B

Sa

b

c

d

a

ARAA

KBSWCNF

1

icf2cnlrw

dmrb

uD

h0

Agricultural Water Management 138 (2014) 37–44

Contents lists available at ScienceDirect

Agricultural Water Management

jou rn al hom ep age: www.elsev ier .com/ locate /agwat

iochar enhances yield and quality of tomato under reduced irrigation

aqib Saleem Akhtara,b, Guitong Li c, Mathias Neumann Andersend, Fulai Liua,b,∗

Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Højbakkegård Allé 13, DK-2630 Tåstrup, DenmarkSino-Danish Center for Education and Research (SDC), Beijing, ChinaCollege of Resources and Environmental Sciences, China Agricultural University, Beijing 10093, ChinaDepartment of Agroecology, Faculty of Science and Technology, Aarhus University, Blichers Allé 20, 8830 Tjele, Denmark

r t i c l e i n f o

rticle history:eceived 6 December 2013ccepted 25 February 2014vailable online 21 March 2014

eywords:iocharoil water contentater use efficiency

hlorophyll content indexitrogen

a b s t r a c t

Biochar is an amendment that can be used for enhancing soil water storage which may increase cropproductivity. The objective of this study was to investigate the effects of biochar on physiology, yield andquality of tomato under different irrigation regimes. From early flowering to fruit maturity stages, theplants were subjected to full irrigation (FI), deficit irrigation (DI) and partial root-zone drying irrigation(PRD) and two levels of biochar (0% and 5% by weight). In FI, the plants were irrigated daily to pot waterholding capacity while in DI and PRD, 70% of FI was irrigated on either the whole or one side of the pots,respectively. In PRD, irrigation was switched between sides when the soil water content of the dry sidedecreased to 15%. The results showed that addition of biochar increased the soil moisture contents in DIand PRD, which consequently improved physiology, yield, and quality of tomato as compared with thenon-biochar control. However, leaf N content and chlorophyll content index (CCI) were decreased signif-

ruit quality icantly in biochar treated plants. Furthermore, given a same irrigation volume, PRD offered advantagesover DI in improving water use efficiency, leaf relative water content, membrane stability index and fruityield. Overall, fruit quality was improved under reduced irrigation (i.e. DI and PRD) as compared with FI.It was concluded that incorporation of biochar under DI and particularly, PRD might be a novel approachto improve water productivity and quality of tomato.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

Biochar, also called black gold for agriculture, is being usedncreasingly in agriculture with an intention to mitigate climatehange by sequestering carbon (C), improving soil properties andunctions and enhancing crop yield (Lehmann, 2007; Sohi et al.,010). It is a C rich material produced by burning any organicompound thorough pyrolysis process. It is very recalcitrant inature (Cheng et al., 2008) due to high degree of aromaticity. In

iterature, the residence time of biochar in soil is reported in theanges of 100–1000 s years. The properties of biochar vary greatlyith different sources of feedstock and with different production

Abbreviations: FI, full irrigation; DI, deficit irrigation; PRD, partial root-zonerying irrigation; CCI, chlorophyll content index; WUE, water use efficiency; gs, sto-atal conductance; An , photosynthetic rate; MSI, membrane stability index; RWC,

elative water content; SA, stomatal aperture; SD, stomatal density; TSS, total solu-le solids; TA, titratable acidity; Vc, vitamin C.∗ Corresponding author at: Department of Plant and Environmental Sciences, Fac-lty of Science, University of Copenhagen, Højbakkegård Allé 13, DK-2630 Tåstrup,enmark. Tel.: +45 3533 3392; fax: +45 3533 3478.

E-mail addresses: lgtong@cau.edu.cn (G. Li), fl@plen.ku.dk (F. Liu).

ttp://dx.doi.org/10.1016/j.agwat.2014.02.016378-3774/© 2014 Elsevier B.V. All rights reserved.

technologies. This is because, elemental composition of each feed-stock is different from the other and their behavior at differenttemperatures varies accordingly.

Recently, research indicated that biochar addition has a poten-tial to enhance water holding capacity of soil (Streubel et al., 2011).This infers that soil amendment with biochar may improve cropproductivity by retaining more water from rainfall in arid regionsand reduce the frequency or amount of irrigation water in irrigatedregions. Basso et al., 2013 applied flash pyrolysis biochar into sandysoil and found a 23% increase of water holding capacity relative tocontrol. Right source and application rate of biochar is extremelyimportant for increasing soil water holding capacity (WHC). Theuse of crop residues to produce biochar has been proposed (Sohiet al., 2010), which on one hand may improve soil fertility and onthe other hand may act as an intelligent way of recycling organicsand reducing CO2 emission.

In addition to soil amendment, deficit irrigation practices arealso used to sustain crop productivity under reduced water appli-

cation. It is because, nowadays, traditional irrigation practice i.e. fullirrigation (FI) is considered as luxury water consumption for cropproduction due to limited irrigation water resources in many placesin the world. On the other hand, deficit irrigation (DI) involves less

3 ater Management 138 (2014) 37–44

whiiwit(iuaDs2tiaewgd

iddtawUai

(ptPetaic

2

2

hls3taBub

toipemwf

Table 1Basic properties of soil and biochar used in the experiment. Means are given withstandard deviations for 3 replicate measurements (where available).

Attribute Soil Biochar

Soil type, FAO system Fluvisols –Soil texture Sandy loam –Bulk density, g/cm3 1.63 0.48<5 mm particles, g/kg – >950Water holding capacity, g/g 0.28 3.7 ± 0.4Organic carbon, g/kg 9.75 623 ± 3Organic nitrogen, g/kg 0.83 10.5 ± 0.8H, g/kg – 43 ± 0.3CaCO3, g/kg 17.3 –pH 8.02 10.64 ± 0.01EC, mS/cm 0.185 1.02 ± 0.05CEC, cmol(+)/kg 10.0 12.5 ± 0.1Available N, mg/kg 56.0 56 ± 3Available P, mg/kg 18.0 469 ± 7.3

8 S.S. Akhtar et al. / Agricultural W

ater than potential evapotranspiration (English et al., 1990) andas received increased attention. Whereas, partial root zone drying

rrigation (PRD) is a further development of DI (Dry et al., 1996) andn this water-saving technique plant receives the same quantity of

ater as DI but only one part of the root-zone is irrigated at eachrrigation event, leaving another part to dry to a certain soil mois-ure content before rewetting by shifting irrigation to the dry sideDavies and Hartung, 2004). Among these irrigation strategies, PRDs considered to be the best for certain crops in improving waterse efficiency (WUE) since it triggers the production of abscisiccid (ABA) in the drying zone of root (Davies and Hartung, 2004;odd, 2007) which results in partial closing of stomata and con-

equently, water loss through transpiration is reduced (Liu et al.,006). At the same time, the plant maintains its water status byaking up water from the wetted zone of the soil. Furthermore, PRDncreased photosynthetic water use efficiency which can be defineds the ratio of photosynthesis (A) to stomatal conductance (gs). Liut al., 2005 observed that, in potato plants gs is more sensitive toater deficit than A and, A/gs increased linearly with decreasing

s up to a certain point (0.2 mol m−2 s−1) after that it decreasedramatically.

In the recent years there has been an increasing interest inntegrated approaches in improving crop production to resist con-itions of nutrient-poor soil, drought, salinization or other forms ofegradation (Ismail and Iberahim, 2003; Zahir et al., 2012). Hence,he combination of biochar and reduced irrigation strategies suchs DI and PRD may substantially save irrigation water and enhanceater productivity compared to their separate implementation.ntil now, no attempt has been made to investigate the effects of DInd PRD under biochar amendment for tomato production whichs one of the major horticultural crop in China.

In this study, we tested the validity of following hypothesis:1) addition of biochar, particularly under reduced water sup-ly will enhance tomato yield and fruit quality in comparison tohe non-biochar controls and (2) given a same irrigation volume,RD irrigation combines with biochar incorporation will furthernhance WUE and fruit quality of tomatoes in relation to the DIreatment. Our objective was to illustrate the underlying mech-nisms by which biochar addition in combination with reducedrrigation may bring about further improvements in water use effi-iency and fruit yield in tomatoes.

. Materials and methods

.1. Experimental setup

The experiment was conducted in April–July, 2012 in a green-ouse at China Agricultural University, Beijing, China. At fifth

eaf stage, tomato (Solanum lycopersicum L. var. No. 2 Hongfen)eedlings were transplanted into plastic pots (25 cm diameter and0 height) containing 20 kg sandy loam soil. The soil was sievedhrough a 2 mm mesh and had soil volumetric water content of 28%nd 5% at field capacity and permanent wilting point, respectively.oth field capacity and permanent wilting point was determinedsing pressure membrane apparatus before biochar incorporationy taking ten soil replicates.

Biochar (made up of a mixture of rice husk and shell of cot-on seed were produced through pyrolysis process at 400 ◦C) wasbtained from China Agriculture University, Beijing. It was grindednto fine powder and mixed thoroughly into soil before filling theot at 5% by weight. Pots without biochar served as controls. To

nsure sufficient nutrients supply during the experiment, recom-ended doses of N, P and K (250, 180, and 180 kg ha−1, respectively)ere applied as urea, diammonium phosphate and potassium sul-

ate, respectively. The basic soil and biochar properties are shown

Available K, mg/kg 93.5 6542 ± 65Ash, g/kg – 126 ± 15

in Table 1. The bulk density of soil was reduced to 1.54 g cm−3 afterbiochar amendment.

2.2. Irrigation treatment

Four weeks after transplanting, plants were subjected to threeirrigation regimes, i.e. full irrigation (FI), deficit Irrigation (DI), andpartial root-zone drying irrigation (PRD). In case of FI, plants wereirrigated daily to pot water holding capacity (28% soil volumetricwater content of control soil) while in DI and PRD, 70% water usedfor FI was irrigated on either the whole or one side of the pots,respectively. In PRD, pots were divided uniformly into two ver-tical sections before soil filling and irrigation was applied to oneside of pots and switched when the soil water content of otherside decreased to 15%. Irrigation was applied using drippers toavoid evaporation and leaching losses. Equal amount of water wasapplied to all biochar and non-biochar treated pots according toirrigation treatments. Total amount of water applied to each FI potwas 32.5 L and to each DI and PRD was 22.7 L during the wholetreatment period. Moisture content was determined with AV-EC5soil moisture sensor (Avalon Scientific Inc., NJ, USA) connectedwith CR23X Micrologger (Campbell Scientific Inc., USA) installedat depth of 15 cm in the pots. Two moisture sensors were installedin each treatment. The data of each point (represents one day) indi-cated the average of 48 reading recorded within 24 h automaticallyby soil moisture sensors connected with digital data logger.

2.3. Fruit yield and quality measurement

At maturity, tomato fruit were harvested and fruit fresh weightwas taken immediately. Total soluble solid (Brix) was assessedby using portable handheld brix refractometer (UV2300, ShanghaiTianmei, China). Titratable acidity and vitamin C contents weredetermined by using standard methods of analysis (AOAC, 1999).

2.4. Physiological measurements

Stomatal conductance (gs) was determined with leaf porometer(Decagon Devices Inc., Pullman, WA, USA) from 7:00 to 17:00 hwith time interval of 2 h for four times (i.e. 14, 21, 28, and 35 daysafter onset of irrigation treatment). Photosynthetic rate was deter-mined at the upper canopy on two mature leaflets per plant from

11:00 to 14:00 h with a portable photosynthesis system (Li-Cormodel 6400, Lincoln, Nebraska, USA). Chlorophyll content index(CCI) were measured six times during the treatment period withportable leaf chlorophyll meter.

ater Management 138 (2014) 37–44 39

t1wt3sIptc

M

ibit(

R

bm

2

twaSam

swi

gsLTSD

S

wl

2

3slpii4m(2

Fig. 1. Daily average soil volumetric moisture content (%) in pots of tomato plantsunder FI, DI, and PRD irrigation treatment whereas, B0 and B5 indicate biochar levels

S.S. Akhtar et al. / Agricultural W

Membrane stability index (MSI) was determined according tohe method of Premchandra et al., 1990 as modified by Sairam,994. Leaf discs (100 mg) were thoroughly washed in running tapater followed by washing with double distilled water, thereafter

he discs were heated in 10 ml of double distilled water at 40 ◦C for0 min. Then the electrical conductivity (C1) of the water was mea-ured by EC meter (Lei-ci DDS-307A, Shanghai Electronics Scientificnstruments Co. Ltd., Shanghai, China). Subsequently the same sam-les were placed in a boiling water bath (100 ◦C) for 10 min andheir electrical conductivity was also recorded (C2). The MSI wasalculated as:

embrane stability index (MSI) =[

1 −(

C1C2

)]× 100 (1)

Relative water contents of the leaves were determined accord-ng to Smart, 1974. Leaf surface was cleaned with a tissue paperefore taking fresh weight (FW) and then put into double deion-

zed water for 4 h under dim light to get turgid weight (TW). Thenhe leaf was dried in an oven at 85 ◦C for 24 h to get dry weightDW). Leaf relative water content was calculated as:

elative water content (RWC) = (FW − DW)(TW − DW)

× 100 (2)

Crop water use efficiency (WUE) was calculated as the ratioetween the fruit yield and the plant water use during the treat-ent period.

.5. Leaf surface imprints and stomatal morphology

Fingernail polish imprints were obtained halfway from the leafip to the base from the abaxial surface of each leaf. The leaf surfaceas cleaned with a soft brush and a thin layer of nail polish was

pplied on the cleaned area and allowed it to dry for 10–15 min.ubsequently, transparent tape was adhered to the respective areand carefully drawn off. Then, the imprint was attached to theicroscopic slide with the help of solution tape.Imprints were photographed through a Leitz DMRD light micro-

cope (Leica Mikroskopie & Systeme GmbH, Wetzlar, Germany)ith an associated camera (Leica DFC 420). A 1 mm2 grid was super-

mposed on the images for calculating the stomatal density (SD).SD and stomatal size parameters including guard cell length (Ls),

uard cell pair width (Ws), stomatal pore aperture length (La), andtomatal pore aperture width (Wa) were measured (data of Ls, Ws,a and Wa is not presented) with the images using UTHSCSA Image-ool software (UTHSCSA ImageTool for Windows version 3.00).tomatal aperture (SA) (�m2) was then calculated according tooheny-Adams et al., 2012 using Eq. (3)

A = � × Wa × La

4(3)

here Wa is the pore aperture width and La is the pore apertureength.

.6. Leaf ABA determination

Two mature leaflets from each treatment were taken from therd leaf from the top and then immediately dipped in liquid N andubsequently stored at −80 ◦C until analysis. Approximately 30 mgeaf sample was weighed and crushed using a precooled mortar andestle by keeping pestle into ice to avoid heating during crushing

n 2 ml deionized water. The samples were homogenized by shak-ng for 24 h at 4 ◦C and then centrifuged at 10,000 × g for 5 min at

◦C. The supernatant was removed, stored on ice and used to deter-ine ABA content through an enzyme linked immunosorbent assay

ELISA) using a monoclonal antibody for ABA (AFRC MAC252) (Asch,000).

and PRD A and PRD B denote left and right sides of the PRD pots. The data of eachpoint (represents one day) indicates the average of 48 reading recorded within 24 hautomatically by soil moisture sensors connected with digital data logger.

2.7. Total leaf nitrogen and carbon content

Total N and C contents (% DW) from finely grounded dry leafsamples were measured directly with a CHNS/O analyzer (Flash2000, Thermo Fisher Scientific, Cambridge, UK) which operatesaccording to the dynamic flash combustion method (modifiedDumas method).

2.8. Statistical analysis

The experiment was conducted in a completely randomizeddesign with four replicates of each treatment. The data werehomogeneous and normal distributed (based on data descriptiveanalysis using “Statistix 8.0” software) and were subjected to two-way analysis of variance (ANOVA) and presented as mean of fourreplicates ± S.E., significance between treatments was checked atP ≤ 0.05.

3. Results

3.1. Soil water status

Dynamics of soil water content in FI, DI and PRD pots during

the experiment under biochar and without biochar treatments areshown in Fig. 1. Soil water content was determined in two potsper treatments so that the difference was only indicative. It wasfound that biochar treatments had comparatively higher soil water

40 S.S. Akhtar et al. / Agricultural Water Management 138 (2014) 37–44

0

600

1200

1800

2400

Fru

it f

resh w

eig

ht

(g p

lant-

1)

B0 B5(a) B:P = 0.004I:P < 0.001

Irriga�on treatments

FI DI PRD

Fi

crbe

3

pBircc

hd

Fato

bcp

3

t

0

10

20

30

FI DI PRD

Vita

min

C c

onte

nt

(mg 1

00 g

-1)

Irriga tion trea tment

(c)

4

4.3

4.6

4.9

5.2

5.5

5.8

To

tal so

luble

so

lids (

Brix) B0 B5(a)

0

0.2

0.4

0.6

Titra

table

acid

ity

(% c

itric a

cid

)

(b)

B:P = 0.232I:P = 0.002

B:P = 0.025I:P = 0.066

B:P = 0.322I:P < 0.001

Fig. 3. Total soluble solids (a), titratable acidity (b) and vitamin C (c) as affected by

Fs

ig. 2. Fruit fresh weight as affected by irrigation and biochar treatment. Error barsndicate S.E. (n = 4).

ontent in DI and PRD with respect to non-biochar treatments,espectively. However, in case of FI there was slight response byiochar amendment. In DI, the soil water content declined consid-rably in the first 15 days after onset of treatment (DAT).

.2. Fruit yield (FY) and quality

Fruit yield (FY) decreased significantly in DI treatments com-ared to FI (Fig. 2). PRD plants produced higher FY than DI plants.iochar amendment increased FY in all irrigation treatments. Max-

mum increase was observed in FI and PRD, i.e. 20% and 13%,espectively, with biochar addition compared with non-biocharontrol. DI plants resulted in only 6% higher fruit yield in the biocharompared to the respective non-biochar control.

The data regarding total soluble solid (TSS) showed that DI fruitsad comparatively higher TSS than FI fruits (Fig. 3a). No significantifference was detected on TSS by biochar amendment.

Titratable acidity (TA) increased under DI and PRD compared toI (Fig. 3b). Significant increase of TA was detected with biocharmendment. Maximum TA was observed in biochar amended PRDreatment which was significantly higher than non-biochar controlf FI and DI.

Vitamin C (Vc) content was affected non-significantly withiochar treatment (P = 0.322). Irrigation treatment affected Vcontent significantly (P < 0.001) and was higher in DI and PRD com-ared to FI (Fig. 3c).

.3. Physiological responses

The influence of biochar on diurnal course of stomatal conduc-ance (gs) under different irrigation regimes is presented in Fig. 4.

ig. 4. Diurnal course of stomatal conductance of tomato plants grown under FI, DI and PRtandard errors of means (S.E.) (n = 3).

irrigation and biochar treatment. Error bars indicate S.E. (n = 4).

The data showed that gs was almost stable from 7 a.m. to 1 p.m. butthereafter, it decreased sharply in all treatments. Maximum gs wasfound in FI treatment which was greater than those of DI and PRDtreatments. Basically, biochar treatment increased gs compared tonon-biochar treatment; however, the increase was significant onlyin DI and PRD. The significant response was only observed from 7

a.m. to 1 p.m. after that no significant differences in gs between thetreatments were evident.

D treatment and biochar levels (B0 and B5) from 7 a.m. to 5 p.m. Error bars indicate

S.S. Akhtar et al. / Agricultural Water Management 138 (2014) 37–44 41

13

16

19

22

FI DI PRD

Ph

oto

syn

the

tic r

ate

(µm

ol m

-2s

-1)

Irriga tion trea tment

B0 B5 B:P = 0.115I:P < 0.0 03

20

30

40

50

60

70

80

90

Ch

loro

phyl

l co

nte

nt

index B0 B5(a) B:P < 0.001

I:P = 0.428

(b)

Fa

be

bPoo

RtPp

awHn

dat

inHP

3

aati

Fig. 6. Relative water contents (RWC) (a), membrane stability index (MSI) (b) andcrop water use efficiency (WUE) (c) of tomato exposed to different irrigation andbiochar treatments. RWC was calculated by using Eq. (2) and MSI was calculated byusing Eq. (1). Error bars indicate S.E. (n = 4).

75

90

105

120

135

150

165

FI DI PRD

Leaf

AB

A c

onte

nt

(ng g

-1F

W)

Irrigation treatment

B0 B5 B:P = 0.075I:P = 0.046

ig. 5. Chlorophyll content index (a) and photosynthetic rate (b) of tomato leavess affected by irrigation and biochar treatment. Error bars indicate S.E. (n = 4).

Chlorophyll content index (CCI) decreased significantly iniochar treated plants. Irrigation treatments had no significantffect on CCI (Fig. 5a).

Leaf photosynthetic rate (An) (Fig. 5b) was affected significantlyy irrigation and insignificantly by biochar treatments. In DI andRD plants, An was lowered compared to FI. Except FI, incorporationf biochar increased An, however greater response was observednly in the PRD treatment.

Both irrigation and biochar treatment had significant effect onWC (Fig. 6a). RWC was decreased under DI and PRD comparedo FI. However, biochar amendment increased RWC in both DI andRD treatment. In addition, greater response was observed in PRDlants compared to DI.

Similar to RWC, membrane stability index (MSI) was alsoffected significantly by both irrigation and biochar treatment. Itas decreased in DI compared to FI and PRD as depicted in Fig. 6b.owever, MSI was greater in all biochar treatments compared toon-biochar control.

Significant increase in crop water use efficiency (WUE) was evi-ent under PRD (35%) and DI (15%) compared to FI (Fig. 6c). Biocharmendment increased WUE in all irrigation treatments comparedo non-biochar control.

Fig. 7 shows the ABA content of tomato leaf as influenced byrrigation and biochar treatment. Leaf ABA content was affected sig-ificantly by irrigation and insignificantly by biochar amendment.owever, biochar addition decreased ABA content in both DI andRD plant compared to their non-biochar control.

.4. Stomatal morphology

Both stomatal density (SD) and stomatal pore aperture (SA) wereffected significantly by irrigation and biochar treatments (Fig. 8a

nd b). Both SD and SA were decreased under DI and PRD comparedo FI; in relations to the non-biochar controls, biochar amendmentncreased SD and SA in all irrigation treatments.

Fig. 7. ABA content of tomato leaves exposed to different irrigation and biochartreatments. Error bars indicate S.E. (n = 4).

42 S.S. Akhtar et al. / Agricultural Water Management 138 (2014) 37–44

0

30

60

90

120

150

180

FI DI PRD

SD

(m

m-2

)

Irriga tion trea tment

(b)0

50

100

150

200S

A (

µm

2)

(a) B:P = 0.003I:P < 0.001

B:P = 0.022I:P = 0.003

Fig. 8. Stomatal pore aperture (SA) (a) and stomatal density (SD) (b) in the abaxialleaf surface of tomato plants exposed to different irrigation and biochar treatments.The data were determined based on 12 leaf impression images (each from one indi-vidual leaf and three leaves per plant of four plants per treatment), and of which 6stomata for each image were measured. SA was calculated by using Eq. (3); SD wasca

3

rstp

lpn

4

smio2htamfiySDacwi

0

0.5

1

1.5

2

2.5

3

3.5

Le

af

N c

onte

nt (%

)

(a)

15

25

35

45

55

Leaf

C c

onte

nt

(%)

(b)

5

10

15

20

25

30

35

FI DI PRD

Le

af

C:N

ra

tio

Irriga tion trea tment

(c)

B:P < 0.001I:P = 0.083

B:P = 0.667I:P = 0.291

B:P = 0.002I:P = 0.577

alculated by counting stomatal number of the 12 images and each image with anrea of 0.00625 mm−2. Error bars indicate S.E.

.5. Total leaf N and C contents

Total leaf N content decreased in all biochar treatments thanespective non-biochar control (Fig. 9a). Irrigation treatment had noignificant effect on total leaf N content. Both biochar and irrigationreatments had no significant effect on total leaf C content in tomatolants (Fig. 9b).

Fig. 9c shows the effect of irrigation and biochar treatment oneaf C/N ratio which was comparatively greater in biochar treatedots than in non-biochar control. Irrigation treatment had no sig-ificant effect on leaf C/N ratio.

. Discussion

The result of present study showed that addition of biochar toandy loam soil increased soil volumetric water content (deter-ined in two pots per treatment therefore the difference was only

ndicative) under both DI and PRD. This is consistent with findingsf other authors (Novak et al., 2009; Artiola et al., 2012; Basso et al.,013) who reported increased water holding capacity because ofigh adsorption capacity and porous structure of biochar. In addi-ion, application of biochar significantly increase plant water statuss exemplified by the improved RWC, MSI and WUE. The improve-ent of plant water status could have contributed to the increased

ruit yield in the biochar treated plants. It was found that the largestncrease in fruit yield was observed in FI and PRD. Furthermore,ield of biochar amended PRD pots was very close to FI control.imilarly, RWC, MSI and WUE were also higher in PRD compared toI. A large body of evidence has suggested that, when irrigated with

same amount of water, PRD is better than DI in term of improvingrop WUE (Dodd, 2009). Here, it is shown that the efficacy of PRDas further enhanced by biochar addition. In accordance with this,

ncreased WUE with biochar addition have also been reported by

Fig. 9. Total leaf N content (a), total leaf C content (b) and leaf C/N (c) of tomatoleaves as affected by irrigation and biochar treatment. Error bars indicate S.E. (n = 4).

e.g. Kammann et al., 2011. Hence, our both hypothesis were ful-filled in term of improved yield and plant water status in tomatoesunder biochar amending.

Plant physiological responses to biochar addition have beenreported in relatively few studies (Kammann et al., 2011). Reducedwater supply impairs crop physiological processes resulting indrastic yield reduction. In the present study, strong reductionin gs and An was observed in DI and PRD compare to FI.Stomatal morphology was also significantly affected by deficitirrigation regimes which may ultimately influence gs and An. Aninteresting finding in this study was the phenomenon of drought-induced reduction in SD (Fig. 8b). This is in contrast to the generalconsensus as, cell growth rate decreases under drought condition,cells become smaller and this results in increased stomatal den-sity (Fraser et al., 2009). Whereas, Shabala et al., 2012 reporteddecreased stomatal density due to increase in leaf succulence andsize of pavement cells under osmotic stress. Some other authorsalso observed decreased stomatal density under drought stress(Nautiyal et al., 1994; Klamkowski and Treder, 2006). Stomatalaperture (SA) also decreases under water stress conditions (Spenceet al., 1986). In general, it is reported that increased SD is oftenaccompanied by a decrease in stomatal size under deficit irrigation

(Spence et al., 1986). However, our results contradicted with thisproposition and were in line with that of Yan et al., 2012 whoreported that in relation to PRD, greater SD is not associated withsmaller stomata for the DI treated potato plants. Nevertheless,

S.S. Akhtar et al. / Agricultural Water Management 138 (2014) 37–44 43

r 2 = 0.76 **

50

70

90

110

130

150

50 70 90 11 0 13 0

SA (µm2)

gs

ml

om

m(-2

s-1

)

(a)

r2 = 0.74 **

50 70 90 11 0 13 0 15 0

SD (mm-2 )

(b)

F matab easu

iiw

taci2g

AsoPlfstHwdaame

i(rcCborNCll(tSaeDni

ment with our finding where increased Vc content under reducedwater supply in relation to full irrigation was evident. MaximumVc content was found in DI. Biochar amendment had no signifi-cant effect on Vc. However, one should be aware that Vc levels in

r2 = 0. 99

50

53

56

59

62

65

1.5 1.7 1.9 2.1 2.3 2.5

Chlo

rophyl

l conte

nt

index

Leaf N conte nt (%)

(FI+B0)

ig. 10. Stomatal conductance (gs) of tomato leaves expressed as a function of stoiochar treatments. The regression lines were made between the mean data of gs (m

ncorporation of biochar increased both SD and SA under deficitrrigation indicating reduced water stress due to improved plant

ater status.Regression analyses between gs and SA and SD indicated that

here were significantly positive correlations between these vari-bles (Fig. 10a and b). Whereas, Yan et al., 2012 found positiveorrelation of gs with SA and negative with SD indicating that gss operated only by SA in DI plants. However, Franks and Farquhar,007 reported enhanced gs is related to increased SD which is inood agreement with our findings.

Decreased SA results in reduced CO2 intake which decreasesn under deficit irrigation. And higher gs sensitivity toward watertress is accompanied with increased level of ABA as observed inur study where leaf ABA contents (Fig. 7) were higher under DI andRD. However, gs and An were higher and leaf ABA content wereower under biochar incorporation which might be attributed toavorite soil water conditions. There is current controversy on thetomatal responding mainly to ABA generated in the leaf, ratherhan to ABA coming from the roots (Wilkinson and Hartung, 2009).owever, it should be noted that a plant under field conditionsith a large root system might have part of their roots under veryifferent soil water conditions, such that no PRD is needed to trigger

root-to-shoot signaling mechanism (e.g. Fernández et al., 2006);nd perhaps a hydraulic signal, rather than a chemical signal is theain actor on a root-to-shoot signaling mechanism (Diaz-Espejo

t al., 2012).A decreased or neutral effect of biochar on chlorophyll content

ndex (CCI) has been reported depending on its source and typeAsai et al., 2009; Ventura et al., 2013). Here we found significanteduction of CCI in biochar treated plants compared to non-biocharontrol (Fig. 5a) but no significant effect of irrigation treatments onCI. There might be two reasons accounting for CCI reduction underiochar treatment. First, it might be attributed to NH4+ adsorptionn the surface of biochar (Lehmann et al., 2002) which may leads toeduced N availability to plant as observed in our study where leaf

content was comparatively reduced in the biochar treated plant.onsistent with this, Kammann et al., 2011 also observed decreased

eaf N content with biochar amendment. Regression analysis ofeaf N contents to CCI indicated that there is significant correlationr2 = 0.99) between the two variables (Fig. 11). Such kind of rela-ionships confirms that CCI reduces with decreasing leaf N content.econd, it might be because of increased soil C/N ratio with theddition of biochar which lead to soil N immobilization (Lehmann

t al., 2002) and consequently, reduced N uptake by the plants.espite the reduced leaf N content, fruit quality was not affectedegatively which might be due to increased uptake of water and

mproved plant physiology with biochar addition.

l aperture (SA) (a) and to stomatal density (SD) (b) under different irrigation andred at 11 a.m.) to SA and SD of all irrigation treatments.

Reduced irrigation treatments (i.e. DI and PRD) in generalimproved fruit quality in relation to FI, as reported by Mitchell et al.,1991. TSS is an important quality parameter for tomato fruit andwas higher in DI compared to PRD plants, however, biochar had nosignificant effect on TSS. Dorji et al., 2005 also found 20% higherTSS in DI compared to other treatments because of reduced watercontents in the fruits. Water deficit resulted in higher starch accu-mulation during first stage of fruit growth (Mitchell et al., 1991)followed by starch conversion to glucose in latter stages (Davisand Cocking, 1965). There are reports for increased fruit acidityunder reduced irrigation (Mitchell et al., 1991; Savic et al., 2011).Whereas, in the present study, increased TA content with biocharwas observed in all irrigation regimes which contradict with thatof Mitchell et al., 1991. However, the largest increase was noticedin PRD compared to DI. Increased organic acid concentrations withincrease in the ratio of inorganic cation:anion uptake has been pro-posed by Davies, 1964 as a means to maintain electro-neutrality intomato fruit tissue. Hence, increased TA with biochar could be dueto that, in order to maintain the C:N ratio in the plants suppliedwith biochar, the extra C may have been used for the production oforganic acids like citric acid and malic acid, which are responsiblefor the acidity of fruit. Vc content is increased under water stresscondition (Sánchez-Rodríguez et al., 2011). This is in good agree-

Fig. 11. Chlorophyll content index (CCI) of tomato leaves expressed as a functionof total leaf N content under different irrigation and biochar treatments. Regressionline was drawn on five points excluding FI + B0 (full irrigation where no biochar wasapplied) because of unexpected value.

4 ater M

vp(i

biaDwmp

A

R

R

A

A

A

A

A

B

C

D

D

D

D

D

D

D

D

D

E

F

4 S.S. Akhtar et al. / Agricultural W

egetables largely depend on various factors, including cultivar,lant nutrition, production practice and the degree of maturityAntonio et al., 2007). Hence, hypotheses #1 and 2 were not fulfilledn term of TSS and Vc increase but in term of improved TA.

In conclusion, our results clearly demonstrated that addingiochar to sandy loam soil under reduced irrigation influence phys-

ology, yield and quality of tomato. Furthermore, under biocharmendment PRD plants produced significantly higher yield thanI which was similar to FI control. Therefore, under limited fresh-ater resources, application of PRD in combination with biocharight be a promising approach for saving water and enhancing

roductivity and quality of tomato.

cknowledgement

Financial support from Sino-Danish Center for Education andesearch (SDC) is highly acknowledged and appreciated.

eferences

ntonio, J.P., Francisco, M.A., Ana, S.M., Marıa, I.F., Estrella, D., 2007. Influence ofagricultural practices on the quality of sweet pepper fruits as affected by thematurity stage. J. Sci. Food Agric. 87, 2075–2080.

rtiola, J.F., Craig, R., Robert, F., 2012. Effects of a biochar-amended alkaline soil onthe growth of Romaine lettuce and bermudagrass. Soil Sci. 177, 561–570.

sai, H., Samson, B., Stephan, H., Songyikhangsuthor, K., Homma, K., Kiyono, Y., Inoue,Y., Shiraiwa, T., Horie, T., 2009. Biochar amendment techniques for upland riceproduction in Northern Laos. Soil physical properties, leaf SPAD and grain yield.Field Crop Res. 111, 81–84, http://dx.doi.org/10.1016/j.fcr.2008.10.008.

sch, F., 2000. Determination of abscisic acid by indirect enzyme linked immunosor-bent assay (ELISA). Technical Report. Laboratory for Agrohydrology andBioclimatology, Department of Agricultural Sciences, The Royal Veterinary andAgricultural University, Taastrup, Denmark.

OAC, 1999. Official Methods of Analysis, 16th Edition, 5th Reversion. AOAC Inter-national, Gaithersburg, MD, method 942.15 and 967.21.

asso, A.S., Miguez, F.E., David, A.L., Robert, H., Westgate, M., 2013. Assessingpotential of biochar for increasing water-holding capacity of sandy soils. GCBBioenergy 5 (2), 132–143, http://dx.doi.org/10.1111/gcbb.12026.

heng, C., Lehmann, J., Thies, J.E., Burton, S.D., 2008. Stability of black carbonin soils across a climatic gradient. J. Geophys. Res. 113, http://dx.doi.org/10.1029/2007JG000642.

avies, W.J., Hartung, W., 2004. Has extrapolation from biochemistry to cropfunctioning worked to sustain plant production under water scarcity? In:Proceeding of the Fourth International Crop Science Congress, Brisbane,Australia, September 26–October 1, 2004.

avies, J.N., 1964. Effect of nitrogen, phosphorous, and potassium fertilizer on thenonvolatile organic acids of tomato fruit. J. Sci. Food Agric. 15, 665–673.

avis, J.N., Cocking, E.C., 1965. Changes in the rbohydrates, proteins and nucleic acidsduring cellular development in tomato fruit locule tissue. Planta 67, 242–246.

iaz-Espejo, A., Buckley, T.N., Sperry, J.S., Cuevas, M.V., de Cires, A., Elsayed-farag,S., Martin-Palomo, M.J., Muriel, J.L., PerezMartin, A., Rodriguez-Dominguez,C., Rubio-Casal, A., Torres-Ruiz, J.M., Fernández, J.E., 2012. Steps towardan improvement in process-based models of water use by fruit trees: acase study in olive. Agric. Water Manage. 114, 37–49, http://dx.doi.org/10.1016/j.agwat.2012.06.027.

odd, I.C., 2007. Soil moisture heterogeneity during deficit irrigation altersroot-to shoot signalling of abscisic acid. Funct. Plant Biol. 34, 439–448,http://dx.doi.org/10.1071/FP07009.

odd, I.C., 2009. Rhizposphere manipulations to maximize ‘crop per drop’during deficit irrigation. J. Exp. Bot. 60, 2454–2459, http://dx.doi.org/10.1093/jxb/erp192.

oheny-Adams, T., Hunt, L., Franks, P.J., Beerling, D.J., Gray, J.E., 2012. Geneticmanipulation of stomatal density influences stomatal size, plant growth andtolerance to restricted water supply across a growth carbon dioxide gradi-ent. Philos. Trans. R. Soc. Lond. B: Biol. Sci. 367, 547–555, http://dx.doi.org/10.1098/rstb.2011.0272.

orji, K., Behboudian, M.H., Zegbe-Domínguez, J.A., 2005. Water relations, growth,yield, and fruit quality of hot pepper under deficit irrigation and partial rootzonedrying. Sci. Hort. 104, 137–149, http://dx.doi.org/10.1016/j.scienta.2004.08.015.

ry, P.R., Loveys, B.R., Botting, D., During, H., 1996. Effects of partial rootzone dryingon grapevine vigour, yield, composition of fruit and use of water. In: Stockley,C.S., Sas, A.N., Johnstone, R.S., Lee, T.H. (Eds.), Proceedings of the 9th Aus-tralian Wine Industry Technical Conference. Winetitles, Adelaide, Australia, pp.126–131.

nglish, M.J., Musick, J.T., Murty, V.V.N., 1990. Deficit irrigation. In: Hoffman, G.J.,Howell, T.A., Solomon, K.H. (Eds.), Management of Farm Irrigation System. Amer-ican Society of Agricultural Engineers, St. Joseph, pp. 631–663.

ernández, J.E., Díaz-Espejo, A., Infante, J.M., Durán, P., Palomo, M.J., Chamorro, V.,Girón, I.F., Villagarcía, L., 2006. Water relations and gas exchange in olive trees

anagement 138 (2014) 37–44

under regulated deficit irrigation and partial rootzone drying. Plant Soil. 284,273–291, http://dx.doi.org/10.1007/s11104-006-0045-9.

Franks, P.J., Farquhar, G.D., 2007. The mechanical diversity of stomata and its signif-icance in gas exchange control. Plant Physiol. 143, 78–87, http://dx.doi.org/10.1104/pp.106.089367.

Fraser, L.H., Greenall, A., Carlyle, C., Turkington, R., Friedman, C.R., 2009. Adaptivephenotypic plasticity of Pseudoroegneria spicata: response of stomatal density,leaf area and biomass changes in water supply and increased temperature. Ann.Bot. 103, 769–775, http://dx.doi.org/10.1093/aob/mcn252.

Ismail, M.R., Iberahim, I., 2003. Towards sustainable management of environmen-tal stress for crop production in the tropics. Food Agric. Environ. 1 (3&4),300–303.

Kammann, C.I., Sebastian, L., Johannes, W.G., Hans-Werner, K., 2011. Influ-ence of biochar on drought tolerance of Chenopodium quinoa Willdand on soil–plant relations. Plant Soil 345, 195–210, http://dx.doi.org/10.1007/s11104-011-0771-5.

Klamkowski, K., Treder, W., 2006. Morphological and physiological responses ofstrawberry plants to water stress. Agric. Conspec. Sci. 71 (4), 59–165.

Lehmann, J., 2007. A handful of carbon. Nature 447, 143–144, http://dx.doi.org/10.1038/447143a.

Lehmann, J., da Silva, J.P., Rondon, M., Cravo, M.S., Greenwood, J., Nehls, T., Steiner, C.,Glaser, B., 2002. Slash-and-char: a feasible alternative for soil fertility manage-ment in the central Amazon? Paper no. 449. In: Proceedings of the 17th WorldCongress of Soil Science, Bangkok, Thailand, 14–21 August 2002.

Liu, F., Jensen, C.R., Shahnazari, A., Andersen, M.N., Jacobsen, S.E., 2005. ABA reg-ulated stomatal control and photosynthetic water use efficiency of potato(Solanum tuberosum L.) during progressive soil drying. Plant Sci. 168, 831–836,http://dx.doi.org/10.1016/j.plantsci.2004.10.016.

Liu, F., Shahnazari, A., Andersen, M.N., Jacobsen, S.E., Jensen, C.R., 2006. Physiologi-cal responses of potato (Solanum tubersum L.) to partial root-zone drying: ABAsignaling, leaf gas exchange, and water use efficiency. J. Exp. Bot. 57, 3727–3735,http://dx.doi.org/10.1093/jxb/erl131.

Mitchell, J.P., Shennan, C., Grattan, S.R., May, D.M., 1991. Tomato fruit yieldsand quality under water deficit and salinity. J. Am. Soc. Sci. Hortic. 116,215–221.

Nautiyal, S., Badola, H.K., Negi, D.S., 1994. Plant responses to water stress: changesin growth, dry matter production, stomatal frequency and leaf anatomy. Biol.Plant. 36, 91–97.

Novak, J.M., Lima, I., Xing, B.S., Gaskin, J.W., Steiner, C., Das, K.C., Ahmedna, M.,Rehrah, D., Watts, D.W., Busscher, W.J., Schomberg, H., 2009. Characterizationof designer biochar produced at different temperatures and their effects on aloamy sand. Ann. Environ. Sci. 3, 195–206.

Premchandra, G.S., Saneoka, H., Ogata, S., 1990. Cell membrane stability, an indicatorof drought tolerance as affected by applied nitrogen in soybean. J. Agric. Sci.Camb. 115, 63–66.

Sairam, R.K., 1994. Effect of moisture stress on physiological activities of two con-trasting wheat genotypes. Ind. J. Exp. Biol. 32, 594–597.

Sánchez-Rodríguez, E., Leyva, R., Constán-Aguilar, C., Romero, L., Ruiz, J.M.,2011. Grafting under water stress in tomato cherry: improving the fruityield and quality. Ann. App. Biol. 161, 302–312, http://dx.doi.org/10.1111/j.1744-7348.2012.00574.x.

Savic, S., Stikic, R., Zaric, V., Vucelic-Radovic, B., Jovanovic, Z., Marjanovic, M., Djord-jevic, S., Petkovic, D., 2011. Deficit irrigation technique for reducing water useof tomato under polytunnel conditions. J. Cen. Eur. Agric. 12 (4), 597–607,http://dx.doi.org/10.5513/JCEA01/12.4.960.

Shabala, L., Mackay, A., Tian, Y., Jacobsen, S.E., Zhou, D., Shabala, S., 2012. Oxidativestress protection and stomatal patterning as components of salinity tolerancemechanism in quinoa (Chenopodium quinoa Willd.). Physiol. Plant. 146 (1),26–38, http://dx.doi.org/10.1111/j.1399-3054.2012.01599.x.

Smart, R.E., 1974. Rapid estimate of relative water content. Plant Physiol. 53,258–260.

Sohi, S.P., Krull, E., Lopez-Capel, E., Bol, R., 2010. A review ofbiochar and its use and function in soil. Adv. Agron. 105, 47–82,http://dx.doi.org/10.1016/j.bbr.2011.03.031.

Spence, R.D., Wu, H., Sharpe, P.J.H., Clark, K.G., 1986. Water stress effects on guardcell anatomy and the mechanical advantage of the epidermal cells. Plant CellEnviron. 9, 197–202, http://dx.doi.org/10.1111/1365-3040.ep11611639.

Streubel, J.D., Collins, H.P., Garcia-Perez, M., Tarara, J., Granatstein, D.,Kruger, C.E., 2011. Influence of contrasting biochar types on five soilsat increasing rates of application. Soil Biol. Biochem. 75, 1402–1413,http://dx.doi.org/10.2136/sssaj2010.0325.

Ventura, M., Sorrenti, G., Panzacchi, P., George, E., Tonon, G., 2013. Biochar reducesshort-term nitrate leaching from A horizon in an apple orchard. J. Environ. Qual.42, 76–82, http://dx.doi.org/10.2134/jeq2012.0250.

Wilkinson, S., Hartung, W., 2009. Food production: reducing water consumptionby manipulating long-distance chemical signalling in plants. J. Exp. Bot. 60,1885–1891, http://dx.doi.org/10.1093/jxb/erp121.

Yan, F., Sun, Y., Song, F., Liu, F., 2012. Differential responses of stoma-tal morphology to partial root-zone drying and deficit irrigation inpotato leaves under varied nitrogen rates. Sci. Hortic. 145, 76–83,

http://dx.doi.org/10.1016/j.bbr.2011.03.031.

Zahir, A.Z., Akhtar, S.S., Ahmad, A., Saifullah, Nadeem, S.M., 2012. Comparative effec-tiveness of Enterobacter aerogenes and Pseudomonas fluorescens for mitigatingthe depressing effect of brackish water on maize. Int. J. Agric. Biol. 14 (3),337–344.