ORIGINAL PAPER

Growth and inorganic composition of ‘Nova’ mandarin plantsgrafted on two commercial rootstocks in response to salinityand silicon

Zacharoula Kostopoulou • Ioannis Therios

Received: 12 March 2013 / Revised: 28 September 2013 / Accepted: 11 March 2014 / Published online: 6 April 2014

� Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2014

Abstract The effects of salinity and its combination with

silicon (Si) were studied in ‘Nova’ mandarin plants grafted

on Citrus aurantium L. or Swingle Citrumelo to determine:

(1) which combination is more tolerant to salt stress and (2)

the impact of Si in limiting the harmful effects of salinity.

Six groups of plants were grown in a greenhouse for

120 days and irrigated with: (1) 50 % Hoagland’s solution

(Control), (2) 50 % Hoagland’s solution plus 80 mM NaCl

(NaCl), and (3) 50 % Hoagland’s solution plus 80 mM

NaCl plus 0.5 mM Si (NaCl ? Si). Grafted plants exhib-

ited accumulation of Na and Cl in their tissues following

exposure to salinity. The ability of S. Citrumelo to retain

the toxic ions in the roots in corroboration with the

observation that the dry weights (DWs) of S. Citrumelo

tissues were not influenced by NaCl treatment indicates

that this rootstock is more tolerant to salinity. Silicon

supplementation into the saline medium promoted the

accumulation of toxic ions, whereas, when compared to

NaCl treatment, it increased the DW of S. Citrumelo roots.

Mineral concentrations were significantly affected by

rootstock, treatment, and their interaction with S. Citru-

melo, which presented better nutrient status than Sour

Orange; and Si which differed depending on citrus tissue. It

appears that S. Citrumelo rootstock is the most tolerant for

‘Nova’ mandarin plants under salinity, whereas salt

tolerance in grafted citrus plants is not improved by Si

application, indicating that the beneficial role of Si depends

on the cultivar or rootstock–scion combinations.

Keywords Citrus aurantium L. � Grafting � Growth �Salt stress � Silicon � Mineral nutrients

Abbreviations

DW Dry weight

LSD Least significant difference

SE Standard error

Introduction

Salinity, already one of the most acute plant growth

stresses, is becoming an even more serious concern as

world desertification is on the increase (Janz et al. 2012).

Under saline conditions, growth reduction (Morais et al.

2012), nutrient imbalance, and ion toxicity are caused by

high Na and Cl concentrations (Daei et al. 2009). One way

to avoid or reduce losses in plant production caused by

adverse soil salinity would be to graft them on rootstocks

capable of reducing the biological impact of external salt

stresses on the aboveground cellular metabolism (Edelstein

et al. 2011). Another way is the exogenous application of

‘defense-promoting compounds’ as an alternative strategy

to enhance salinity tolerance of crops which will eventually

improve crop productivity under high salinity. Although

silicon (Si) is the second most abundant element on earth,

its role in plant biology has not yet been fully understood.

Recent evidence suggests that Si is a beneficial element for

the growth of higher plants, especially for those grown

Communicated by L. Bavaresco.

Z. Kostopoulou (&) � I. Therios

Laboratory of Pomology, Department of Horticulture, School of

Agriculture, Aristotle University of Thessaloniki,

54124 Thessalonıki, Greece

e-mail: zrkostopoulou@gmail.com

I. Therios

e-mail: therios@agro.auth.gr

123

Acta Physiol Plant (2014) 36:1363–1372

DOI 10.1007/s11738-014-1515-y

under stressed environments (Ma and Yamaji 2008),

including salinity (Epstein 1999). It has also been shown

that Si supply improves salinity tolerance in many plant

species, such as barley (Liang et al. 1996, 2003; Liang

1999), broad bean (Shahzad et al. 2013), canola (Farshidi

et al. 2012), common bean (Zuccarini 2008), cucumber

(Zhu et al. 2004), maize (Wang et al. 2004), rice (Matoh

et al. 1986; Yeo et al. 1999), spinach (Eraslan et al. 2008),

sugarcane (Ashraf et al. 2009), sunflower (Saqib et al.

2011), tomato (Al-aghabary et al. 2004; Romero-Aranda

et al. 2006), and wheat (Ahmad et al. 1992; Tuna et al.

2008; Ali et al. 2012; Tahir et al. 2012). In the present

study, an important horticultural crop, citrus tree, was

selected as the test plant material as it is relatively sensitive

to salinity (Dambier et al. 2011). In addition, citrus trees

represent an interesting experimental grafting model for

salinity studies. In fact, it is believed that the physiological

basis for citrus tolerance to salt stress is mostly related to

the Cl exclusion capacity, or to the plant ability to restrict

Cl uptake and transport from roots to leaves, a mechanism

whose efficiency is particularly dependent upon rootstock

performance (Brumos et al. 2010). Although citrus is not

considered as a Si-accumulating plant (Wutcher 1989), a

reaction to Si fertilizer of citrus grown under biotic and

abiotic stresses has been recorded (Matichenkov et al.

1999, 2001). On this basis, the aim of the present study was

to investigate whether grafting of ‘Nova’ mandarin plants

onto the rootstocks Sour Orange and Swingle Citrumelo

could improve salinity tolerance. In addition, the impact of

exogenous Si application to salinized substrate on

enhancing salt acclimation of the ‘Nova’, a valuable

commercial mandarin cultivar, was investigated.

Materials and methods

Plant material and treatments

The experiment was conducted in a greenhouse at the

Experimental Farm of the Aristotle University of Thessa-

loniki (Northern Greece) in 2011. One-year-old ‘Nova’

hybrid [C. clementina hort. ex Tan. 9 (C. reticulata

Blanco 9 C. paradisi Macf.)] grafted on Sour Orange

(Citrus aurantium L.) or on Swingle Citrumelo [C. paradisi

Macf. 9 Poncirus trifoliata (L.) Raf.] was used. All plants

were uniform in stem diameter and height. All lateral

shoots of the scion and the major part of the root system

were cut, while the main shoot of the scion was defoliated

and decapitated 5 cm above the grafting union. Subse-

quently, the grafted plants were transplanted into 10 L

plastic pots filled with sand:perlite (1:1, v/v) mixture. After

transplanting, the plants were transferred to a greenhouse

and were irrigated for a month with good quality tap water,

until new shoots appeared. Experimental plants were then

divided into three groups per rootstock–scion combina-

tions, randomly distributed in the greenhouse, and each day

were irrigated with 200 mL of half-strength Hoagland

nutrient solution (Hoagland and Arnon 1938). The exper-

imental design was completely randomized and consisted

of the following treatments for the ‘Nova’ mandarin graf-

ted on two rootstocks: (1) Control: only 50 % Hoagland’s

nutrient solution, (2) NaCl treatment: 50 % Hoagland’s

nutrient solution plus 80 mM NaCl, and (3) NaCl ? Si

treatment: 50 % Hoagland’s nutrient solution plus 80 mM

NaCl and 0.50 mM Si. Sodium metasilicate (Na2SiO3) was

used as the source of Si. To avoid accumulation of salt in

the substrate, the pots were irrigated with distilled water

every 15 days to the point of draining. The experiment

lasted for 120 days until visible typical leaf salt toxicity

symptoms appeared. Afterwards, the grafted plants were

divided into shoots and leaves of the scion, as well as shoot

and root of the rootstock.

Growth and mineral content analysis

The collected samples (shoots and leaves of the scion as

well as shoots and roots of the rootstocks) were washed

initially with tap and afterwards with distilled water, oven-

dried at 75 �C for 2 days, weighed [dry weight (DW)] and

milled to a fine powder so as to pass a 30-mesh screen. The

Cl concentration was measured by titration with AgNO3

according to Kolthoff and Kuroda (1951). Na, K, Ca, Mg,

Fe, Mn, and Zn concentrations were determined by atomic

absorption spectrometry (Perkin-Elmer 2380, Norway)

following standard procedure and P was measured spec-

trophotometrically at 470 nm using the phosphovanado-

molybdate yellow method (Page et al. 1982).

Statistical analysis

Data were analysed with the statistical program SPSS

v15.0 (SPSS Inc., Chicago, IL, USA) and means compared

by the least significant difference (LSD) test at the 0.05

level of confidence. Means of ionic composition were also

compared at 0.01 and 0.001 level of confidence. The LSD

test was used to analyse differences among treatment

means according to two-way ANOVA test.

Results

Growth parameters

To investigate the impact of rootstock, NaCl and Si

application on the growth of grafted citrus trees, the DW of

the different tissues was measured (Table 1). Treatment

1364 Acta Physiol Plant (2014) 36:1363–1372

123

with 80 mM NaCl for 120 days caused a significant

reduction in the DW of ‘Nova’ mandarin leaves grafted on

Sour Orange (73.9 %) and on Swingle Citrumelo (43.1 %)

compared to the control. However, ‘Nova’ plants grown

under NaCl stress showed less DW reduction in their

leaves when grafted on S. Citrumelo than on Sour Orange.

In addition, salinity reduced the DW of mandarin shoots

grafted on Sour Orange by 70.9 %, whereas the DW of

mandarin shoots grafted on S. Citrumelo was similar to the

control. The DW of mandarin leaves grafted on both

rootstocks was not affected by Si supply under saline

conditions compared to NaCl-treated plants, although those

grafted on S. Citrumelo exhibited higher DW (Table 1).

Similarly, the shoots of S. Citrumelo presented higher DW

values in the saline treatments than those of Sour Orange

(Table 1). In response to NaCl treatment, the DW of the

shoots and roots of Sour Orange was decreased (57.8 and

52.6 %, respectively), whereas the DW of the corre-

sponding tissues of S. Citrumelo remained unaffected

compared to the control. The addition of Si did not influ-

ence the DW of the shoots in either of the rootstocks. As a

result of Si application to the saline medium, the DW of the

S. Citrumelo roots was increased (23.9 %) in comparison

to NaCl treatment. In contrast, the DW of Sour Orange

roots exposed to Si supply remained at a similar level with

the salt-stressed roots. The DW of S. Citrumelo roots

exhibited higher values than those of Sour Orange in saline

conditions, whereas the opposite result was obtained under

non-saline conditions.

Na and Cl concentration

Na and Cl were examined to discover if there is a differ-

ence in their concentration in the tissues of grafted citrus

plants. Treatment with NaCl resulted in an accumulation of

Na in ‘Nova’ mandarin tissues, irrespectively of the

rootstock. However, mandarin leaves grafted on Sour

Orange accumulated more Na than those grafted on S.

Citrumelo (Fig. 1a), while mandarin shoots presented

almost the same Na concentration when grafted on both

rootstocks (Fig. 1b). Exogenously applied Si caused further

Na accumulation in mandarin leaves grafted on both

rootstocks with a higher leaf Na level on Sour Orange.

Meanwhile, Na concentration was increased by 25.1 % in

‘Nova’ mandarin shoots grafted on S. Citrumelo and

exposed to Si and NaCl, whereas Si had no effect on the Na

level in mandarin shoots grafted on Sour Orange (Fig. 1b).

Under saline conditions, the Na concentrations in the

shoots of both rootstocks were higher compared to the

controls, with the Sour Orange shoots exhibiting higher Na

values than the S. Citrumelo ones (Fig. 1c). Although the

interaction between NaCl and Si promoted Na accumula-

tion in the shoots of Sour Orange, the Na concentration in

S. Citrumelo shoots was not affected by Si treatment.

When compared to the control, it was found that the Na

concentration in the roots of both Sour Orange and S. Ci-

trumelo (23.8 and 54.4 %, respectively) increased due to

salinity. Na concentrations, however, showed no difference

between NaCl and NaCl ? Si treatment in the roots of

either rootstock (Fig. 1d). On the other hand, Cl concen-

tration increased in the salt-stressed mandarin leaves

grafted on both rootstocks, with those grafted on Sour

Orange having higher values than those grafted on S. Ci-

trumelo. Compared to NaCl treatment, while the level of Cl

concentration in Si-treated mandarin leaves grafted on Sour

Orange remained the same, an increase of 27.6 % was

observed in those grafted on S. Citrumelo. Also, salinity

seems to have had the same level of increase on Cl accu-

mulation in mandarin shoots grafted on both rootstocks

(Fig. 2b) in comparison to the control. The combination of

Si and NaCl in the nutrient solution did not modify the

level of Cl in mandarin shoots of the ‘Nova’ grafted on

Table 1 The effect of NaCl and Si additions to the nutrient media on the DW of leaves and stems of ‘Nova’ mandarin scion as well as the shoots

and roots of Sour Orange and Swingle Citrumelo rootstocks

Rootstock Treatment Dry weight of grafted plants (g plant-1)

Tissues

Leaves Stem Shoot of rootstock Roots

Sour Orange Control 16.22 ± 0.33c 10.53 ± 0.48c 23.35 ± 3.55b 38.25 ± 3.03d

NaCl 4.22 ± 0.35a 3.06 ± 0.42a 9.85 ± 1.06a 18.13 ± 0.27a

NaCl ? Si 3.56 ± 0.12a 4.84 ± 1.15a 11.75 ± 0.76a 20.49 ± 1.2ab

Swingle Citrumelo Control 15.17 ± 0.27c 10.11 ± 0.64bc 22.31 ± 0.26b 24.32 ± 0.81b

NaCl 8.63 ± 0.33b 7.69 ± 0.6b 19.39 ± 0.49b 23.83 ± 1.11b

NaCl ? Si 8.06 ± 1.28b 9.12 ± 0.99bc 21.93 ± 0.64b 31.35 ± 1.6c

Dry weight was evaluated in the leaves and stems of ‘Nova’ mandarin scion as well as in the shoots and roots of the rootstocks

Different letters in each tissue indicate significant differences according to the LSD test (P \ 0.05). Values are the means of three replicate

samples ± SE

Acta Physiol Plant (2014) 36:1363–1372 1365

123

Sour Orange, although it did increase Cl concentration

(25.2 %) in mandarin shoots grafted on S. Citrumelo

(Fig. 2b). Furthermore, there was a striking increase in Cl

concentration in NaCl-treated shoots of both Sour Orange

and S. Citrumelo (Fig. 2c), however, Cl concentration in

the shoots of the rootstocks did not differ on exposure to

NaCl treatment (Fig. 2c). In addition, while Cl concentra-

tion in Sour Orange shoots was unaffected by Si applica-

tion, an increase of 14.5 % was observed in the shoots of S.

Citrumelo (Fig. 2c). The roots of S. Citrumelo accumulated

more Cl than those of Sour Orange when exposed to

salinity. Finally, it appears that Si application did not affect

the Cl concentration in the roots of either species grown

under saline conditions (Fig. 2d).

K, Ca, Mg, P, Fe, Mn, and Zn concentration

The findings showed that mineral nutrient concentrations in

the tissues of grafted plants were altered depending on the

rootstock and salt treatments. It was found that the treat-

ment parameter had a significant influence on the Mg, P,

Mn, and Zn in mandarin ‘Nova’ leaves. More specifically,

it appears that NaCl treatment alone had the following

effects on mandarin leaves: reduced Mg concentration

when leaves were grafted on S. Citrumelo, increased P

concentration when grafted on Sour Orange and it likewise

increased Zn concentration in leaves grafted on both

rootstocks. Mandarin leaves exhibited lower values of P

under control conditions, lower level of Mn and Zn under

non-saline and saline conditions but higher Mg concen-

tration under NaCl treatment when grafted on Sour Orange.

On the other hand, when Si was added, the findings showed

that in the mandarin leaves there were higher levels of Mg

and Mn concentrations when grafted on S. Citrumelo;

lower Zn concentrations on Sour Orange; and lower P on

both rootstocks. Furthermore, the rootstock parameter

significantly influenced the K, P, Fe, Mn, and Zn concen-

trations in mandarin leaves. More specifically, ‘Nova’

leaves grafted on S. Citrumelo showed higher K, Mn, and

Zn concentrations under both non-saline and saline con-

ditions, whereas, there were higher P levels under

unstressed conditions and higher Fe levels under saline

conditions compared to Sour Orange. The interaction

treatment 9 rootstock in ‘Nova’ mandarin leaves was

significant for K, Ca, Mg, Fe, and Mn.

The effect of treatment on nutrient accumulation in

grafted mandarin ‘Nova’ shoots was significant for all the

elements with the exception of Fe. Exposing mandarin

Rootstock x scion combination

0.0

0.5

1.0

1.5

2.0

a

d

e

b

a

c

Nova/Sour Orange

Nova/Citrumelo

Na

conc

entr

atio

n (%

DW

)

a

0.0

0.1

0.2

0.3

0.4

a

c

d

b

a

bc

Nova/Sour Orange

Nova/Citrumelo

Na

conc

entr

atio

n (%

DW

)

c

0.0

0.5

1.0

1.5

abc

c

d

ab

d

Nova/Sour Orange

Nova/Citrumelo

Na

conc

entr

atio

n (%

DW

)

d

0.0

0.5

1.0

1.5

2.0

a

bb b

a

c

Nova/Sour Orange

Nova/Citrumelo

Na

conc

entr

atio

n (%

DW

)

bControl

NaCl

NaCl + Si

Fig. 1 The effect of NaCl and Si additions to the nutrient solution on

Na concentration of ‘Nova’ mandarin plants grafted on Sour Orange

and Swingle Citrumelo. Measurements were contacted in the leaves

(a) and shoots (b) of ‘Nova’ mandarin scion as well as in the shoots

(c) and roots (d) of the rootstocks. Bars are mean ± standard error of

three replicates. Means with different letters above bars were

significantly different at P B 0.05 level applying LSD test

1366 Acta Physiol Plant (2014) 36:1363–1372

123

shoots to NaCl alone decreased the concentrations of K,

Ca, Mg, Mn when grafted on Sour Orange and Mg when

grafted on S. Citrumelo. However, NaCl treatment had the

effect of increasing Ca and Zn concentrations in ‘Nova’

shoots grafted on S. Citrumelo and on both rootstocks,

respectively. Concerning supplementary Si in the saline

medium, this had the effect of increasing the Ca concen-

tration in mandarin shoots when grafted on Sour Orange,

the Mg concentration when grafted on S. Citrumelo, and

the Mn concentration when grafted on both rootstocks but

decreasing the P concentration when grafted on both

rootstocks, when compared to NaCl treatment alone. Fur-

thermore, in the shoots of the scion, the concentrations of

K, Ca, Mg, Fe, Mn, and Zn were affected by the rootstock

parameter, whereas the interaction treatment 9 rootstock

influenced Ca, Fe, and Mn levels.

The shoots of the rootstocks displayed differences in K,

Ca, P, Fe, and Mn concentrations in both the control and

saline treatments. More specifically, salinized shoots of

both rootstocks exhibited a reduction in K and P concen-

trations, whereas there was an increase in Ca and Fe con-

centrations in Sour Orange shoots. The addition of Si in the

nutrient solution reduced the Fe level in Sour Orange but

increased the levels of K, Fe, and Mn in S. Citrumelo.

Finally, K, Ca, Mg, Fe, and Zn levels were significantly

affected by the rootstock parameter, whereas the interac-

tion treatment 9 rootstock influenced only Ca, Mg, and Fe

concentrations in the shoots of the rootstocks.

The nutrient status of Sour Orange and S. Citrumelo

roots was significantly influenced by the treatment

parameter. More specifically, salinity caused a reduction in

the concentrations of K, Mg, and P in the roots of Sour

Orange and Mg and Mn in those of S. Citrumelo. Si

addition in the salinized roots decreased the P level in both

rootstocks and Mn level in S. Citrumelo roots, but

increased Zn in the Sour Orange roots. The rootstock

parameter affected the concentrations of K, Ca, Mg, P, and

Mn in roots, where the highest values were found in those

of S. Citrumelo. Lastly, the interaction treatment 9 root-

stock affected Ca, P, Mn, and Zn levels.

Discussion

Because of their potential to influence entire food webs and

ecosystems, defense interactions between scions and root-

stocks in fruit trees are highly relevant from an ecological

point of view. As citrus plants are relatively salt sensitive

(Sykes 2011), the present study investigated whether the

grafting of ‘Nova’ mandarin scion on Sour Orange or on

0

1

2

3

4

a

dcd

b

a

c

Nova/Sour Orange

Nova/Citrumelo

Cl c

once

ntra

tion

(% D

W)

a

0.0

0.5

1.0

1.5

2.0

a

bb b

a

c

Nova/Sour Orange

Nova/Citrumelo

Cl c

once

ntra

tion

(% D

W)

b

0.0

0.2

0.4

0.6

0.8

a

bb b

a

c

Nova/Sour Orange

Nova/Citrumelo

Cl c

once

ntra

tion

(% D

W)

c

0.0

0.5

1.0

1.5

2.0

2.5

a

b b

c

a

c

Nova/Sour Orange

Nova/Citrumelo

Cl c

once

ntra

tion

(% D

W)

d

Control

NaCl

NaCl + Si

Rootstock x scion combination

Fig. 2 The effect of NaCl and Si additions to the nutrient solution on

Cl concentration of ‘Nova’ mandarin plants grafted on Sour Orange

and Swingle Citrumelo. Measurements were contacted in the leaves

(a) and shoots (b) of ‘Nova’ mandarin scion as well as in the shoots

(c) and roots (d) of the rootstocks. Bars are mean ± standard error of

three replicates. Means with different letters above bars were

significantly different at P B0.05 level applying LSD test

Acta Physiol Plant (2014) 36:1363–1372 1367

123

Swingle Citrumelo rootstocks could improve salinity tol-

erance. It is generally accepted that the first symptom of

salt-stressed plants is a reduction in growth resulting in

yield loss (Garcia-Legaz et al. 2005). The growth of

mandarin leaves expressed as DW, under our experimental

conditions, declined in response to 80 mM NaCl treatment,

irrespectively of the rootstock (Table 1). Nevertheless,

there were significant differences in the growth pattern

between the two types of grafted plants. In fact, the root

growth of Sour Orange was reduced by NaCl treatment,

whereas that of S. Citrumelo was unaffected (Table 1),

indicating that the former is more sensitive to salinity. This

finding is supported by the observation that the reduction of

growth was significantly higher in the mandarin leaves

grafted on Sour Orange than in those grafted on S. Citru-

melo (Table 1). This implies that the combination of

Swingle Citrumelo 9 Nova is less sensitive to salt stress

than that of Sour Orange 9 Nova.

The differential responses of fruit yield among graft

combinations were tightly linked to the different abilities of

rootstocks to regulate the transport of saline ions, as it

appears that the grafted plants that were most productive

maintained relatively low levels of leaf Na and Cl concen-

trations (often termed ‘salt exclusion mechanism’) (Brumos

et al. 2010; Garcia-Sanchez et al. 2006a, b; Melgar et al.

2008; Gimeno et al. 2009, 2010; Forner-Giner et al. 2011). In

the present study, the concentrations of Na and Cl in the

tissues of grafted citrus plants increased under salinity

(Figs. 1, 2, respectively). Furthermore, ‘Nova’ mandarin

plants grown under NaCl treatment contained less Na in

their leaves when grafted on S. Citrumelo than on Sour

Orange (Fig. 1a). Also the NaCl-treated S. Citrumelo roots

accumulate more Na than those of Sour Orange (Fig. 1d),

whereas the exact opposite results were observed in the Na

level in the shoots of the rootstocks (Fig. 1c). It has been

established that the effect of salinity on citrus is associated

with the accumulation of Cl in photosynthetic tissues (Sto-

rey and Walker 1999). In the present study, it was found that

S. Citrumelo roots treated with NaCl have a relatively higher

Cl accumulation (Fig. 2d), and the ‘Nova’ leaves of the S.

Citrumelo rootstock contained significantly lower Cl

(Fig. 2a) than those of Sour Orange. Thus, it can be noticed

that S. Citrumelo limits Cl transport to the leaves storing

more of it in the roots. Sour Orange, on the other hand, was

found to have transported the two toxic ions in the leaves of

‘Nova’. Overall, it appears then that S. Citrumelo rootstocks

are able to reduce the NaCl toxicity effects on the ‘Nova’

mandarin scion.

On account of the growing evidence which suggests that

Si application is involved in salinity stress tolerance

(Matoh et al. 1986; Ahmad et al. 1992; Liang et al. 1996;

Liang 1999; Yeo et al. 1999; Liang et al. 2003; Al-ag-

habary et al. 2004; Wang et al. 2004; Zhu et al. 2004;

Romero-Aranda et al. 2006; Eraslan et al. 2008; Tuna et al.

2008; Zuccarini 2008; Ashraf et al. 2009; Saqib et al. 2011;

Ali et al. 2012; Farshidi et al. 2012; Tahir et al. 2012;

Shahzad et al. 2013), we decided to also investigate whe-

ther an exogenous Si supply could change grafted citrus

plant behavior in relation to salinity. The findings showed

that even though the tissue growth of grafted plants was

generally unaffected by Si application under salinity in

comparison to NaCl treatment, surprisingly, there was an

increase in the root growth of S. Citrumelo when Si was

combined with NaCl (Table 1). This unexpected Si-medi-

ated positive effect on root growth could be attributed to its

ability to modify cell wall metabolism by improving its

extensibility and consequently enhancing root cell

enlargement (Romero-Aranda et al. 2006; Kafi and Rahimi

2011). An increase in root fresh weight has been reported

in grapefruit seedlings grown under salt stress with the

addition of silica slag (Matichenkov et al. 2001). It has

been demonstrated that Si also ameliorated the negative

effects of salinity by inhibiting the uptake of toxic ions by

plants (Yeo et al. 1999; Ma and Yamaji 2008; Ashraf et al.

2009; Ali et al. 2012). The present results, however, do not

confirm this. In comparison to the salt-stressed sample

alone, Si application seems to in many cases have pro-

moted the Na and Cl build up in grafted plant tissue

(Figs. 1, 2). This finding, which was also observed by

Romero-Aranda et al. (2006) in tomato plants and Eraslan

et al. (2008) in spinach, both treated with Si upon salinity,

may indicate that exogenous application of Si stimulates

the absorption and translocation of toxic ions in some plant

species. We assume that, particularly in the case of S.

Citrumelo, such an effect could be due to the higher root

growth of Si-treated plants under salinity (Table 1) which

facilitates the uptake of toxic ions and their transport to

shoots and leaves (Figs. 1a, b, 2a, b). The significance of

this data warrants further study.

NaCl salinity causes the presence of Na and Cl to be

disproportionate in both cellular and extra-cellular com-

partments, having a negative impact on the homeostasis of

essential nutrients (Hu et al. 2006). The general approach

taken in the present research to characterize the way treat-

ment, rootstock and the interaction between them has

affected nutrient concentrations, revealed significant chan-

ges in nutrient concentration in the tissues of citrus plants in

response to NaCl treatment alone (Table 2). Interestingly,

there was an increase in Ca in the mandarin shoots grafted

on S. Citrumelo and an increase in Zn on both rootstocks,

whereas there was a decrease in K, Mg, P in the roots of

Sour Orange, as well as a decrease in Mg, Mn in the roots of

S. Citrumelo under NaCl treatment alone. This confirms

that salt stress induces ion imbalances through complex

competitive interactions (Lutts et al. 1999; Huang et al.

2010; Forner-Giner et al. 2011). Another interesting point

1368 Acta Physiol Plant (2014) 36:1363–1372

123

Ta

ble

2T

he

effe

cto

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and

Si

add

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ns

toth

en

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ien

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edia

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ion

so

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g,

P,

Fe,

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KC

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gP

Fe

Mn

Zn

Lea

ves

So

ur

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ng

eC

on

tro

l1

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±0

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±0

.00

c0

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±0

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b1

10

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cd1

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1.0

0b

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7±

0.3

3a

NaC

l1

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bc

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ab1

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l?

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±1

.15

a

Cit

rum

elo

Co

ntr

ol

1.7

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1b

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±0

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c1

01

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±2

.73

bc

25

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±0

.58

c1

3.0

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0c

NaC

l1

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±0

.04

c3

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±0

.07

bc

0.2

6±

0.0

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1±

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2c

98

.00

±3

.46

bc

29

.00

±2

.00

c1

4.6

7±

0.3

3d

NaC

l?

Si

1.6

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0.0

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4±

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5c

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1b

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±0

.02

ab1

27

.00

±1

2.3

4d

42

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±2

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0.0

0d

Tre

atm

ent

ns

ns

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1.7

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Sh

oo

to

fth

esc

ion

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Ora

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l?

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3b

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0.6

7ab

Cit

rum

elo

Co

ntr

ol

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0.2

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0.0

5a

0.1

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0.0

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0.0

9±

0.0

1b

44

.67

±1

.85

b9

.33

±0

.67

c7

.00

±0

.00

b

NaC

l1

.09

±0

.05

a2

.18

±0

.02

b0

.09

±0

.01

a0

.10

±0

.01

b5

3.3

3±

1.8

5c

9.0

0±

0.0

0c

9.0

0±

0.5

8c

NaC

l?

Si

1.0

4±

0.0

2a

2.1

7±

0.0

4b

0.1

2±

0.0

0b

0.0

6±

0.0

1a

54

.33

±4

.90

c1

2.0

0±

0.0

0d

7.6

7±

0.6

7b

c

Tre

atm

ent

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ns

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ent

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sn

s*

**

*n

s

LS

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0.2

10

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0.0

38

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1.3

31

.57

Sh

oo

to

fth

ero

ots

tock

So

ur

Ora

ng

eC

on

tro

l0

.34

±0

.04

cd0

.97

±0

.02

a0

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±0

.00

b0

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±0

.00

b3

0.6

7±

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3a

6.0

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0a

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3ab

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l0

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±0

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a1

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l?

Si

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0.0

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3±

0.0

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13

3.6

7±

14

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±0

.88

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±0

.67

ab

Cit

rum

elo

Co

ntr

ol

0.3

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0.0

2d

0.9

0±

0.0

5a

0.0

4±

0.0

0b

0.0

4±

0.0

0b

61

.00

±3

.05

b5

.67

±0

.88

a2

.67

±0

.33

ab

NaC

l0

.20

±0

.01

b0

.84

±0

.03

a0

.06

±0

.00

d0

.02

±0

.00

a5

5.0

0±

4.1

6ab

6.0

0±

0.5

8a

3.3

3±

0.3

3b

NaC

l?

Si

0.2

9±

0.0

2c

0.8

2±

0.0

2a

0.0

5±

0.0

0c

0.0

4±

0.0

0ab

10

4.6

7±

12

.33

c8

.33

±0

.33

b3

.33

±0

.33

b

Tre

atm

ent

**

**

*n

s*

**

**

**

ns

Ro

ots

tock

**

**

**

**

*n

s*

**

ns

*

Tre

atm

ent

9ro

ots

tock

ns

**

**

*n

s*

**

ns

ns

LS

D0

.06

0.1

60

.01

0.0

22

5.1

01

.83

1.1

9

Acta Physiol Plant (2014) 36:1363–1372 1369

123

concerns the impact of Si supplementation in the homeo-

stasis of nutrients in grafted tissues. The addition of Si, for

instance, generally promoted Mn accumulation in the

majority of tissues (Table 2), indicating that Si has a

positive impact on Mn discrimination in the process of

absorption and transportation in citrus plants. The addition

of Si seems to have the opposite effect on other nutrients.

For example, the reduction of P in the mandarin tissues and

roots of both rootstocks indicates the negative impact of Si

on P accumulation.

The third interesting point concerns the difference in the

ability of the two citrus rootstocks to absorb, retain, and

distribute the ions in the tissues of grafted plants. S. Ci-

trumelo exhibited a higher capacity than Sour Orange in

retaining certain nutrients in the roots (e.g., K, P, Mn, Zn)

and/or in translocating nutrients to the upper tissues (e.g.,

K, Mn, Zn in mandarin leaves and Ca, Fe, Mn, Zn in

mandarin shoots) (Table 2). This behavior could be an

effort to maintain ion homeostasis in order for the grafted

plants to survive against salinity stress, thus, further sup-

porting the hypothesis that Swingle Citrumelo is more

tolerant to salinity than Sour Orange.

In conclusion, the findings in the present study dem-

onstrate that ‘Nova’ mandarin is more tolerant to salinity

when grafted on S. Citrumelo than on Sour Orange, as

evidenced by the growth reduction and the distribution of

toxic ions. Also, it appears that external Si application was

insufficient to improve the performance of the grafted

citrus plants in relation to the saline environment. Finally,

the data seem to suggest that Sour Orange and Swingle

Citrumelo rootstocks quite possibly exclude Si absorption

and/or transport it to the upper parts of the plant, indicating

that the behavior of Si in enhancing salinity tolerance is not

universal but is dependent on the plant species, the root-

stock, the scion, and their combination.

Author contribution Z. Kostopoulou and I. Therios

designed the experiment. Z. Kostopoulou made all the

measurements and the manuscript preparation. I. Therios

contributed to the manuscript with valuable comments and

notes.

Acknowledgments We would like to express our sincere gratitude

to Maria Vitsiou for kindly providing the grafted ‘Nova’ mandarin

plants; also our thanks to S. Kouti and V. Tsakiridou for technical

assistance. The authors gratefully acknowledge the financial support

of the Aristotle University of Thessaloniki.

Conflict of interest The authors declare that they have no conflict

of interest.

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nti

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.91

2.1

0

Mac

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utr

ien

ts(K

,C

a,M

g,

P)

exp

ress

edas

%D

Ww

hil

em

icro

nu

trie

nts

(Fe,

Mn

,Z

n)

asp

pm

Wit

hin

each

colu

mn

,d

iffe

ren

tle

tter

sin

dic

ate

sig

nifi

can

td

iffe

ren

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atP

<0

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(LS

Dte

st).

ns,

*,

**

and

**

*in

dic

ate

no

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ign

ifica

nt

or

sig

nifi

can

td

iffe

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ces

atP

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or

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Val

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are

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of

thre

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cate

s±

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