamics,pathogen suppression and growth promotion of tomato amended with biochar

Effective soil-borne disease control by biochar amendment may result from its varied influences on the rhizosphere soil microenvironment–microorganisms–pathogen–plant system (Graberet al.2014).To date,no comprehensive relationship has been established between biochar-induced changes in rhizosphere compound composition,microbial activity and the concomitant suppression ofR.solanacearumdevelopment and enhanced plant performance.Therefore,correlation analysis,clustering analysis and redundancy analysis were performed here to explore these relationships.

The correlation analysis revealed significant negative relationships between pathogen density in tomato stem and plant biomass,C and N contents,rhizosphere soil Nmic,Pmicand respiration.Plant biomass,C,N,and Si contents,as well as soil pH and Si content showed significant positive correlations with rhizosphere soil Nmic,Pmicand respiration.TheqCO2was also significantly positively correlated with plant C,N,and Si contents,as well as soil pH and Si content.Additionally,rhizosphere soil Nmicand Pmicwere significantly positively correlated with respiration andqCO2(Table 5).

Table 4 Organic acids and amino acids identified in tomato rhizosphere soils with or without biochar amendment that was eitherpathogen-free or inoculated with Ralstonia solanacearum1)

Clustering analysis grouped the plant and soil microbial parameters (response variables in redundancy analysis)into three groups.Group 1 only comprised Rs-stem,while group 2 comprised MBC and MQ,and group 3 consisted of plant biomass,plant C/N/Si,rhizosphere soil Nmic,Pmic,soil respiration andqCO2(Appendix D).Additionally,there was a clear separation of plant and soil microbial parameters in NI-BC from those in the other treatments (Appendix D).The rhizosphere organic compounds (explanatory variables in redundancy analysis) were also clustered into three groups.Group 1 only comprised salicylic acid,while group 2 comprised malic acid,valine,tartaric acid,alanine,threonine,succinic acid,histidine,and group 3 consisted of fumaric acid,methionine,arginine,lysine,citric acid and phenylalanine.Furthermore,there was a clear separation of rhizosphere compounds between treatments with (NI-BC and I-BC) and without (NI-CK and I-CK) biochar amendment(Appendix E).

Redundancy analysis revealed that the biochar treatment(CKvs.BC) was clearly separated by the first axis,while the inoculation treatment (NIvs.I) was separated by the second axis (Fig.3).The first and second axes explained 64.3 and 22.3% of the species–environment variation,respectively.Furthermore,rhizosphere citric acid (F=11.76,P=0.002),methionine (F=14.33,P=0.002),lysine (F=3.39,P=0.02) and salicylic acid (F=2.72,P=0.02) were the most important compounds which affected plant growth and disease development,while the other compounds did not affect them (P>0.05).Citric acid and lysine were significantly positively correlated with plant biomass,plant C,plant N,rhizosphere soil Nmic,Pmicand respiration,and negatively correlated with Rs-stem.Salicylic acid was significantly positively correlated with Rs-stem,but negatively correlated with plant biomass,plant C,plant N,rhizosphere soil Nmic,Pmicand respiration (Fig.3).

Fig.3 Redundancy analysis between plant and microbial attributes (species variables,solid lines) and rhizosphere organic compounds composition (environment variables,dashed lines) for different treatments.NI-CK,non-inoculation of R.solanacearum without biochar amendment;NI-BC,noninoculation of R.solanacearum with 2% biochar amendment;I-CK,inoculation of R.solanacearum without biochar amendment;I-BC,inoculation of R.solanacearum with 2%biochar amendment.Rs-stem,the population density of Ralstonia solanacearum in tomato stem;plant C,shoot C concentration;plant N,shoot N concentration;plant Si,shoot Si concentration;Cmic,soil microbial biomass C;Nmic,soil microbial biomass N;Pmic,soil microbial biomass P;qCO2,respiration per unit of Cmic;MQ,the ratio between Cmic and Corg.

4.Discussion

Biochar has been reported to suppress the diseases caused by several soil-borne pathogens,which may result from its direct and indirect impacts on the complex interactions among soil environment,host plant,pathogen and rhizosphere microbiome (Graberet al.2014).In the present study,we provided direct evidence that biochar soil amendment effectively suppressed tomato bacterial wilt caused byR.solanacearum.Here,enhanced plant nutrients and growth,increased microbial biomass/activity and changes of exudate composition in the rhizosphere of tomato were discussed in view of their potential roles in bacterial wilt control.

In this study,inoculation ofR.solanacearumresulted in a significant decrease of rhizosphere pH,which was consistent with previous studies that suggested a close correlation between soil acidification and the occurrence of bacterial wilt (Liet al.2017a;Zhanget al.2017).In contrast,biochar amendment significantly increased rhizosphere pH,which would,in turn,suppress the growth ofR.solanacearumby enhancing the activity of antagonistic microorganisms such asPseudomonas fluorescensandBacillus cereus(Liet al.2017a;Shenet al.2018).However,we found no correlation between soil pH and the population density ofR.solanacearumin this study (Table 5).One possible explanation for this discrepancy is that the influence of pH on the pathogen itself was insufficient to cause pathogen decrease in a short time period.In addition,many types of biochar,including the wheat straw biochar used here,contain inhibitory elements that can increase plant resistance to pathogen attack (Graberet al.2014).For instance,Si has been proven to improve plant resistance toR.solanacearum,and it can be associated with the formation of a cuticle-Si double layer in plant leaves that can impede pathogen penetration (Diogo and Wydra 2007;Chenet al.2015).In this study,rhizosphere available Si was significantly increased by biochar amendment,but there was no difference in the tissue Si concentration of inoculated tomato in response to biochar amendment (Tables 1 and 2).The lack of correlations between plant Si and the population density ofR.solanacearum/tomato biomass growth may suggest a non-significant role of the Si supply in bacterial wilt control in this study.Furthermore,the improvement of soil quality (as indicated by increased soil pH,available Si,Corgand TN) by biochar amendment could improve plant growth,and hence influence the susceptibility of the plants to the pathogen (Graberet al.2014).Additionally,root system development is critical to plant growth and survival due to its role in resource acquisition (Badri and Vivanco 2009).In this study,inoculation ofR.solanacearumsuppressed tomato root development by decreasing root length,surface area and volume (Table 1).Similar results have been reported previously,in which infection with a specificR.solanacearumstrain inhibited root elongation in petunia plants (Zolobowska and Van Gijsegem 2006).In contrast,biochar amendment significantly improved root surface area,diameter and volume,which may improve the root system’s efficiency in absorbing soil resources (water,nutrients,etc.) and consequently stimulate plant biomass growth (Xianget al.2017).

Table 5 Pearson’s correlation coefficients between pathogen population density,plant growth and rhizosphere soil properties for different treatments1)

Biochar has been found to alter soil microbial biomass,activity and community structure (Graberet al.2014),and such changes may well have effects on nutrient cycles and pathogen control,and thereby indirectly affect plant growth(Jaiswalet al.2017;Gaoet al.2019).General suppression of soil-borne diseases is often enhanced by stimulating soil microbial activity (Jaiswalet al.2017).This is supported by our study,where the rhizosphere soil respiration was significantly negatively correlated with the population density ofR.solanacearum(Table 5),and biochar application significantly increased soil respiration.As substrates for soil microorganisms,C or mineral nutrients released from biochar could contribute to increasing soil microbial activity,and thereby enhance the likelihood of competitive effects in the soil (Yuliaret al.2015).In addition,the enhancement of soil respiration as well as Corgby biochar amendment did not coincide with an increase in Cmic,resulting in a significant increase ofqCO2(soil basal respiration per unit of Cmic) but a decrease of MQ (the ratio between Cmicand Corg) (Table 2).In this study,theqCO2and MQ should be viewed as indicators of disturbance,rather than stress or deterioration of soil quality (Omirouet al.2013).The increase ofqCO2with a concurrent decrease of MQ under biochar amendment could be interpreted as positive priming on the decomposition of the labile pools of soil organic matter (Kuzyakovet al.2000).Although on the long-term biochar addition may retard the decomposition of soil organic matter,it is believed that the labile fraction in biochar may trigger increased microbial activity in the short term,and therefore induce a positive priming effect (Maestriniet al.2015).The nutrients which were made more available during this process,as well as nutrients supplied directly by biochar,could improve plant vigor,and thereby enhance the likelihood of resistance to diseases (Graberet al.2014).This mechanism was supported by the significant negative correlations between plant N and the population density ofR.solanacearumin this study.

Rhizosphere compounds,such as organic acids and amino acids,are widely known to be involved in the interactions between plants and microorganisms (Baiset al.2006).Altering the composition of rhizosphere compounds could influence the suitability of the rhizosphere for the development of the pathogen,and may thereby offer an alternative strategy for protecting plants from infection by pathogens (Akhteret al.2015;Wuet al.2017).Organic acids and amino acids are non-stable organic compounds in the rhizosphere,which are subject to influences by microbial degradation,re-uptake by roots and sorption processes caused by the soil solid phase and biochar amendment (Chenget al.2018;Taherymoosaviet al.2018).Therefore,the estimations of rhizosphere organic acids and amino acids given here are not absolute values,but should be seen as a snapshot of a dynamic system.We found that rhizosphere extractions from different treatments showed diverse effects on the growth and development ofR.solanacearum.Rhizosphere extractions from treatments without biochar amendment (NI-CK and I-CK) promoted the growth ofR.solanacearum,and this is supported by previous findings thatR.solanacearumcould be specifically attracted to several organic acids and amino acids present in root exudates of tomato (Yao and Allen 2006).It was also suggested that oxalic acid,malic acid and citric acid present in the root exudates of tobacco could increase the chemotaxis abilityin vitroand the recruitment ofR.solanacearumto tobacco root (Wuet al.2015).In contrast,we found that the rhizosphere extractions of tomato amended with biochar effectively suppressed the growth ofR.solanacearum,indicating biochar-induced qualitative or quantitative changes in rhizosphere compounds (Fig.2;Table 3).It has also been demonstrated that wood biochar amendment can influence the quality and composition of tomato root exudates tomato and suppress thein vitrogrowth and development ofF.oxysporum(Akhteret al.2015).Nonetheless,the chemical differences in root exudates induced by biochar amendment and their potential influences on pathogen growth and development are still unknown.

Since low molecular weight compounds such as organic acids and amino acids account for many of the compounds present in the plant rhizosphere (Baiset al.2006),the composition of organic acids and amino acids in the tomato rhizosphere was explored in this study.As expected,most of the detected organic acids and amino acids (except for arginine,histidine and phenylalanine)were dramatically altered by biochar amendment (Table 4).It has been estimated that up to 50% of the total fixed C produced from photosynthesis may be released into the rhizosphereviaroot excretion (Spokaset al.2011).In this study,we found a significant improvement of tomato growth by biochar amendment,which may consequently influence the quantitative and qualitative composition of rhizosphere organic compounds.This possibility is supported by our result that tomato biomass growth was significantly correlated with rhizosphere citric acid,salicylic acid,methionine,arginine,phenylalanine and lysine(Fig.3;Appendix F).Another possibility is that the biochar induced changes in rhizosphere microbial activity,as lowmolecular organic compounds in the rhizosphere have been shown to serve as an important source of energy and nutrients to microbes (Baiset al.2006).Furthermore,the alterations in rhizosphere organic acids and amino acids induced by biochar amendment were shown to be significantly correlated with soil microbial biomass and activity (Appendix F;Fig.3).Other explanations,including differential adsorption of certain rhizosphere compounds by biochar (Graber and Elad 2013),plant re-uptake and losses through leaching,may also possibly account for the observed differences (Taherymoosaviet al.2018).

Redundancy analysis further revealed that rhizosphere citric acid,lysine,methionine and salicylic acid are the most important parameters influencing the plant-pathogen interactions.Among them,citric acid,lysine and salicylic acid showed contrasting responses (increasing or decreasing)toR.solanacearuminoculation and biochar amendment,respectively.Citric acid and lysine were negatively correlated with Rs-stem,but positively correlated with rhizosphere microbial activity and tomato biomass,indicating that elevated citric acid and lysine in the tomato rhizosphere may support suppression of pathogens and promotion of tomato growth.Previous studies reported that citric acid was able to promote root colonization by antagonistic bacterial strain such asBacillus cereus,therefore,the competitive use of citric acid by a bacterial strain that is antagonistic withR.solanacearumcould decrease the pathogenic population density and effectively control tomato bacterial wilt (Wuet al.2017).Lysine soil amendment has also been proven to suppress tomato bacterial wilt by shifting the rhizosphere soil microbial community structure,leading to the more rapid death of the pathogen (Posas and Toyota 2010).Salicylic acid,one of the naturally occurring plant hormones,has been considered as an important signaling molecule involved in regulating plant defense responses against various pathogens (Bari and Jones 2009).Other hormone-related signaling pathways,such as jasmonic acid and ethylene,as well as plant antioxidant enzymes,such as phenylalanineammonia-lyase and lipoxygenase,are also widely known to play important roles in regulating plant defense responses against various pathogens (Bari and Jones 2009;Songet al.2015).Recent studies have suggested that the salicylic acid,jasmonic acid and ethylene signaling pathways are engaged synergistically in the defense response of tomato toR.solanacearum(Chenet al.2009).In this study,we observed a significant increase in rhizosphere salicylic acid when tomato was inoculated withR.solanacearum,and consequently a positive correlation between salicylic acid and Rs-stem (Table 2;Fig.3).These effects may result from enhanced root exudation or debris decay,as accumulation of large quantities of salicylic acid is only one among the various mechanisms which plants have developed in order to defend themselves against pathogens,includingR.solanacearum(Bari and Jones 2009;Lowe-Poweret al.2016).The decreased rhizosphere salicylic acid under biochar amendment may thus suggest a relatively lower disease stress condition under these circumstances.Recent results in our lab also demonstrated a down-regulation of the expression of salicylic acid pathway related genes in inoculated tomato when biochar was amended (unpublished data),suggesting that biocharinduced responses (as discussed above),rather than salicylic acid-mediated plant defense,may play a significant role in the control of bacterial wilt.Although exogenously applied salicylic acid has proven beneficial in controlling various plant diseases,some studies found no effects of salicylic acid on biological control against pepper damping off (Rajkumaret al.2008).In addition,the bacterial class ofRalstoniahas been demonstrated to be enriched in salicylic acid amended microcosms (Luoet al.2008).

On the other hand,changes of rhizosphere compound composition are expected to alter microbial community structure (Baiset al.2006),and such changes may contribute to the suppression of soil-borne diseases (Graberet al.2014).In our previous study,biochar amendment significantly increased the densities of bacteria and actinomycetes,while it decreased fungal densities in the tomato rhizosphere (Luet al.2016).Previous studies have identified correlations where higher bacterial densities and lower fungal densities were associated with increasing suppression of a series of soil-borne diseases (Bonanomiet al.2010;Posas and Toyota 2010),and actinomycetes are known to be strong producers of antibiotic compounds that directly influence disease suppression (Mazzolaet al.2001).Furthermore,recent data in this area revealed a clear alteration in rhizosphere bacterial community structures by biochar amendment,with significant stimulation of Bacteroidetes (Chitinophaga,for instance) that are known to be antibiotic producers (Gaoet al.2019).

5.Conclusion

Biochar amendment effectively suppressed tomato bacterial wilt caused byR.solanacearumand simultaneously improved plant growth.The ‘biochar effect’ here could be associated with alterations in rhizosphere compound composition and enhanced microbial activity.The elevated citric acid and lysine and reduced salicylic acid in tomato rhizosphere soils that were induced by biochar amendment may stimulate microbial activity and render the rhizosphere unsuitable for pathogen development.The nutrients which were made more available by the stimulated microbial activity or supplied by the biochar may improve plant vigor and resistance to diseases.Future research is needed to investigate the biochar induced plant defense to pathogens from the aspect of the secondary metabolism.

Ackonwledgements

This work was supported by the National Natural Science Foundation of China (31870420 and 41807084),the Natural Science Foundation of Guangdong Province,China(2017A030313177 and 2018A030310214),and the Science and Technology Project of Guangdong Province,China(2019B030301007).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendicesassociated with this paper are available on http://www.ChinaAgriSci.com/V2/En/appendix.htm