Ece Turhan, Ş.Şebnem Ellialtıoğlu, Sevinç Kıran, Volkan Alveroğlu, Şebnem Kuşvuran, Çağla Ateş

Turkey

Over 20% of irrigated land is affected by salinity (Rozema and Flowers, 2008)

In arid and semiarid regions of the world, Also less extreme environments

Osmotic effects Nutritional effects Toxic effects

Secondary stress

Primary stress

Salt stress is complex and imposes a water deficit because of osmotic effects on a wide variety of metabolic activities

the formation of reactive oxygen species (ROS) in plants

However, ROS are also used by cells as secondary messengers involved in

the stress-response signal transduction pathway.

ROS accumulate during stress and may lead to cell injury and death.

Plants use two major strategies to control the level of ROS and prevent cellular damage:

Scavenge of ROS by anti-oxidants and anti-oxidative enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and glutathione reductase (GR) (Mittler et al 2004).

Lower the rate of ROS production in cells by supressing ROS-producing reactions and by different stress avoidance strategies.

Under salinity stress the amount of 1-aminocyclopropane-1-carboxylate (ACC), a precursor for ethylene production in plants, increases.

seed germination and root growth may decrease (Belimov et al., 2001; Saravanakumar and Samiyappan, 2007).

Traditional breeding programmes Genetic transformation Use of saline water in irrigated agriculture Tolerant cultivars, Several cultural practices, Some of the proposed practices, The application of chemical amendments or

leaching salts to deeper soil layers One environment-friendly technique is grafting The use of plant growth promoting bacteria

(PGPRs)

better growth and yield, higher photosynthesis and leaf water content, greater root-to-shoot ratio, higher accumulation of compatible osmolytes,

abscisic acid and polyamines in leaves, greater antioxidant capacity in leaves, lower accumulation of Na+ and/or Cl- in shoots

than ungrafted or self-grafted plants (Colla et al.

2010).

(Maheshvari, 2012)

•Many researchers have noted the positive effects of the ACC deaminase-producing bacteria in the rhizosphere in alleviating different stresses including salinity on plant growth (Cheng et al., 2007; Saravanakumar and Samiyappan, 2007).

•PGPR includes bacteria belonging to the genera Azotobacter, Azospirillum, Arthrobacter, Bacillus, Burkholderia, Klebsiella, Pseudomonas and Serratia (Vessey 2003).

Plant Growth-Promoting Rhizobacteria (PGPR)

Rhizobacteria mediated metabolic responses in plants

(Maheshvari, 2012)

The objective of this work was to evaluate the effect of grafting on a salt-tolerant rootstock and inoculation with the 1-aminocyclopropane-1-carboxylate (ACC)-deaminase (EC 4.1.99.4) containing bacteria on eggplant growth, mineral uptake and activities of the antioxidant enzymes including SOD, CAT, APX and GR of plant leaves under salinity stress.

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A salt-tolerant eggplant rootstock: cv.Vista-306

As scions: Two salt-tolerant eggplant genotypes;‘Mardin Kızıltepe’, ‘Burdur Merkez’ and two salt-sensitive eggplant genotypes; ‘Artvin Hopa’, ‘Kemer’.

ACC Deaminase containing bacteria: Serratia marcescens

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The bacteria were isolated from the rhizosphere soil samples from graminae plants growing in Eskisehir, Turkey according to Dworkin and Foster (1958).

The selected strains of the bacteria were stored at −20 °C with 20% glycerol.

ACC-deaminase activity was determined as described by Honma and Shimomura (1978).

PGPR were identified by using the Vitek 2 microbial identification system in Nevşehir Public Health Laboratory. 14

Grafted and non-grafted seedlings with 4-5 true leaves were transferred to pots filled with a mixture of peat and perlite (2:1) in a controlled greenhouse (23-25 °C, approximately 50-55% relative humidity).

Treatment Rootstock Genotype

Control Non-grafted Mardin Kızıltepe

Burdur Merkez

Artvin Hopa

Kemer

Vista-306 Mardin Kızıltepe

Burdur Merkez

Artvin Hopa

Kemer

NaCl Non-grafted Mardin Kızıltepe

Burdur Merkez

Artvin Hopa

Kemer

Vista-306 Mardin Kızıltepe

Burdur Merkez

Artvin Hopa

Kemer

NaCl+S Non-grafted Mardin Kızıltepe

Burdur Merkez

Artvin Hopa

Kemer

Vista-306 Mardin Kızıltepe

Burdur Merkez

Artvin Hopa

Kemer

After 10 days from planting, 20 mL bacteria solution (cell density of 3x108 cells/mL) per plant was applied to root region of the seedlings as inoculum (Alveroğlu, 2014).

Salt solution; 200 mM NaCl was applied, after 5 days from inoculation of the bacteria.

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Shoot fresh weight, shoot dry weight (Dasgan and Koç, 2009; Kuşvuran, 2010)

Stem length, leaf area and SPAD values Lipid peroxidation was estimated by

determining the malondialdehyde (MDA) content in the leaves according to the method of Lutts et al. (1996). The MDA concentration was determined using an extinction coefficient of 155 mM.

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Na+ K+, Ca ++ and Cl− contents of the dried samples digested in concentrated 0.1 N nitric acid (HNO3) and perchloric acid (HClO) (4:1) were determined by flame photometry (Kusvuran

2004). Chloride (Cl) was determined by titration method with 0.1 N AgNO3 (Kacar ve İnal, 2008).

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SOD activity was assayed using NBT method (measured at 560 nm) (Giannapolitis and Ries, 1977).

CAT was assayed from the crude extract by monitoring the consumption of H2O2 at 240 nm (Rao et.al. 1996).

APX activity was determined by measuring decrease in absorbance of the oxidised ascorbate at 290 nm, according to Nakano and Asada (1980).

GR activity was determined by following the oxidation of β-nicotinamide adenine dinucleotide phosphate (NADPH) at 340 nm (extinction coefficient 6.2 mM/cm) (Cakmak and Marschener, 1992).

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The data were subjected to an analysis of variance using the MSTAT-C package program (Freed et al.,1989) and mean separation was accomplished using the LSD test at p ≤ 0.05.

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Mardin Kızıltepe Burdur Merkez Artvin Hopa Kemer

Sh

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(mg

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Control NaCl NaCl+S

Treatment Rootstock Genotype Shoot Fresh Weight

(g plant-1)

Shoot Dry Weight

(mg plant-1)

Control Non-grafted Mardin Kızıltepe 25.28 de 60.25 de

Burdur Merkez 20.36 gh 51.36 j

Artvin Hopa 22.03 fg 55.20 gh

Kemer 27.36 d 59.28 de

Vista-306 Mardin Kızıltepe 21.95 fg 51.14 j

Burdur Merkez 23.86 ef 54.32 g-ı

Artvin Hopa 22.32 fg 62.00 d

Kemer 17.21 ı 53.24 h-j

NaCl Non-grafted Mardin Kızıltepe 19.03 hı 58.36 ef

Burdur Merkez 21.98 fg 61.36 d

Artvin Hopa 17.52 ı 52.92 h-j

Kemer 19.42 hı 56.28 fg

Vista-306 Mardin Kızıltepe 32.76 c 73.21 a

Burdur Merkez 18.01 hı 36.36 n

Artvin Hopa 22.14 fg 41.56 m

Kemer 34.23 bc 71.70 ab

NaCl+S Non-grafted Mardin Kızıltepe 20.09 gh 37.24 n

Burdur Merkez 25.78 de 42.96 lm

Artvin Hopa 36.92 a 69.23 bc

Kemer 19.35 hı 41.59 m

Vista-306 Mardin Kızıltepe 23.95 ef 45.06 kl

Burdur Merkez 35.24 ab 68.39 c

Artvin Hopa 20.11 gh 46.46 k

Kemer 23.57 ef 52.24 ıj

LSD (%5) 2.46 2.83

Treatment Rootstock Genotype Stem length (cm) Leaf area (cm2)

Control Non-grafted Mardin Kızıltepe 30.30 a-e 38.21 a

Burdur Merkez 25.12 g-ı 30.36 fg

Artvin Hopa 29.53 b-f 31.26 d-f

Kemer 34.20 a 38.45 a

Vista-306 Mardin Kızıltepe 28.21 c-h 31.69 c-f

Burdur Merkez 31.51 a-c 34.87 bc

Artvin Hopa 30.42 a-e 38.36 a

Kemer 26.56 e-ı 30.12 fg

NaCl Non-grafted Mardin Kızıltepe 27.84 c-h 31.60 d-f

Burdur Merkez 30.25 a-e 37.56 ab

Artvin Hopa 26.54 e-ı 30.53 e-g

Kemer 28.73 c-g 33.90 cd

Vista-306 Mardin Kızıltepe 34.22 a 38.29 a

Burdur Merkez 24.53 hı 26.32 hı

Artvin Hopa 27.36 d-h 27.98 gh

Kemer 32.86 ab 37.69 ab

NaCl+S Non-grafted Mardin Kızıltepe 24.75 g-ı 30.68 e-g

Burdur Merkez 29.20 b-f 33.61 c-e

Artvin Hopa 33.77 a 38.54 a

Kemer 26.23 f-ı 20.31 k

Vista-306 Mardin Kızıltepe 28.02 c-h 23.32 ı-k

Burdur Merkez 31.00 a-d 37.50 ab

Artvin Hopa 23.10 ı 23.02 jk

Kemer 28.51 c-h 25.62 h-j

LSD (%5) 13.44 3.20

Treatment Rootstock Genotype Chlorophyll

(SPAD value)

MDA

(µ mol/g FW)

Control Non-grafted Mardin Kızıltepe 38.21 a 1.84 mn

Burdur Merkez 30.36 cd 2.39 l

Artvin Hopa 32.26 b-d 2.26 lm

Kemer 38.45 a 1.71 n

Vista-306 Mardin Kızıltepe 31.69 b-d 1.71 n

Burdur Merkez 32.87 bc 1.58 n

Artvin Hopa 38.36 a 1.94 mn

Kemer 30.12 d 1.84 mn

NaCl Non-grafted Mardin Kızıltepe 31.60 b-d 5.23 ıj

Burdur Merkez 37.56 a 5.90 g

Artvin Hopa 30.53 cd 8.61 c

Kemer 33.90 b 9.68 a

Vista-306 Mardin Kızıltepe 38.29 a 4.84 jk

Burdur Merkez 26.32 e 5.13 ıj

Artvin Hopa 26.98 e 7.74 de

Kemer 37.69 a 7.26 f

NaCl+S Non-grafted Mardin Kızıltepe 30.85 cd 5.42 hı

Burdur Merkez 33.61 b 5.81 gh

Artvin Hopa 38.54 a 8.03 d

Kemer 20.31 g 9.06 b

Vista-306 Mardin Kızıltepe 22.49 fg 4.65 k

Burdur Merkez 37.50 a 5.29 l

Artvin Hopa 23.02 f 7.29 f

Kemer 26.62 e 7.48 ef

LSD(%5) 2.55 0.43

Treatment Rootstock Genotype Na(%) Cl(%) K(%) Ca(%)

Control Non-grafted Mardin Kızıltepe 0.57 kl 0.11 j 2.28 0.93

Burdur Merkez 0.57 kl 0.21 b 2.23 1.02

Artvin Hopa 0.47 o 0.15 f 2.34 1.11

Kemer 0.50 n 0.11 j 2.25 1.16

Vista-306 Mardin Kızıltepe 0.53 m 0.15 f 1.91 1.23

Burdur Merkez 0.48 o 0.13 h 1.94 0.95

Artvin Hopa 0.50 n 0.11 j 2.30 1.33

Kemer 0.68 ef 0.21 b 2.22 1.36

NaCl Non-grafted Mardin Kızıltepe 0.69 f 0.16 e 2.12 0.84

Burdur Merkez 0.72 d 0.06 l 2.11 0.80

Artvin Hopa 0.66 gh 0.17 d 1.96 0.89

Kemer 0.82 a 0.16 e 1.98 0.93

Vista-306 Mardin Kızıltepe 0.67 fg 0.12 ı 1.82 1.11

Burdur Merkez 0.56 l 0.21 b 1.80 0.85

Artvin Hopa 0.78 c 0.16 e 1.75 1.07

Kemer 0.80 b 0.12 ı 1.82 1.05

NaCl+S Non-grafted Mardin Kızıltepe 0.64 ıj 0.20 c 2.20 0.88

Burdur Merkez 0.65 hı 0.14 g 2.18 0.86

Artvin Hopa 0.64 ıj 0.09 k 2.07 1.02

Kemer 0.67 fg 0.16 e 2.12 0.99

Vista-306 Mardin Kızıltepe 0.58 k 0.14 g 1.85 1.15

Burdur Merkez 0.54 m 0.12 ı 1.90 0.89

Artvin Hopa 0.65 hı 0.24 a 1.95 1.24

Kemer 0.63 j 0.21 b 1.95 1.11

LSD (%5) 0.016 0.005 - -

0500

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Mardin Kızıltepe Burdur Merkez Artvin Hopa Kemer

Control NaCl NaCl+S

APX

(µ m

ol/

min

/mg

FW

)

Treatment Rootstock Genotype SOD

(U/min/mg FW)

CAT

(µ mol/ min/mg

FW)

GR

(µ mol/ min/mg

FW)

APX

(µ mol/min/mg

FW)

Control Non-grafted Mardin Kızıltepe 174.55 293.29 n-p 14.81 m 2266.67

Burdur Merkez 164.44 319.80 lm 18.67 h-m 2641.98

Artvin Hopa 89.81 274.11 pq 17.22 j-m 1908.50

Kemer 162.22 328.93 ı 19.91 f-k 3346.41

Vista-306 Mardin Kızıltepe 112.89 304.57 m-o 24.72 c-e 2588.24

Burdur Merkez 160.22 284.26 o-q 15.14 lm 2370.37

Artvin Hopa 126.67 269.04 q 15.83 k-m 2222.22

Kemer 190.22 307.61 l-n 18.33 ı-m 2411.11

NaCl Non-grafted Mardin Kızıltepe 295.32 494.37 d-f 24.11 c-f 2433.33

Burdur Merkez 270.71 489.70 ef 22.08 d-ı 2875.00

Artvin Hopa 155.02 399.32 k 19.51 g-l 2341.88

Kemer 180.32 410.15 jk 20.65 e-j 3511.11

Vista-306 Mardin Kızıltepe 340.00 552.74 c 27.04 bc 2962.96

Burdur Merkez 313.68 507.61 de 18.52 ı-m 2518.52

Artvin Hopa 217.17 423.01 ıj 19.72 f-l 2862.75

Kemer 206.35 456.85 gh 19.61 f-l 2784.31

NaCl+S Non-grafted Mardin Kızıltepe 305.88 545.69 c 31.90 a 3142.86

Burdur Merkez 289.23 512.69 d 25.33 cd 2822.22

Artvin Hopa 200.73 418.78 ı-k 21.22 d-j 2481.48

Kemer 171.26 406.09 jk 21.78 d-j 2611.11

Vista-306 Mardin Kızıltepe 395.86 636.83 a 30.33 ab 3263.89

Burdur Merkez 342.96 609.14 b 23.89 c-g 2300.00

Artvin Hopa 261.82 436.55 hı 21.44 d-j 2814.81

Kemer 281.48 477.16 fg 23.11 c-h 2566.67

LSD(%5) - 21.44 4.59 -

Shoot FW and DW, stem length, leaf area and SPAD values were adversely affected by salt stress, but generally salt-tolerant genotypes were comparatively less affected.

Sodium (Na+) and Cl− contents in the leaves were significantly enhanced.

Malondialdehyde (MDA) content, the activities of CAT, GR, APX and SOD in the leaf tissues increased in salt stress conditions.

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Significant differences in cultivar level. The grafted seedlings induced salt tolerance Inoculation of the bacteria significantly

counteracted the salt-induced adverse effects on growth characteristics, leaf K+, Ca++, Cl−, Na+, MDA content and antioxidative enzyme activities.

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To conclude, grafted and Serratia marcescens-inoculated eggplants have a positive effect on growth of eggplant seedlings under salt stress.

Based on these findings, beside grafting method, the biological treatments may help alleviate the negative effect of salinity on the growth of eggplant seedlings.

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There are no results about yield and fruit quality.

In addition, almost no molecular studies regarding the effects of salt on Serratia-eggplant interactions have been carried out so far.

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