research article effects of biochar amendment on tomato...

Research ArticleEffects of Biochar Amendment on Tomato Bacterial WiltResistance and Soil Microbial Amount and Activity

Yang Lu1 Shuang Rao1 Fei Huang1 Yixia Cai1 Guoping Wang2 and Kunzheng Cai1

1Key Laboratory of Tropical Agro-Environment Ministry of Agriculture South China Agricultural UniversityGuangzhou 510642 China2College of Horticulture South China Agricultural University Guangzhou 510642 China

Correspondence should be addressed to Kunzheng Cai kzcaiscaueducn

Received 11 April 2016 Accepted 3 August 2016

Academic Editor Allen Barker

Copyright copy 2016 Yang Lu et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Bacterial wilt is a serious soilborne disease of Solanaceae crops which is caused by Ralstonia solanacearum The importantrole of biochar in enhancing disease resistance in plants has been verified however the underlying mechanism remains notfully understood In this study two different biochars made from peanut shell (BC1) and wheat straw (BC2) were added toRalstonia solanacearum-infected soil to explore the interrelation among biochar tomato bacterial wilt and soilmicrobial propertiesThe results showed that both BC1 and BC2 treatments significantly reduced the disease index of bacterial wilt by 286 and657 respectively The populations of R solanacearum in soil were also significantly decreased by biochar application Ralstoniasolanacearum infection significantly reduced the densities of soil bacteria and actinomycetes and increased the ratio of soilfungibacteria in the soil By contrast BC1 and BC2 addition to pathogen-infected soil significantly increased the densities ofsoil bacteria and actinomycetes but decreased the density of fungi and the ratios of soil fungibacteria and fungiactinomycetesBiochar treatments also increased soil neutral phosphatase and urease activity Furthermore higher metabolic capabilities ofmicroorganisms by biochar application were found at 96 and 144 h in Biolog EcoPlates These results suggest that both peanutand wheat biochar amendments were effective in inhibiting tomato bacterial wilt caused by R solanacearum The results suggest arelationship between the disease resistance of the plants and the changes in soil microbial population densities and activity

1 Introduction

Bacterial wilt is a serious soilborne disease caused by Ral-stonia solanacearum [1] This disease is difficult to controlbecause the pathogen can survive within a large temperaturerange (10∘C to 41∘C) and in diverse environments [2] Rsolanacearum can induce persistent latent infection in nurs-ery plants even at low populations in soil or irrigation water[3] Traditional control methods including host resistancecrop rotation and chemical methods may be limited or elicitnegative effects on food safety and environment [4] There-fore effective and eco-friendly approaches should be devel-oped to reduce this disease

Biochar a product of the thermal degradation of organicmaterials in the absence of air (pyrolysis) is distinguishedfrom charcoal in terms of usage In particular as a soil

amendment biochar can exhibit long-term carbon seques-tration potential and reduce greenhouse gas emission and insoil [5] Biochar can also improve soil tilth [6] and increasecrop productivity and competitive ability [7 8] Biocharapplication can also enhance crop response to disease [9] andthis enhancement can be attributed to an increase in soil pH[10] nutrient retention [6 11] cation exchange capacity insoil [11] transformations and turnover of P and S [12] andneutralization of phytotoxic compounds in soil [13]

It is reported that biochar can increase plant resistanceto disease For instance biochar can reduce fungal foliar dis-eases caused byBotrytis cinerea andOidiopsis sicula in tomato(Solanum lycopersicum L) and pepper (Capsicumannuum L)[14] Harel et al suggested that strawberry defense responsesmediated by biochars are functionally similar to inducedsystemic resistance [15] Moreover biochar can reduce

Hindawi Publishing CorporationInternational Journal of AgronomyVolume 2016 Article ID 2938282 10 pageshttpdxdoiorg10115520162938282

2 International Journal of Agronomy

Table 1 Basic properties of biochars used in the experiment

Type Feedstock Temperature (∘C) pH Ca () Na () Available P (mg kgminus1) Available K (mg kgminus1) Ashb () CN ratio ()BC1 Peanut shell 500 times 2 h 989 1741 060 6113 3872 7070 2902BC2 Wheat straw 500 times 2 h 1002 4738 098 3250 3820 4760 4835aMolar ratios analyzed with an elemental analysis apparatus vario TOC cube Elementar Analysensysteme GmbH GermanybMass ww analyzed by dry combustion in a muffle furnace at 600∘C for 2 h

soilborne diseases caused by bacteria and fungi [16] Neromeet al found that bacterial wilt (R solanacearum) in tomatowas reduced by adding biochar derived from municipalbiowaste [17] Suppressions of plant diseases by biocharwere attributed to several mechanisms [18ndash20] including thefollowing The chemical components of biochar may providedirect inhibition of pathogens and the porous structure ofbiochar may provide microbial habitats which is good forbacterial abundance Biochar can also promote plant growthby providing nutrients and improving nutrient solubilizationand uptake To some extent biochar sorption may changethe mobility and activity of pathogens or modify signalingbetween pathogens and plants [19]

Densities activity and diversity of soil microorganismscan be important indicators to evaluate soil health statusand soil quality [21 22] Pathogen infection may changesoil microbial properties thus soil can be converted froma highly fertile bacterial type to poorly fertile fungal type[23 24] Biochar amendments to soils may alter soil functionand fertility in various ways including through inducedchanges in the microbial community Kelly et al found asignificant decline in the fungibacteria ratio in the Coloradosoil with switchgrass biochar [25] Considering that biocharis implicated in soilborne disease reduction we hypothesizedthat disease resistance mediated by biochar may be relatedto changes in microbial properties This study aimed toinvestigate the interrelation among biochar bacterial wiltresistance and soil microbial components and activity

2 Materials and Methods

21 Plant Materials and Soil Conditions Tomato cv TaiwanRed cherry (tomato genotype produced by Kefeng Seed CoLtd Changchun Jilin China) which is susceptible to Rsolanacearum was used in this experiment Tomato seedswere stored in a fridge at 4∘C and steeped in water at roomtemperature for 2 h before use The seeds were surface-sterilized in water at 50∘C for 15min and then germinatedon moist filter paper in Petri dishes After 2 d the seeds weresown in nursery soil (tomato farm soil disinfected at 150∘Cfor 4 h) in a growth chamber with the following conditions30∘C25∘C (daynight) 14 h light and 200120583moLsdotmminus2sdotsminus1 lightintensity After five weeks tomato plants were transplantedto a polyethylene plastic pot (170mm in diameter 165mmin height) filled with 2 kg of soil each pot with 2 plants Theplants weremaintained at 28∘C in a controlled greenhouse for15 months until the end of the experiment

The soil collected from a continuous cropping cultivationtomato field (Zhucun Village Zengcheng City Guangdong

China) was sandy loam where the amount of bacterial wilt isrelatively high the proportions of sand silt and clay particlesat 0ndash20 cm soil layer were 73 22 and 5 respectivelyThe contents of soil organic matter soil-available N P andK and soil-total N P and Kwere 1630 g kgminus1 11547mg kgminus115110mg kgminus1 8254mg kgminus1 0948 g kgminus1 1347 g kgminus1 and292 g kgminus1 respectively Soil pH was 589 and soil CN ratiowas 982 The soil samples were sieved at lt2mm and storedat 4∘C until analysis to characterize relevant physical andbiochemical attributes of the soil Biochar was amended tosoil before transplantation of tomato plants

22 Biochar Two kinds of biochar (Sanli New EnergyResources Co Ltd Shangqiu Henan China) were used inthis experiment biochar made from peanut shell (BC1) andwheat straw (BC2) Biochar was prepared via pyrolysis in avertical kiln with a temperature at 500∘C and a resistancetime of about 2 h in an anaerobic condition (Sanli NewEnergy Resources Co Ltd) The basic properties of the twobiochar treatments are shown in Table 1 Scanning electronmicroscope images are shown in Figure 1

23 Experimental Design Four treatments were assessed inthis experiment no biochar and no R solanacearum inocu-lation (CK) R solanacearum inoculation (Rs) peanut shellbiochar (BC1) amendment and R solanacearum inoculation(BC1 + Rs) and wheat straw biochar (BC2) amendment andR solanacearum inoculation (BC2 + Rs) The experimentwas arranged in a completely randomized design with fourreplications each replication had one pot with 2 tomatoplants Our preliminary experiment showed that 2 ww ofthe two biochar amendments had the best effects in inhibitingbacterial wilt of tomato Thus 2 ww ratio was used in thisexperiment 2 ww ratio of peanut and wheat biochar wasused in this experiment 28 d after R solanacearum inocula-tion when the plants of R solanacearum treatment all diedsoil from all treatment groups was collected to determine theamount of R solanacearum in soil soil microbial populationdensities (bacteria fungi and actinomycetes) and soilmicro-bial activities (soil sucrase urease and neutral phosphatase)Biolog EcoPlate experiment was also conductedwhen the soilwas collected

24 R solanacearum Inoculation R solanacearum strainbiovar 3 (provided by College of Horticulture South ChinaAgricultural University Guangzhou 510642 China) wasused to inoculate tomato plants This strain is a highlyaggressive species The isolate was cultured on 235-triphenyltetrazolium chloride (TTC) medium and incubated

International Journal of Agronomy 3

(a) (b)

Figure 1 Scanning electron microscope images of two biochar used in this study (a) Biochar made by peanut shell (b) biochar made bywheat straw

at 30∘C for 48 h cell density was adjusted to 108 CFUsdotmLminus1before the isolate was inoculated As the sixth euphyllia ofthe tomato plant appeared the roots of each tomato plantwere lightly stabbed and inoculated with R solanacearum bypouring 10mL of the bacterial suspension into each pot CKplants were also stabbed but the same volume of deionizedwater was added After all of the plants of the Rs treatmentdied the plants were harvested and the soil was collected

25 Pathogen Evaluation Data collection was conducted atan interval of 2 d after pathogen infection by using a diseasescore [26] based on 10 plants per treatmentThe investigationbegan when the tomato leaf exhibited symptoms of wilt Thefollowing scoring method was used 0 no symptom 1 oneleaf wilted 3 two or three leaves wilted 5 all except thetop shooting leaves wilted 7 all leaves wilted and 9 stemscollapsed or plants died

Disease index was calculated using the following equa-tion disease index () = [sum(119903 times119873119903)(119877 times 119899)] times 100 where119903 is the rating value119873119903 is the number of infected leaves witha rating of 119903119877 is the value of themost serious disease severityand 119899 is the total number of tested plants [26]

26 Determination of the Amount of R solanacearum inSoil The amount of R solanacearum in soil was determinedaccording to the method described by Wang et al [27] withminor modification At 25 d after pathogen inoculation 10 gof fresh soil was collected added to flasks with 90mL ofsterile water and diluted to 10minus5 The soil-suspending liquidwas spread with TTC medium (bacterial general mediumwith 100 120583LsdotmLminus1 235-triphenyltetrazolium chloride) andthen incubated for 2 d at 30∘C in an incubator (GXZ Intel-ligent Jiangnan Instrument Plant) Plate culture count wasperformed to record the amount of R solanacearum in soil

27 Determination of Soil Microbial Population Densities Thepopulation densities of bacteria fungi and actinomyceteswere determined using the dilution method described byMartin [28] The media (Guangdong Huankai Microbial Sciamp Tech Co Ltd China) for cultivating bacteria fungiand actinomycetes were nutrient agar rose Bengal agar andgauzersquos medium number 1 respectively Fresh soil (10 g) was

added to a flask with 90mL of sterile water and shaken in ashaker (TaiCang Experimental Factory amp Suzhou Bing LabEquipment Co Ltd Suzhou China) for 30min at 150 rpmAfterward 100120583L of supernatant fluid from each sample wasextracted into a 2mL sterile centrifuge tube with 900 120583L ofsterile water Vortex Genius was used to mix the solution inthe centrifuge tube The solution was diluted to 10minus5 10minus3and 10minus5 for bacterial fungal and actinomycetes analysesrespectively The dilution was spread in the correspondingmedium and then placed in an incubator (GXZ IntelligentJiangnan Instrument Plant) at 30∘C Bacteria fungi andactinomycetes were cultured for 2 5 and 5 d respectivelyAfter these microorganisms were incubated the number ofcolonies was recorded to determine the densities of differentmicrobial populations

28 Determination of Soil Enzyme Activity Soil urease activ-ity was determined using the method described by Yaoand Huang [29] Air-dried finely sifted soil (5 g passedthrough a 2mm sieve) was placed in a 50mL conical flaskApproximately 1mL of toluene was added to the flask After15min 10mL of 10 urea solution and 20mL of citratebuffer (pH 67) were added The flasks were immediatelyshaken and placed in an incubator (GXZ Intelligent JiangnanInstrument Plant) at 37 plusmn 1∘C for 24 h The sample wasincubated and filtered using a filter paper Soil urease activitywas colorimetrically determined at 578 nm with a UVVisspectrophotometer (T90 UVVis spectrophotometer PGen-eral Beijing China) for 1 h

Considering that soil pHwas mostly neutral in this studywe determined soil neutral phosphatase enzyme activitysoil neutral phosphatase is the main phosphatase enzyme[30] Neutral phosphatase was determined using a previouslydescribed chemical method [27] similar to urease measure-ment Soil neutral phosphatase activity was colorimetricallydetermined at 660 nm with a UVVis spectrophotometer(T90 UVVis spectrophotometer PGeneral Beijing China)

Soil sucrase activity was tested according to the methoddescribed by Ling and Zhang [31] In brief 2 g of air-dried soil(sieved through lt1mm) 15mL of 8 glucose solution 5mLof 02M phosphate buffer (pH 55) and 1mL of toluene wereadded to a 50mL conical flask The solution was incubated


CK Rs BC1 + Rs BC2 + Rs

(a)

0

20

40

60

80

100

Dise

ase i

ndex

()

12 14 16 18 20 2210Inoculation time (day)

RsBC1 + Rs

BC2 + Rs

(b)

Figure 2 Suppressive effects of biochar amendment on R solanacearum of tomato plant (a) and effects of biochar amendment and Rsolanacearum inoculation on the disease index () of bacterial wilt in tomato plants (b) CK no biochar and no R solanacearum inoculationRs R solanacearum inoculation BC1 + Rs peanut biochar amendment and R solanacearum inoculation BC2 + Rs wheat biochar (BC2)amendment and R solanacearum inoculation

for 24 h at 37 plusmn 1∘C and filtered afterward 1mL of aliquotwas transferred to a test tube with 3mL of 35-dinitrosalicylicacid (DNS) solution The sample was heated in boiled waterfor 5min cooled in water for 3min and colorimetricallyquantified at 508 nm by using a spectrophotometer (T90UVVis spectrophotometer PGeneral Beijing China)

29 Microbial Community Analysis with Biolog EcoPlatesThe intensity and the diversity of bacterial metabolism wereevaluated using Biolog EcoPlates [32] Ten grams of freshsoil aliquots was added to 90mL of sterile saline solution(085 wv NaCl) and diluted to 10minus3 with the same solutionAfterward 150120583L of diluted solution was added to eachwell of Biolog EcoPlate (Biolog CA) and incubated at 30∘Cfor 168 h Absorbance at 590 nm was recorded with BiologMicroStation (Bio Tec Instruments Inc CA USA) at aninterval of 24 h data were analyzed using average well colordevelopment (AWCD)

Biolog data obtained after 168 h of incubation in AWCDwere subjected to Shannon diversity (1198671015840) analysis to deter-mine microbial functional diversity1198671015840 was calculated usingthe following equation [33]

1198671015840

= minus

31

sum

119894=1

(119875119894times ln119875

119894)

119875119894=

(119862119894minus 119877)

sum31

119894=1

(119862119894minus 119877)

(1)

where119862119894is the color production within each well and119877 is the

absorbance value of the platersquos control well

210 Soil pH Analysis As the soil was acidic soil fromsouthern part of China soil pH was determined with twodifferent kinds of solutions (1M KCl for CK and Rs treat-ments aqueous solution for BC1 and BC2 treatments) Theratio of soil and solution was 1 25 Briefly 10 g aliquots of

air-dry soil from the four treatments were each added to25mL solution The mix solution was shaken for 30min at150 rpm and then stood for 1 h Subsequently soil pH wasmeasured respectively by PHS-3C PHMeter (Shanghai REXInstrument Factory Shanghai China)

211 Statistical Analysis Data in the figures were expressedas mean plusmn standard error of four replicates and analyzed byone-way ANOVA in SPSS170 (Statistical Analysis SystemsInstitute SPSS Inc Chicago IL USA) Statistical differencesamong treatments were determined by Duncanrsquos test (119875 lt005) Graphs were constructed using SigmaPlot 125 (SystatSoftware Inc San Jose CA USA)

3 Results

31 Effects of Biochar Application on Disease Severity Thesymptoms of bacterial wilt in R solanacearum-inoculatedtreatment were observed at 4 d postinoculation (dpi) By con-trast these symptoms in biochar-amended treatment wereobserved only at 10 dpi indicating that biochar amendmentdelayed pathogen development Both biochar treatmentssignificantly suppressed disease development and increaseddisease resistance of tomato plants (Figure 2) Comparedwith Rs treatment BC1 + Rs treatment reduced the diseaseindex of bacterial wilt by 6000 3061 3226 and 3056at 10 14 20 and 24 dpi respectively BC2 + Rs treatmentreduced the disease index by 6000 7959 6774 and6667 respectively These results showed that BC2 wascould effectively reduce bacterial wilt to a greater extent thanBC1

32 Effects of Biochar Application on the Density of Rsolanacearum in Soil Compared with CK treatment thedensity ofR solanacearumwasmarkedly increased by 8043after pathogen inoculation However biochar treatmentssignificantly decreased the density of R solanacearum by


A

B BC

C

0

2

4

6

8

10

12

14

The d

ensit

y of

R so

lana

cear

umin

soiltimes107

(CFU

middotgminus1)


Figure 3 Effects of biochar amendment and R solanacearum inoc-ulation on R solanacearum density in soil CK no biochar and noR solanacearum inoculation Rs R solanacearum inoculation BC1+ Rs peanut biochar amendment and R solanacearum inoculationBC2 + Rs wheat biochar (BC2) amendment and R solanacearuminoculation Different letters on the columns denote a significantdifference at 119875 lt 005 using the DMRT (Duncanrsquos new multiplerange tests) method

5163 (BC1) and 6822 (BC2) compared with Rs treatmentalone (Figure 3)

33 Effects of Biochar Application on Soil Microbial Popu-lation Densities Biochar amendment and R solanacearuminoculation significantly influenced the density of soil micro-bial population (Figure 4) Compared with CK treatmentR solanacearum inoculation significantly decreased theamounts of soil bacteria and actinomycetes by 5755 and2645 respectively By contrast R solanacearum inocula-tion increased the amount of fungi by 27258 Howeverbiochar in BC1 + Rs and BC2 + Rs treatments significantlyincreased soil bacteria by 5739 and 9642 and soil acti-nomycetes by 4247 and 6333 and reduced soil fungi by6587 and 2973 respectively

R solanacearum inoculation significantly increasedsoil fungiactinomycetes ratio and fungibacteria ratio by41844 and 73514 respectively However these ratioswere decreased by 7681 and 7857 in BC1 + Rs and by5676 and 6334 in BC2 + Rs treatments respectivelycompared with Rs treatment (Figure 5)

34 Effects of Biochar Application on Soil Enzyme ActivitySoil neutral phosphatase and urease activities were signif-icantly affected by R solanacearum inoculation (Figure 6)Compared with CK treatment R solanacearum infectionreduced soil neutral phosphatase activity by 8339 and soilurease activity by 452 Biochar amendments increased soilenzyme activities In particular BC1 + Rs and BC2 + Rstreatments increased soil neutral phosphatase by 45870and 32915 respectively (Figure 6(a)) likewise these treat-ments increased soil urease activity by 1536 and 1408respectively (Figure 6(b)) R solanacearum inoculation didnot significantly affect soil sucrase activity however biochar

amendments decreased soil sucrase activity (Figure 6(c)) by2994 (BC1 + Rs) and 2797 (BC2 + Rs)

35 Effects of Biochar Application on Physiological Profiles ata Microbial Community Level Biolog assay was originallydeveloped to identify microbial isolates based on substrateutilization profiles This assay is commonly performed toobtain substrate utilization profiles at a community level [32]Color intensity was determined by calculating the AWCD ofeach plateTheAWCDvalues of different treatments at differ-ent stages are shown in Figure 7 Biochar treatments yieldedhigher AWCD than nonbiochar treatments especially after72 h this result indicated that the testedmetabolic capabilitiesof biochar treatments were higher than those of nonbiochartreatments The physiological profiles of biochar treatmentssignificantly differed at specific representative stages (96 144and 168 h after incubation)

We selected 144 h after cultivation as a specific timeto identify differences in AWCD value of each treatmentbecause 144 h is the logarithmic period of soil microbes Theresults showed that Rs treatment decreased the AWCD valueby 329 compared with CK treatment by contrast biochartreatments significantly increased AWCD by 1521 (BC1 +Rs treatment) and 1553 (BC2 + Rs treatment) comparedwith Rs treatment

36 Effects of Biochar Application on Soil pH Soil pH wassignificantly increased after biochar amendment regardlessof pathogen inoculation (Figure 8) Pathogen inoculationdecreased soil pH by 316 compared with CK treatmentsBiochar amendment treatments increased soil pH by 2753(BC1) and 2267 (BC2) compared with Rs treatmentrespectively The soil was increased to around neutral pH bybiochar amendment (Figure 8)

4 Discussion

The beneficial role of biochar inducing plant diseases resis-tance has been investigated in several foliar and soilbornepathosystems [14 15 34] In the present study 2ww peanutand wheat biochar amendment delayed R solanacearumdevelopment significantly reduced the severity of diseaseincidence and increased tomato plant resistanceThe densityof R solanacearum in soil was also significantly decreased bybiochar application And also due to biochar amendment soilpH got back to the neutral level which is good for bacteriagrowth

The soil microbial population structure is critical to soilfunction and ecosystem services which affect soil structureand stability nutrient cycling aeration water use efficiencydisease resistance and C storage capacity [35] In previousreports the increase in bacterial densities is associated withthe enhanced resistance of amended soils against southernblight of processing tomatoesPhytophthora root rot of alfalfaand potato scab [36 37] Beneficial microbiota can competewith pathogens for space and nutrients or produce microbialagents thereby improving plant health [38] Our resultsshowed that the density of soil bacteria and actinomycetes


A

B

AB

C

0

2

4

6

8

10

The d

ensit

y of

bac

teria

in so

iltimes107

(CFU

middotgminus1)


(a)

A A

A

B

CK Rs BC1 + Rs BC2 + Rs0

2

4

6

8

10

12

The d

ensit

y of

actin

omyc

etes

in so

iltimes107

(CFU

middotgminus1)

(b)

A

B

C

C


2

4

6

8

10

The d

ensit

y of

fung

i in

soiltimes105

(CFU

middotgminus1)

(c)

Figure 4 Effects of biochar amendment and R solanacearum on the amount of soil bacteria (a) actinomycetes (b) and fungi (c) CK nobiochar and no R solanacearum inoculation Rs R solanacearum inoculation BC1 + Rs peanut biochar amendment and R solanacearuminoculation BC2 + Rs wheat biochar (BC2) amendment and R solanacearum inoculation Different letters on the columns denote asignificant difference at 119875 lt 005 using the DMRT (Duncanrsquos new multiple range tests) method

was significantly decreased after R solanacearum was inocu-lated by contrast the amount of soil fungi increased Thussoil was converted from ldquobacterial typerdquo to ldquofungal typerdquoand this result is similar to that of Larkin [23] Interestinglybiochar addition to R solanacearum-infected soil couldincrease the amount of soil bacteria and actinomycetes con-versely biochar addition could decrease the amount of soilfungi soil fungibacteria ratio and fungiactinomycetes ratioto reverse the change in soil microorganism composition thatresulted from pathogen infection And this finding is similarto that in a previous study in which silicon supply resulted

in the change of soil microbial components under pathogeninoculation [27]

Our results also showed that higher metabolic capabili-ties were found in the two biochar amendment treatmentsat 96 and 144 h (two sensitive stages during cultivation)indicating that biochar addition resulted in high substrateutilization capability of microorganism What is more agood relationship is observed between soil fertility and soilmicroorganisms In plants soil microbes interact to mediateand influence various exchanges that contribute to plantgrowth and productivity [21] The extent of these effects


Fungiactinomycetes

soil

mic

roor

gani

sms (

Fungibacteria

0

2

4

6

8

10

12

14

The p

ropo

rtio

n of

the a

mou

nt o

ftimes10minus2)


Figure 5 Effects of biochar amendment andR solanacearum on the ratio of soilmicroorganism fungiactinomycetes and fungibacteria CKno biochar and no R solanacearum inoculation Rs R solanacearum inoculation BC1 + Rs peanut biochar amendment and R solanacearuminoculation BC2 + Rs wheat biochar (BC2) amendment and R solanacearum inoculation

000

100

200

300

400

500

600

700

800

Neu

tral

pho

spha

tase

activ

ity (120583

gmiddotgminus1)


A

B

CC

(a)CK Rs BC1 + Rs BC2 + Rs

ABC C

000

005

010

015

020

025

030U

reas

e act

ivity

(mgmiddotgminus1)

(b)


A A

B B

000

200

400

600

800

1000

1200

1400

Sucr

ase a

ctiv

ity (m

gmiddotgminus1)

(c)

Figure 6 Effects of biochar amendment and R solanacearum inoculation on the activity of soil neutral phosphatase (a) urease (b) andsucrase (c) CK no biochar and no R solanacearum inoculation Rs R solanacearum inoculation BC1 + Rs peanut biochar amendment andR solanacearum inoculation BC2 + Rs wheat biochar (BC2) amendment and R solanacearum inoculation Different letters on the columnsdenote a significant difference at 119875 lt 005 using the DMRT (Duncanrsquos new multiple range tests) method


00

05

10

15

20

25

30

AWCD

48 72 96 120 144 16824Incubation time (h)

CKRs

BC1 + Rs

BC2 + Rs

Figure 7 Effects of biochar amendment and R solanacearumon soil microorganism community-level substrate utilization pro-files CK no biochar and no R solanacearum inoculation Rs Rsolanacearum inoculation BC1 + Rs peanut biochar amendmentand R solanacearum inoculation BC2 + Rs wheat biochar (BC2)amendment and R solanacearum inoculation

CD

AB

50

55

60

65

70

75

80

Soil

pH


Figure 8 Effects of treatment on soil pH CK no biochar and noR solanacearum inoculation Rs R solanacearum inoculation BC1+ Rs peanut biochar amendment and R solanacearum inoculationBC2 + Rs wheat biochar (BC2) amendment and R solanacearuminoculation Different letters on the columns denote a significantdifference at 119875 lt 005 using the DMRT (Duncanrsquos new multiplerange tests) method

likely depends on biochar production conditions and feed-stock which control macrostructures and microstructuresof biochar particles [12] Brussaard et al suggested thatorganic amendments are possibly among the most importantstrategies of soil biodiversity management [39]

Soil enzymes are directmediators of biological catabolismof soil organic and mineral components As an integral partof nutrient cycling in soil soil-specific enzyme activitiesincluding dehydrogenase and phosphatase activities can beused to estimate soil microbial activity and evaluate soil

health [40] Soil microbial activity is implicated in quantify-ing soil function such as C and N cycles and organic matterdecomposition [41 42] Some enzymes (eg hydrolase andglucosidase) facilitate the breakdown of organicmatter otherenzymes (eg amidase urease phosphatase and sulfatase)are involved in nutrient mineralization Urease phosphataseand arylsulphatase are important in the mineralization ofnitrogen phosphorous and sulfur compounds [40] More-over soil enzymes may exhibit a strong relationship withdisease suppression Some enzymes (eg chitinases andorglucanases) may reinforce plant resistance to pathogensby breaking down polysaccharides chitin and 120573-glucansresponsible for the rigidity of fungal cell walls therebydestroying cell wall integrity [43] Baek et al found that chiti-nase enzyme activity is positively correlated with suppressionability of Rhizoctonia solani-incited cotton seedling disease[44] Woo et al reported that biocontrol activity against Bcinerea on bean leaves was reduced when chitinase activityis disrupted [45] In this study R solanacearum infectionsignificantly reduced soil neutral phosphatase and ureaseactivities by contrast biochar amendment increased enzymeactivities and maintained a relatively higher soil microbialactivityThe activity of microorganisms related to soil neutralphosphatase and urease was enhanced

In conclusion our study indicates that both peanutand wheat biochar applications significantly reduced theseverity of bacterial wilt caused by R solanacearum Biocharamendment could increase the population densities of soilbacteria and actinomycetes modify soil fungibacteria andfungiactinomycetes ratio increase soil microbial activityand suppress R solanacearum distribution to establish ahealthy soil environment The pathogen resistance of tomatoafter biochar amendment is closely related to the changesin soil microbial activity and community structure Fur-ther studies could be conducted by molecular microbiol-ogy and sequencing technologies to identify specific soilmicrobial group or species with antagonistic roles against Rsolanacearum

Abbreviations

TTC 235-Triphenyltetrazolium chlorideRs R solanacearum

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

This study was financially supported by grants fromthe National Key Basic Research Funds of China(2011CB100400) the National Natural Science Foundation ofChina (31370456) Doctoral Foundation of the Ministryof Education of China (20124404110007) and theNatural Science Foundation of Guangdong Province(S2012010010331) They would like to thank ProfessorShiming Luo at South China Agricultural University China


for reviewing this paper and giving useful suggestions andcomments

References

[1] E Yabuuchi Y Kosako I Yano H Hotta and Y Nishi-uchi ldquoTransfer of two Burkholderia and an Alcaligenes speciesto Ralstonia gen nov proposal of Ralstonia pickettii (Ral-ston Palleroni and Doudoroff 1973) comb nov Ralstoniasolanacearum (Smith 1896) comb nov and Ralstonia eutropha(Davis 1969) comb novrdquoMicrobiology and Immunology vol 39no 11 pp 897ndash904 1995

[2] JMuthoni H Shimelis andRMelis ldquoManagement of bacterialwilt (Rhalstonia solanacearumYabuuchi et al 1995) of potatoesopportunity for host resistance in Kenyardquo The Journal ofAgricultural Science vol 4 no 9 pp 64ndash78 2012

[3] R Kubota B G Vine AM Alvarez andDM Jenkins ldquoDetec-tion of Ralstonia solanacearum by loop-mediated isothermalamplificationrdquo Phytopathology vol 98 no 9 pp 1045ndash10512008

[4] N A S Messiha A D Van Diepeningen M Wenneker et alldquoBiological Soil Disinfestation (BSD) a new control methodfor potato brown rot caused by Ralstonia solanacearum race 3biovar 2rdquo European Journal of Plant Pathology vol 117 no 4 pp403ndash415 2007

[5] A R Zimmerman ldquoAbiotic and microbial oxidation of lab-oratory-produced black carbon (biochar)rdquo Environmental Sci-ence and Technology vol 44 no 4 pp 1295ndash1301 2010

[6] K Y Chan L Van Zwieten I Meszaros A Downie andS Joseph ldquoAgronomic values of greenwaste biochar as a soilamendmentrdquo Soil Research vol 45 no 8 pp 629ndash634 2007

[7] E R Graber Y M Harel M Kolton et al ldquoBiochar impact ondevelopment and productivity of pepper and tomato grown infertigated soilless mediardquo Plant and Soil vol 337 no 1 pp 481ndash496 2010

[8] S Jeffery F G A Verheijen M van der Velde and A C BastosldquoA quantitative review of the effects of biochar application tosoils on crop productivity using meta-analysisrdquo AgricultureEcosystems and Environment vol 144 no 1 pp 175ndash187 2011

[9] Y Elad E Cytryn Y M Harel B Lew and E R Graber ldquoThebiochar effect plant resistance to biotic stressesrdquo Phytopatholo-gia Mediterranea vol 50 no 3 pp 335ndash349 2011

[10] J M Novak W J Busscher D L Laird M Ahmedna D WWatts and M A S Niandou ldquoImpact of biochar amendmenton fertility of a southeastern coastal plain soilrdquo Soil Science vol174 no 2 pp 105ndash112 2009

[11] C SteinerWG Teixeira J Lehmann et al ldquoLong term effects ofmanure charcoal and mineral fertilization on crop productionand fertility on a highly weathered Central Amazonian uplandsoilrdquo Plant and Soil vol 291 no 1-2 pp 275ndash290 2007

[12] J Lehmann S Joseph F Casanoves et al Biochar for Environ-mental Management Science and Technology 2010

[13] D Wardle O Zackrisson and M Nilsson ldquoThe charcoal effectin Boreal forests mechanisms and ecological consequencesrdquoOecologia vol 115 no 3 pp 419ndash426 1998

[14] Y Elad D R David Y M Harel et al ldquoInduction of systemicresistance in plants by biochar a soil-applied carbon sequester-ing agentrdquo Phytopathology vol 100 no 9 pp 913ndash921 2010

[15] Y M Harel Y Elad D Rav-David et al ldquoBiochar mediatessystemic response of strawberry to foliar fungal pathogensrdquoPlant and Soil vol 357 no 1 pp 245ndash257 2012

[16] A K Jaiswal Y Elad E R Graber andO Frenkel ldquoRhizoctoniasolani suppression and plant growth promotion in cucumber asaffected by biochar pyrolysis temperature feedstock and con-centrationrdquo Soil Biology and Biochemistry vol 69 pp 110ndash1182014

[17] M Nerome K Toyota T Islam et al ldquoSuppression of bacterialwilt of tomato by incorporation of municipal biowaste charcoalinto soilrdquo Soil Microorganisms vol 59 pp 9ndash14 2005

[18] H A Hoitink and P C Fahy ldquoBasis for the control ofsoilborne plant pathogens with compostsrdquo Annual Review ofPhytopathology vol 24 no 1 pp 93ndash114 1986

[19] J Lehmann M C Rillig J Thies C A Masiello W CHockaday and D Crowley ldquoBiochar effects on soil biotamdashareviewrdquo Soil Biology and Biochemistry vol 43 no 9 pp 1812ndash1836 2011

[20] R Noble and E Coventry ldquoSuppression of soil-borne plantdiseases with composts a reviewrdquo Biocontrol Science and Tech-nology vol 15 no 1 pp 3ndash20 2005

[21] J M Chaparro A M Sheflin D K Manter and J M VivancoldquoManipulating the soil microbiome to increase soil health andplant fertilityrdquo Biology and Fertility of Soils vol 48 no 5 pp489ndash499 2012

[22] C Pankhurst B Hawke H McDonald et al ldquoEvaluation of soilbiological properties as potential bioindicators of soil healthrdquoAustralian Journal of Experimental Agriculture vol 35 no 7 pp1015ndash1028 1995

[23] R P Larkin ldquoCharacterization of soil microbial communitiesunder different potato cropping systems by microbial popula-tion dynamics substrate utilization and fatty acid profilesrdquo SoilBiology and Biochemistry vol 35 no 11 pp 1451ndash1466 2003

[24] M C C Leon A Stone and R P Dick ldquoOrganic soil amend-ments impacts on snap bean common root rot (Aphanomyeseuteiches) and soil qualityrdquo Applied Soil Ecology vol 31 no 3pp 199ndash210 2006

[25] C N Kelly F C Calderon V Acosta-Martınez et al ldquoSwitch-grass biochar effects on plant biomass and microbial dynamicsin two soils from different regionsrdquo Pedosphere vol 25 no 3pp 329ndash342 2015

[26] Z Fang Research Methods of Plant Pathology China Agricul-ture Press Beijing China 1998

[27] L Wang K Cai Y Chen and G Wang ldquoSilicon-mediatedtomato resistance against Ralstonia solanacearum is associatedwith modification of soil microbial community structure andactivityrdquo Biological Trace Element Research vol 152 no 2 pp275ndash283 2013

[28] J P Martin ldquoUse of acid rose bengal and streptomycin in theplate method for estimating soil fungirdquo Soil Science vol 69 no3 pp 215ndash232 1950

[29] H Yao and C Huang Soil Microbial Ecology and Its Experimen-tal Technique Science Press Beijing China 2006

[30] F Eivazi and M A Tabatabai ldquoPhosphatases in soilsrdquo SoilBiology and Biochemistry vol 9 no 3 pp 167ndash172 1977

[31] L Ling and W-X Zhang ldquoSequestration of arsenate in zero-valent iron nanoparticles visualization of intraparticle reac-tions at angstrom resolutionrdquo Environmental Science amp Technol-ogy Letters vol 1 no 7 pp 305ndash309 2014

[32] J L Garland and A L Mills ldquoClassification and character-ization of heterotrophic microbial communities on the basisof patterns of community-level sole-carbon-source utilizationrdquoApplied and EnvironmentalMicrobiology vol 57 no 8 pp 2351ndash2359 1991


[33] H Zheng Z Y Ouyang X KWang Z G Fang T Q Zhao andH Miao ldquoEffects of regenerating forest cover on soil microbialcommunities a case study in hilly red soil region SouthernChinardquo Forest Ecology and Management vol 217 no 2-3 pp244ndash254 2005

[34] W H Elmer and J J Pignatello ldquoEffect of biochar amendmentson mycorrhizal associations and Fusarium crown and root rotof asparagus in replant soilsrdquo Plant Disease vol 95 no 8 pp960ndash966 2011

[35] L Brussaard V M Behan-Pelletier D E Bignell et al ldquoBiodi-versity and ecosystem functioning in soilrdquoAMBIO A Journal ofthe Human Environment vol 26 no 8 pp 563ndash570 1997

[36] L R Bulluck III and J B Ristaino ldquoEffect of synthetic andorganic soil fertility amendments on southern blight soilmicrobial communities and yield of processing tomatoesrdquoPhytopathology vol 92 no 2 pp 181ndash189 2002

[37] B E Wiggins and L L Kinkel ldquoGreen manures and cropsequences influence alfalfa root rot and pathogen inhibitoryactivity among soil-borne streptomycetesrdquo Plant and Soil vol268 no 1 pp 271ndash283 2005

[38] S E Lindow and J H J Leveau ldquoPhyllosphere microbiologyrdquoCurrent Opinion in Biotechnology vol 13 no 3 pp 238ndash2432002

[39] L Brussaard P CDeRuiter andGG Brown ldquoSoil biodiversityfor agricultural sustainabilityrdquo Agriculture Ecosystems amp Envi-ronment vol 121 no 3 pp 233ndash244 2007

[40] L Gianfreda M A Rao A Piotrowska G Palumbo and CColombo ldquoSoil enzyme activities as affected by anthropogenicalterations intensive agricultural practices and organic pollu-tionrdquo Science of the Total Environment vol 341 no 1ndash3 pp 265ndash279 2005

[41] J H J R Makoi and P A Ndakidemi ldquoSelected soil enzymesexamples of their potential roles in the ecosystemrdquoAfrican Jour-nal of Biotechnology vol 7 no 3 pp 181ndash191 2008

[42] R Pavel J Doyle and Y Steinberger ldquoSeasonal patterns of cel-lulase concentration in desert soilrdquo Soil Biology amp Biochemistryvol 36 no 3 pp 549ndash554 2004

[43] C R Howell ldquoMechanisms employed by Trichoderma speciesin the biological control of plant diseases the history andevolution of current conceptsrdquo Plant Disease vol 87 no 1 pp4ndash10 2003

[44] J-M Baek C R Howell and C M Kenerley ldquoThe role of anextracellular chitinase from Trichoderma virens Gv29-8 in thebiocontrol of Rhizoctonia solanirdquo Current Genetics vol 35 no1 pp 41ndash50 1999

[45] S L Woo B Donzelli F Scala et al ldquoDisruption of the ech42(Endochitinase-Encoding) gene affects biocontrol activity inTrichoderma harzianum P1rdquo Molecular Plant-Microbe Interac-tions vol 12 no 5 pp 419ndash429 2007

Submit your manuscripts athttpwwwhindawicom

Nutrition and Metabolism

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Food ScienceInternational Journal of

Agronomy


International Journal of



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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Applied ampEnvironmentalSoil Science

Volume 2014

AgricultureAdvances in


PsycheHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BiodiversityInternational Journal of


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GenomicsInternational Journal of


Plant GenomicsInternational Journal of


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Forestry ResearchInternational Journal of


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EcologyInternational Journal of


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Cell BiologyInternational Journal of


Evolutionary BiologyInternational Journal of



Table 1 Basic properties of biochars used in the experiment

Type Feedstock Temperature (∘C) pH Ca () Na () Available P (mg kgminus1) Available K (mg kgminus1) Ashb () CN ratio ()BC1 Peanut shell 500 times 2 h 989 1741 060 6113 3872 7070 2902BC2 Wheat straw 500 times 2 h 1002 4738 098 3250 3820 4760 4835aMolar ratios analyzed with an elemental analysis apparatus vario TOC cube Elementar Analysensysteme GmbH GermanybMass ww analyzed by dry combustion in a muffle furnace at 600∘C for 2 h

soilborne diseases caused by bacteria and fungi [16] Neromeet al found that bacterial wilt (R solanacearum) in tomatowas reduced by adding biochar derived from municipalbiowaste [17] Suppressions of plant diseases by biocharwere attributed to several mechanisms [18ndash20] including thefollowing The chemical components of biochar may providedirect inhibition of pathogens and the porous structure ofbiochar may provide microbial habitats which is good forbacterial abundance Biochar can also promote plant growthby providing nutrients and improving nutrient solubilizationand uptake To some extent biochar sorption may changethe mobility and activity of pathogens or modify signalingbetween pathogens and plants [19]

Densities activity and diversity of soil microorganismscan be important indicators to evaluate soil health statusand soil quality [21 22] Pathogen infection may changesoil microbial properties thus soil can be converted froma highly fertile bacterial type to poorly fertile fungal type[23 24] Biochar amendments to soils may alter soil functionand fertility in various ways including through inducedchanges in the microbial community Kelly et al found asignificant decline in the fungibacteria ratio in the Coloradosoil with switchgrass biochar [25] Considering that biocharis implicated in soilborne disease reduction we hypothesizedthat disease resistance mediated by biochar may be relatedto changes in microbial properties This study aimed toinvestigate the interrelation among biochar bacterial wiltresistance and soil microbial components and activity

2 Materials and Methods

21 Plant Materials and Soil Conditions Tomato cv TaiwanRed cherry (tomato genotype produced by Kefeng Seed CoLtd Changchun Jilin China) which is susceptible to Rsolanacearum was used in this experiment Tomato seedswere stored in a fridge at 4∘C and steeped in water at roomtemperature for 2 h before use The seeds were surface-sterilized in water at 50∘C for 15min and then germinatedon moist filter paper in Petri dishes After 2 d the seeds weresown in nursery soil (tomato farm soil disinfected at 150∘Cfor 4 h) in a growth chamber with the following conditions30∘C25∘C (daynight) 14 h light and 200120583moLsdotmminus2sdotsminus1 lightintensity After five weeks tomato plants were transplantedto a polyethylene plastic pot (170mm in diameter 165mmin height) filled with 2 kg of soil each pot with 2 plants Theplants weremaintained at 28∘C in a controlled greenhouse for15 months until the end of the experiment

The soil collected from a continuous cropping cultivationtomato field (Zhucun Village Zengcheng City Guangdong

China) was sandy loam where the amount of bacterial wilt isrelatively high the proportions of sand silt and clay particlesat 0ndash20 cm soil layer were 73 22 and 5 respectivelyThe contents of soil organic matter soil-available N P andK and soil-total N P and Kwere 1630 g kgminus1 11547mg kgminus115110mg kgminus1 8254mg kgminus1 0948 g kgminus1 1347 g kgminus1 and292 g kgminus1 respectively Soil pH was 589 and soil CN ratiowas 982 The soil samples were sieved at lt2mm and storedat 4∘C until analysis to characterize relevant physical andbiochemical attributes of the soil Biochar was amended tosoil before transplantation of tomato plants

22 Biochar Two kinds of biochar (Sanli New EnergyResources Co Ltd Shangqiu Henan China) were used inthis experiment biochar made from peanut shell (BC1) andwheat straw (BC2) Biochar was prepared via pyrolysis in avertical kiln with a temperature at 500∘C and a resistancetime of about 2 h in an anaerobic condition (Sanli NewEnergy Resources Co Ltd) The basic properties of the twobiochar treatments are shown in Table 1 Scanning electronmicroscope images are shown in Figure 1

23 Experimental Design Four treatments were assessed inthis experiment no biochar and no R solanacearum inocu-lation (CK) R solanacearum inoculation (Rs) peanut shellbiochar (BC1) amendment and R solanacearum inoculation(BC1 + Rs) and wheat straw biochar (BC2) amendment andR solanacearum inoculation (BC2 + Rs) The experimentwas arranged in a completely randomized design with fourreplications each replication had one pot with 2 tomatoplants Our preliminary experiment showed that 2 ww ofthe two biochar amendments had the best effects in inhibitingbacterial wilt of tomato Thus 2 ww ratio was used in thisexperiment 2 ww ratio of peanut and wheat biochar wasused in this experiment 28 d after R solanacearum inocula-tion when the plants of R solanacearum treatment all diedsoil from all treatment groups was collected to determine theamount of R solanacearum in soil soil microbial populationdensities (bacteria fungi and actinomycetes) and soilmicro-bial activities (soil sucrase urease and neutral phosphatase)Biolog EcoPlate experiment was also conductedwhen the soilwas collected

24 R solanacearum Inoculation R solanacearum strainbiovar 3 (provided by College of Horticulture South ChinaAgricultural University Guangzhou 510642 China) wasused to inoculate tomato plants This strain is a highlyaggressive species The isolate was cultured on 235-triphenyltetrazolium chloride (TTC) medium and incubated


(a) (b)













(a)

0

20

40

60

80

100

Dise

ase i

ndex

()


RsBC1 + Rs

BC2 + Rs

(b)





1198671015840

= minus

31

sum

119894=1

(119875119894times ln119875

119894)

119875119894=

(119862119894minus 119877)

sum31

119894=1

(119862119894minus 119877)

(1)






3 Results




A

B BC

C

0

2

4

6

8

10

12

14

The d

ensit

y of

R so

lana

cear

umin

soiltimes107

(CFU

middotgminus1)











4 Discussion




A

B

AB

C

0

2

4

6

8

10

The d

ensit

y of

bac

teria

in so

iltimes107

(CFU

middotgminus1)


(a)

A A

A

B


2

4

6

8

10

12

The d

ensit

y of

actin

omyc

etes

in so

iltimes107

(CFU

middotgminus1)

(b)

A

B

C

C


2

4

6

8

10

The d

ensit

y of

fung

i in

soiltimes105

(CFU

middotgminus1)

(c)






Fungiactinomycetes

soil

mic

roor

gani

sms (

Fungibacteria

0

2

4

6

8

10

12

14

The p

ropo

rtio

n of

the a

mou

nt o

ftimes10minus2)



000

100

200

300

400

500

600

700

800

Neu

tral

pho

spha

tase

activ

ity (120583

gmiddotgminus1)


A

B

CC


ABC C

000

005

010

015

020

025

030U

reas

e act

ivity

(mgmiddotgminus1)

(b)


A A

B B

000

200

400

600

800

1000

1200

1400

Sucr

ase a

ctiv

ity (m

gmiddotgminus1)

(c)



00

05

10

15

20

25

30

AWCD


CKRs

BC1 + Rs

BC2 + Rs


CD

AB

50

55

60

65

70

75

80

Soil

pH







Abbreviations


Competing Interests


Acknowledgments




References

















































Journal of




Agronomy





Microbiology




Volume 2014

























(a) (b)













(a)

0

20

40

60

80

100

Dise

ase i

ndex

()


RsBC1 + Rs

BC2 + Rs

(b)





1198671015840

= minus

31

sum

119894=1

(119875119894times ln119875

119894)

119875119894=

(119862119894minus 119877)

sum31

119894=1

(119862119894minus 119877)

(1)






3 Results




A

B BC

C

0

2

4

6

8

10

12

14

The d

ensit

y of

R so

lana

cear

umin

soiltimes107

(CFU

middotgminus1)











4 Discussion




A

B

AB

C

0

2

4

6

8

10

The d

ensit

y of

bac

teria

in so

iltimes107

(CFU

middotgminus1)


(a)

A A

A

B


2

4

6

8

10

12

The d

ensit

y of

actin

omyc

etes

in so

iltimes107

(CFU

middotgminus1)

(b)

A

B

C

C


2

4

6

8

10

The d

ensit

y of

fung

i in

soiltimes105

(CFU

middotgminus1)

(c)






Fungiactinomycetes

soil

mic

roor

gani

sms (

Fungibacteria

0

2

4

6

8

10

12

14

The p

ropo

rtio

n of

the a

mou

nt o

ftimes10minus2)



000

100

200

300

400

500

600

700

800

Neu

tral

pho

spha

tase

activ

ity (120583

gmiddotgminus1)


A

B

CC


ABC C

000

005

010

015

020

025

030U

reas

e act

ivity

(mgmiddotgminus1)

(b)


A A

B B

000

200

400

600

800

1000

1200

1400

Sucr

ase a

ctiv

ity (m

gmiddotgminus1)

(c)



00

05

10

15

20

25

30

AWCD


CKRs

BC1 + Rs

BC2 + Rs


CD

AB

50

55

60

65

70

75

80

Soil

pH







Abbreviations


Competing Interests


Acknowledgments




References

















































Journal of




Agronomy





Microbiology




Volume 2014


























(a)

0

20

40

60

80

100

Dise

ase i

ndex

()


RsBC1 + Rs

BC2 + Rs

(b)





1198671015840

= minus

31

sum

119894=1

(119875119894times ln119875

119894)

119875119894=

(119862119894minus 119877)

sum31

119894=1

(119862119894minus 119877)

(1)






3 Results




A

B BC

C

0

2

4

6

8

10

12

14

The d

ensit

y of

R so

lana

cear

umin

soiltimes107

(CFU

middotgminus1)











4 Discussion




A

B

AB

C

0

2

4

6

8

10

The d

ensit

y of

bac

teria

in so

iltimes107

(CFU

middotgminus1)


(a)

A A

A

B


2

4

6

8

10

12

The d

ensit

y of

actin

omyc

etes

in so

iltimes107

(CFU

middotgminus1)

(b)

A

B

C

C


2

4

6

8

10

The d

ensit

y of

fung

i in

soiltimes105

(CFU

middotgminus1)

(c)






Fungiactinomycetes

soil

mic

roor

gani

sms (

Fungibacteria

0

2

4

6

8

10

12

14

The p

ropo

rtio

n of

the a

mou

nt o

ftimes10minus2)



000

100

200

300

400

500

600

700

800

Neu

tral

pho

spha

tase

activ

ity (120583

gmiddotgminus1)


A

B

CC


ABC C

000

005

010

015

020

025

030U

reas

e act

ivity

(mgmiddotgminus1)

(b)


A A

B B

000

200

400

600

800

1000

1200

1400

Sucr

ase a

ctiv

ity (m

gmiddotgminus1)

(c)



00

05

10

15

20

25

30

AWCD


CKRs

BC1 + Rs

BC2 + Rs


CD

AB

50

55

60

65

70

75

80

Soil

pH







Abbreviations


Competing Interests


Acknowledgments




References

















































Journal of




Agronomy





Microbiology




Volume 2014

























A

B BC

C

0

2

4

6

8

10

12

14

The d

ensit

y of

R so

lana

cear

umin

soiltimes107

(CFU

middotgminus1)











4 Discussion




A

B

AB

C

0

2

4

6

8

10

The d

ensit

y of

bac

teria

in so

iltimes107

(CFU

middotgminus1)


(a)

A A

A

B


2

4

6

8

10

12

The d

ensit

y of

actin

omyc

etes

in so

iltimes107

(CFU

middotgminus1)

(b)

A

B

C

C


2

4

6

8

10

The d

ensit

y of

fung

i in

soiltimes105

(CFU

middotgminus1)

(c)






Fungiactinomycetes

soil

mic

roor

gani

sms (

Fungibacteria

0

2

4

6

8

10

12

14

The p

ropo

rtio

n of

the a

mou

nt o

ftimes10minus2)



000

100

200

300

400

500

600

700

800

Neu

tral

pho

spha

tase

activ

ity (120583

gmiddotgminus1)


A

B

CC


ABC C

000

005

010

015

020

025

030U

reas

e act

ivity

(mgmiddotgminus1)

(b)


A A

B B

000

200

400

600

800

1000

1200

1400

Sucr

ase a

ctiv

ity (m

gmiddotgminus1)

(c)



00

05

10

15

20

25

30

AWCD


CKRs

BC1 + Rs

BC2 + Rs


CD

AB

50

55

60

65

70

75

80

Soil

pH







Abbreviations


Competing Interests


Acknowledgments




References

















































Journal of




Agronomy





Microbiology




Volume 2014

























A

B

AB

C

0

2

4

6

8

10

The d

ensit

y of

bac

teria

in so

iltimes107

(CFU

middotgminus1)


(a)

A A

A

B


2

4

6

8

10

12

The d

ensit

y of

actin

omyc

etes

in so

iltimes107

(CFU

middotgminus1)

(b)

A

B

C

C


2

4

6

8

10

The d

ensit

y of

fung

i in

soiltimes105

(CFU

middotgminus1)

(c)






Fungiactinomycetes

soil

mic

roor

gani

sms (

Fungibacteria

0

2

4

6

8

10

12

14

The p

ropo

rtio

n of

the a

mou

nt o

ftimes10minus2)



000

100

200

300

400

500

600

700

800

Neu

tral

pho

spha

tase

activ

ity (120583

gmiddotgminus1)


A

B

CC


ABC C

000

005

010

015

020

025

030U

reas

e act

ivity

(mgmiddotgminus1)

(b)


A A

B B

000

200

400

600

800

1000

1200

1400

Sucr

ase a

ctiv

ity (m

gmiddotgminus1)

(c)



00

05

10

15

20

25

30

AWCD


CKRs

BC1 + Rs

BC2 + Rs


CD

AB

50

55

60

65

70

75

80

Soil

pH







Abbreviations


Competing Interests


Acknowledgments




References

















































Journal of




Agronomy





Microbiology




Volume 2014

























Fungiactinomycetes

soil

mic

roor

gani

sms (

Fungibacteria

0

2

4

6

8

10

12

14

The p

ropo

rtio

n of

the a

mou

nt o

ftimes10minus2)



000

100

200

300

400

500

600

700

800

Neu

tral

pho

spha

tase

activ

ity (120583

gmiddotgminus1)


A

B

CC


ABC C

000

005

010

015

020

025

030U

reas

e act

ivity

(mgmiddotgminus1)

(b)


A A

B B

000

200

400

600

800

1000

1200

1400

Sucr

ase a

ctiv

ity (m

gmiddotgminus1)

(c)



00

05

10

15

20

25

30

AWCD


CKRs

BC1 + Rs

BC2 + Rs


CD

AB

50

55

60

65

70

75

80

Soil

pH







Abbreviations


Competing Interests


Acknowledgments




References

















































Journal of




Agronomy





Microbiology




Volume 2014

























00

05

10

15

20

25

30

AWCD


CKRs

BC1 + Rs

BC2 + Rs


CD

AB

50

55

60

65

70

75

80

Soil

pH







Abbreviations


Competing Interests


Acknowledgments




References

















































Journal of




Agronomy





Microbiology




Volume 2014


























References

















































Journal of




Agronomy





Microbiology




Volume 2014








































Journal of




Agronomy





Microbiology




Volume 2014
























research article effects of biochar amendment on tomato...

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