Journal of Applied Botany and Food Quality 89, 89 - 97 (2016), DOI:10.5073/JABFQ.2016.089.011 1Department of Horticulture, Faculty of Agriculture and Natural Sciences, Bozok University, Yozgat, Turkey 2Department of Horticulture, Faculty of Agriculture and Natural Sciences, Recep Tayyip Erdogan University, Rize, Turkey 3Department of Horticulture, Faculty of Agriculture, Ataturk University, Erzurum, Turkey Influence of arbuscular mycorrhizae and plant growth promoting rhizobacteria on proline content, membrane permeability and growth of strawberry (Fragaria × ananassa Duch.) under salt stress Aysen Koc1, Gulden Balci1, Yasar Erturk1, Hakan Keles1, Nalan Bakoglu2, Sezai Ercisli3* (Received November 30, 2015) * Corresponding author Summary Salinity is one of the most important factors negatively effecting the yield in crop species. In this study the effect of plant growth promoting rhizobacteria (PGPR) and arbuscular mycorrhizae on proline content, membrane permeability and growth of strawberry cv. ‘San Andreas’ were studied under different salt treatments (0, 30 and 60 mM/L NaCl). The leaf area was measured 0, 15, 30, 45 and 60 days after saline solution applications on the plants. The results showed that increasing concentrations of NaCl decreased all growth parameters. Increased salt concentration led to increased proline level compared to the control. Bacterial application at 60 mM/L NaCl concentration provided the highest ameliorative effect and therefore determined the most effective protection of the plant against salt stress. It was observed that the anthocyanin content increased in line with the increasing salt concentration. In general, the salt applied on the plants causes an increase in membrane permeability and thus disrupts membrane stability and becomes a significant factor damaging the plant. Membrane permeability increased at applications with 30 mM/L and 60 mM/L NaCl. Our results revealed that bacteria application can have an ameliorative effect that helps the plant to tolerate the negative effects of salt stress by increasing proline and anthocyanin levels. Introduction Strawberries (Fragaria × ananassa Duch.) dominate the world berry production and are cultivated in Europe, Asia, North and South America with a big commercial importance (ALKAN TORUN et al., 2014). The total strawberry production of the world is more than 4.516.000 tons with USA taking the first places as highest strawberry producing country (1.367.000 tons), followed by Mexico (360.426 tons), and Turkey (353.173 tons), respectively) (FAO, 2012). Strawberry cultivation is suitable for both greenhouse and open field condition and it has high capability to adapt to diverse ecologic con- ditions. Strawberries are relatively low salt tolerant plants and one of the main problem for their cultivation is salinity that restrict plant growth (KEUTGEN and PAWELZIK, 2009). In particular in greenhouse conditions, salinization is serious problem due to the fact that a certain area of space is used continuously and intensively with in- tense use of salt included fertilizers. It is reported that soil and water salinity decrease the usefulness of ground water and thus affect plant growth negatively (TANJI, 1990). One of the major effects of salinity on plants is the ethylene accu- mulation in their roots, which decrease root growth and finally re- duce the yield of crops. PGPRs are able to produce ACC-deaminase in plants rhizosphere and they can consume pre-produced ethy- lene (ACC) and convert it to α-ketobutyrate and ammonium, so they are able to reduce ethylene level in plants and hence, increase their growth (PENROSE and GLICK, 2003). Arbuscular mycorrhizal (AM) fungi were reported by several researchers as enhancer of root systems and they are known to support stronger, healthier, higher- yielding plants through increased nutrient acquisition (MILLER et al., 2010), reduce levels of water stress (AUGE, 2001), and increase phy- tohormone production (SHAUL-KEINAN et al., 2002). Hence, mycor- rhizal plants are able to increase their tolerance to salinity stress due to their high soil exploration conferred by their hypha structure and growth (MIRANSARI et al., 2008). Anthocyanins are a large class of water soluble pigments in the flavonoid group found in all plant tissues, are largely responsible for coloration in higher plants (IWASHINA, 2000). These substances accumulate in different plant tissues under the influence of various environmental stimuli (GOULD et al., 2000). Previously, it is reported that anthocyanins in plant tissue is increased with elevated salt stress (CHALKER-SCOTT, 1999). It is known that under salt stress conditions, plants increase cellular osmotic pressure by producing secondary metabolites, various che- micals and particularly stress proteins (such as proline, etc.), and thus they sustain their existence by balancing the high osmotic pressure that emerges in the nutrient medium. Physiological reactions of plant species cultivated in media with increasing salinity vary and to avoid negative effects of the osmotic pressure due to increasing salinity, plants increase their proline content. Plants and species capable of generating more proline are more likely to resist the stress and grow healthily (EDREVA, 1998). In this study, the ameliorative effects of some rhizobacteria and mychorriza applications on growth and the biochemical changes of strawberry plants under salt stress were investigated. Material and methods Plant material and site description The day-neutral strawberry cultivar ‘San Andreas’ was used in this study. First class health cold-stored frigo plants (seedlings) were planted in pots (25 × 18 × 20 cm) filled with perlite: turf media (1:1) (SAHIN et al., 2002) at May 6th 2013. The experiment was established with 1 cultivar × 3 salt concentrations (0, 30, 60 mM/L NaCl) × 4 treatments (control, mychorriza, bacteria and mychorizza + bacteria) × 3 repetitions × 20 seedlings in each repetition = 720 seedlings in factorial randomized block design. The experiment was conducted at the Bozok University Gedikhasan experimental farm. Bacterial application The bacteria used in the study were isolated from rhizosphere soils of a total 56 tea growing orchards located different agroclimatic regions in Rize and Trabzon provinces in Black Sea region in Turkey and were identificated according to their fatty acid methyl esters (FAMEs) analysis conducted in Sherlock Microbial Identification System and confirmed with BIOLOG system. Among bacteria 90 A. Koc, G. Balci, Y. Erturk, H. Keles, N. Bakoglu, S. Ercisli isolated two bacteria of them (Bacillus cereus RCP 3/1 + Rhizobium radiobacter RCR 11/2) were selected for their ability to grow in a saline culture medium (10 % sodium chloride (NaCl). The phosphate dissolving and nitrogen fixing capacities of bacteria were determined previously (Tab. 1) (CAKMAKCI et al., 2010). For this experiment, the bacterial strains were grown on nutrient agar. A single colony was transferred to 250-mL flasks containing nutrient broth and grown aerobically in flasks on a rotating shaker (95 rpm) for 24 h at 27 °C. Inoculation of bacterial treatments was performed using a dipping method in which plant roots in celled trays were inoculated with the bacterial suspensions of the concentration of 108 colony-forming units/mL in sterile water for 30 min before planting. Control plants were dipped into sterile water. Mychorriza application In mychorriza application, preparates in commercial powder form containing 9 different Glomus fungi were used. In the preparation fungi types and ratios were; Glomus intraradices (21 %), Glomus aggregatum (20 %), Glomus mosseage (20 %), Glomus clarum (1 %), Glomus monosporus (1 %), Glomus deserticola (1 %), Glomus bra- silianum (1 %), Glomus etunicatum (1 %), and Gigaspora margarita (1 %). Mychorrizal applications were performed by inoculating the plant roots by fungi in the solution prepared by mixing 10-liter water and 250 gr powder in packages before planting. Salt application 40 days after planting (when 3-4 leaves were formed, 17.06.2013) salt solution including 0, 30 and 60 mM/L NaCl were applied with an amount of 100 ml, 2 times a week. Also, in order to ensure plants are nourished, 100 gram of 12-2-14 fertilizer solution (11.7 % nitrate, 0.3 % ammonium, 2 % phosphoric acid, 14 % potassium, 6 % calci- um, 3 % magnesium and trace elements) per pot was applied three times a week. Growth parameters Growth parameters were measured after plants rooted out on 0, 15th, 30th, 45th and 60th days after salt application on the plants. Leaves surface area (LSA) were measured in cm2 by ADC BioScientific Area Meter AM300. The roots and shoot dry weight (RDW and SDW) were determined in a drying cabin immediately after their fresh weights were determined. They were kept in drying cabin until their weights did not change at 65 °C and then, their weights were measured. Weighing operations were performed by balances sensi- tive to 0.001 g. Proline analysis Proline content was determined according to BATES et al. (1973). Leaves were ground and homogenized in sulfosalicylic acid. The homogenate was filtered through filter paper. After the addition of ninhydrin reagent, the mixture was heated to 100 °C for one hour. The reaction was then stopped in ice. The mixture was extracted with 4 ml toluene, and the sample was vigorously shaken for 15 s. Sample absorbance of the toluene layer was read at 520 nm. Proline concen- tration was determined by using a calibration curve and expressed as μM per 100 mg fresh weight (FW). Anthocyanin content The ACM-200 plus Anthocyanin Content Meter provides a fast esti- mate of anthocyanin content on the intact leaves of plants. The non- destructive technique allows researchers to monitor anthocyanin without the costly and time consuming extraction. For each plant, measurements were taken at four locations on each leaf; two on each side of the mid rib on all fully expanded leaves and then averaged (KHAN et al., 2003). Electrolyte leakage (membrane permeability) For measurement of electrolyte leakage, 10 leaf discs (10 mm in diameter) from the young fully expanded leaves from two plants per replicate were placed in 50 mL glass vials, rinsed with distilled water to remove electrolytes released during leaf disc excision. Vials were then filled with 30 mL of distilled water and allowed to stand in the dark for 24 h at room temperature. Electrical conductivity (EC1) of the bathing solution was determined at the end of the incubation period. Vials were heated in a temperature-controlled water bath at 95 ºC for 20 min and then cooled to room temperature and the elec- trical conductivity (EC2) was again measured. Electrolyte leakage (membrane permeability – MP) was calculated as a percentage of EC1/EC2 (SHI et al., 2006). Data analysis The experiment was arranged in a randomized blocks in factorial design with three replications. Data were tested by SPSS 20.0 for Windows program. The differences between the means were compa- red using the Duncan test (P < 5 %). Results and discussion Salt stress is complex and has toxic effects on plants and lead to metabolic changes (LATRACH et al., 2014). Strawberry is considered as a NaCl salinity sensitive species and it has been shown to reduce number of runners, runner length, leaf number, fresh and dry root weight (SAIED et al., 2005). Within the scope of our study, it was determined that effects of salt concentrations, applications, and uprooting days on leaf area are statistically significant (p < 0.05, Fig. 1). Leaf areas were observed to decrease with the increasing concentrations of salt. While the leaf area with 60 mM/L NaCl concentration was determined to be 33.37 cm2, with 0 mM/L NaCl concentration leaf area was measured as 38.43 cm2. In terms of applications, it was observed that mycor- rhiza and bacteria applications have positive impact on leaf area. Tab. 1: Laboratory test results on the used isolates MIS Results Oxidase Test Catalase Test N-free media Sucrose Test NBRIP-BPB Amylase Test development media development Bacillus cereus RCP3/1 Rhizobium radiobacter 11/2 S+ = Strong positive; W+ = weak positive - W+ S+ - S+ W+ S+ + S+ - + - Arbuscular mycorrhizae and plant growth promoting rhizobacteria affects strawberry (Fragaria × ananassa Duch.) growth under salt stress 91 While leaf area increased up to 40.50 cm2 in plants that were sub- jected to bacteria application, the plants of the control group, had an average leaf area of 32.76 cm2. In terms of uprooting days, while the largest leaf area was determined at the plants uprooted on the 45th day, it was observed that leaf areas decrease at the uprootings of the 60th day (Fig. 1). In salt × application interaction while the largest leaf area was measured at plants with 30 mM/L NaCl concentra- tion and bacteria application, the smallest areas were determined at the plants subjected to Mycorrhiza+Bacteria combined application (Tab. 2). Bacteria application at 0 mM/L NaCl concentration on the plants uprooted on the 45th day gave the highest average leaf area value (Fig. 1). Salinity stress is reduction in the rate of leaf surface expansion leading to cessation of expansion as salt concen- tration increases (WANG and NIL, 2000). Salt stress also results in a considerable decrease in the dry weights of leaves, stems, and roots (CHARTZOULAKIS and KLAPAKI, 2000). While the effects of salt con- centration, applications and salt × application interaction were de- termined to be statistically insignificant on offshoot dry weight, the effects of uprooting dates and day × salt × application interaction on the same were determined to be significant. Examining the uprooting dates showed that the average shoot dry weight reached its highest value on the plants uprooted on the 45th day, while the weight started to decrease on the uprootings of the 60th day (Tab. 3). As for the day × salt × application interaction, mycorrhiza + bacteria application at 0 mM/L NaCl concentration on the plants uprooted on the 45th day gave the highest average value (Tab. 3). It was further determined that the applications, uprooting dates and the day × salt × application interaction had significant effects in terms of root dry weight. While the control and bacteria application caused an increase in root dry weight, mycorrhiza and myc+bac application caused a decrease. In terms of uprooting days, the uprootings of the 30th and the 45th days gave the highest root dry weight (Fig. 3). As for the day × salt × application interaction, control application at 30 mM/L NaCl concentration on the plants uprooted on the 30th day gave the highest average dry root weight (Tab. 3). Shoot dry weight, and root dry weight of strawberry plants were lower at salt stress treatment as compared to non-saline conditions (p < 0.05). Similar result has been shown (SAIED et al., 2005). Previous study indicating that the use of mycorrhiza and PGPR that produce ACC (1-amino- cyclopropane-1-carboxylate) deaminase (by Glomus intraradices) enhanced phytoremediation in saline soils, and the study also show- ed that the use of PGPR and/or mycorrhiza increase the volume of plants (CHANG, 2007). In another study Pseudomonas mendocina Palleroni alone or in combination with either Glomus intraradices (Schenk & Smith) or Glomus mosseae (Nicol & Gerd) applied on lettuce and revealed that the plants inoculated with P. mendocina had higher offshot volume than the control plants at both salinity levels, and that mycorrhiza applications increased offshoot volume only at Fig. 1: Surface areas of leaves of strawberry ‘San Andreas’ plants under salt (0, 30 and 60 mM/L NaCl) stress and applications (control, mycorrhiza, bacteria and myc+bac) for the periods of 0, 15, 30, 45 and 60 days. Fig. 2: Shoot dry weights of strawberry ‘San Andreas’ plants under salt (0, 30 and 60 mM/L NaCl) stress and applications (control, mycorrhiza, bacteria and myc+bac) for the periods of 0, 15, 30, 45 and 60 days. 92 A. Koc, G. Balci, Y. Erturk, H. Keles, N. Bakoglu, S. Ercisli Tab. 2: Surface areas of leaves, shoot and root dry weights, proline, anthocyanins and membrane permeability in the leaf tissues of strawberry ‘San Andreas’ plants under salt (0, 30 and 60 mM/L NaCl) stress and applications (control, mycorrhiza, bacteria and myc+bac). Salt Applications Control Mycorrhiza Bacteria Myc+Bac Means Surface areas of leaves (cm2) 0 mM/L NaCl 34,35 ac 40,55 ac 42,62 ab 36,18 ac 38,43 a 30 mM/L NaCl 31,52 bc 39,83 ac 44,71 a 30,95 c 36,75 ab 60 mM/L NaCl 32,40 bc 32,83 bc 34,16 ac 34,09 ac 33,37 b Means 32,76 b 37,74 ab 40,50 a 33,74 b Shoot dry weights (g) 0 mM/L NaCl 5,15 NS 5,12 5,08 5,48 5,21 30 mM/L NaCl 4,64 4,37 5,44 5,13 4,90 60 mM/L NaCl 4,30 4,54 4,50 5,11 4,61 Means 4,70 4,68 5,01 5,24 Root dry weights (g) 0 mM/L NaCl 3,37 NS 3,26 3,57 2,86 3,27 30 mM/L NaCl 3,89 2,69 3,29 3,09 3,24 60 mM/L NaCl 3,42 2,82 2,76 2,78 2,95 Means 3,56 2,92 3,21 2,91 Proline (μmol/100 mg) 0 mM/L NaCl 0,582 df 0,558 ef 0,627 df 0,541 f 0,577 c 30 mM/L NaCl 0,835 b-f 0,776 c-f 1,139 b 0,685 c-f 0,858 b 60 mM/L NaCl 1,023 bc 0,933 b-d 1,459 a 0,912 b-e 1,082 a Means 0,813 b 0,756 b 1,075 a 0,713 b Anthocyanins 0 mM/L NaCl 8,364 cd 7,597 d 8,735 b-d 7,373 d 8,017 c 30 mM/L NaCl 10,013 a-d 9,653 b-d 10,454 a-d 8,871 b-d 9,748 b 60 mM/L NaCl 12,297 ab 11,746 a-c 13,543 a 9,336 b-d 11,731 a Means 10,224 b 9,665 b 10,911 a 8,527 c Membrane Permeability 0 mM/L NaCl 9,217 bc 8,886 cd 8,538 d 8,496 d 8,784 b 30 mM/L NaCl 9,563 ab 9,349 b 9,438 b 9,262 bc 9,403 a 60 mM/L NaCl 9,914 a 9,404 9,372 b 9,419 b 9,527 a Means 9,565 a 9,213 b 9,116 b 9,059 b *Values within by the same letter are not significantly different at P < 0.05 by Duncan NS Not significant medium level of salinity (KOHLER et al., 2009). It is known that under salt stress conditions plants increase cellular osmotic pressure by producing secondary metabolites, various che- micals and particularly stress proteins (such as proline, etc.), and thus they sustain their existence by balancing the high osmotic pres- sure that emerges in the nutrient medium (EDREVA, 1998). In present study, the effects of salt concentrations, applications, uprooting days, the interaction between salt and application, and the interaction between day, salt and application were determined to be statistically significant (p < 0.05) on proline content. Salt application, in general, caused an increase in the proline content of plants. While the proline content was 1.082 μmol/100 mg at 60 mM/L NaCl con- centration, it was determined to be 0.577 μmol/100 mg at 0 mM/L NaCl. Examining the effects of the applications showed that the bac- teria application caused the proline content to reach its maximum value. In terms of uprooting days, on the other hand, the highest proline content was determined to be in the first uprooting day, and it decreased on the following uprooting days (Fig. 4). In salt × ap- plication interaction, bacteria application at 60 mM/L NaCl con- centration provided the highest proline content as for the day × salt × application interaction, bacteria application at 60 mM/L NaCl con- centration on the plants uprooted on the 45th day gave the highest average value (Tab. 2, 4). Similarly, a study conducted on tomato (AZIZ et al., 1999) reported that introducing salt concentrations in- creases plants’ proline content. In another study were used six NaCl levels (0, 25, 50, 75, 100 and 150 mM) and two strawberry culti- vars, ‘Camarosa’ and ‘Albino’. Elevated salinity level significant- ly increased leaf proline content of both cultivars. ‘Albino’ leaves accumulated higher proline compared with ‘Camarosa’ at salinized and non-salinized treatments (AL-SHORAFA et al., 2014). KEUTGEN Arbuscular mycorrhizae and plant growth promoting rhizobacteria affects strawberry (Fragaria × ananassa Duch.) growth under salt stress 93 Tab. 3: Surface areas of leaves, shoot and root dry weights in the leaf tissues of strawberry ‘San Andreas’ plants under salt (0, 30 and 60 mM/L NaCl) stress and applications (control, mycorrhiza, bacteria and myc+bac) for the periods of 0, 15, 30, 45 and 60 days. Salt Applications 0 15 30 45 60 Surface areas of leaves (cm2) 0 mM/lt NaCl Control 25,340 f-j 29,510 d-j 29,730 d-j 45,067 a-h 42,097 a-j Mycorrhiza 29,160 d-j 44,253 a-i 44,180 a-i 53,780 a-c 31,353 c-j Bacteria 31,497 c-j 30,613 c-j 44,873 a-h 60,527 a 45,603 a-h Myc+Bac 24,743 g-j 33,230 c-j 36,737 b-j 48,980 a-f 37,230 b-j 30 mM/lt NaCl Control 30,693 c-j 20,933 ij 33,873 c-j 46,507 a-h 25,580 f-j Mycorrhiza 36,733 b-j 35,127 b-j 32,297 c-j 57,847 ab 37,123 b-j Bacteria 31,500 c-j 50,647 a-e 49,973 a-e 52,543 a-d 38,890 a-j Myc+Bac 23,103 h-j 25,980 f-j 30,203 c-j 46,737 a-h 28,747 e-j 60 mM/lt NaCl Control 37,043 b-j 26,237 f-j 36,620 b-j 42,360 a-j 19,757 j Mycorrhiza 29,563 d-j 31,437 c-j 33,673 c-j 49,920 a-e 19,533 j Bacteria 32,057 c-j 30,117 d-j 24,793 g-j 48,327 a-g 35,513 b-j Myc+Bac 20,643 ij 35,900 b-j 28,903 d-j 51,730 a-e 33,273 c-j Shoot dry weights (g) 0 mM/lt NaCl Control 1,817 kl 3,237 e-l 6,577 b-l 8,283 b-e 5,850 b-l Mycorrhiza 2,283 h-l 3,810 c-l 6,907 b-k 8,353 b-d 4,243 b-l Bacteria 2,793 g-l 4,263 b-l 5,540 b-l 5,860 b-l 6,920 b-k Myc+Bac 3,350 d-l 2,840 g-l 4,440 b-l 13,330 a 3,460 d-l 30 mM/lt NaCl Control 2,947 g-l 3,557 d-l 6,300 b-l 5,763 b-l 4,613 b-l Mycorrhiza 2,470 h-l 2,300 h-l 3,810 c-l 8,847 bc 4,443 b-l Bacteria 3,207 e-l 2,730 g-l 7,063 b-j 7,693 b-g 6,520 b-l Myc+Bac 1,720 l 3,053 g-l 4,237 b-l 8,177 b-f 8,437 b-d 60 mM/lt NaCl Control 2,800 g-l 2,717 g-l 4,767 b-l 6,570 b-l 4,623 b-l Mycorrhiza 2,077 j-l 3,923 c-l 5,983 b-l 7,257 b-i 3,437 d-l Bacteria 2,750 g-l 1,920 kl 7,337 b-h 7,343 b-h 3,140 f-l Myc+Bac 2,143 i-l 3,110 f-l 3,910 c-l 9,033 b 7,360 b-h Root dry weights (g) 0 mM/lt NaCl Control 1,510 g 3,353 b-g 4,220 b-g 4,290 b-g 3,477 b-g Mycorrhiza 1,627 fg 5,683 ab 3,970 b-g 2,547 c-g 2,493 d-g Bacteria 2,070 d-g 4,647 a-e 3,983 b-g 2,853 b-g 4,283 b-g Myc+Bac 2,583 c-g 2,113 d-g 2,480 d-g 5,450 a-c 1,650 fg 30 mM/lt NaCl Control 1,573 g 4,037 b-g 7,130 a 2,777 b-g 3,950 b-g Mycorrhiza 2,520 c-g 1,693 e-g 2,463 d-g 3,730 b-g 3,023 b-g Bacteria 2,897 b-g 2,983 b-g 3,927 b-g 3,730 b-g 2,930 b-g Myc+Bac 1,627 fg 2,823 b-g 3,263 b-g 4,077 b-g 3,667 b-g 60 mM/lt NaCl Control 2,643 c-g 3,937 b-g 4,540 a-f 3,187 b-g 2,773 b-g Mycorrhiza 1,827 e-g 2,203 d-g 4,160 b-g 2,957 b-g 2,940 b-g Bacteria 2,007 d-g 2,513 c-g 4,250 b-g 3,117 b-g 1,917 e-g Myc+Bac 1,797 e-g 1,673 fg 2,547 c-g 4,880 a-d 3,013 b-g *Values within by the same letter are not significantly different at P < 0.05 by Duncan and PAWELZİK (2009) reported that free amino acids and proline ac- cumulated in salt conditions in cultivars, ‘Elsanta’ and ‘Korona’, but the latter revealed higher free amino acid content. However, higher accumulation of proline has been linked with better osmotic adjust- ment and, consequently, higher adaptation to salt conditions (SAIED et al., 2005). In strawberry cultivars, a dramatic accumulation of pro- line following salt stress was observed (KEUTGEN and PAWELZIK, 2009). The effects of salt concentrations, applications, uprooting days, the interaction between salt and application, and the interaction between day, salt and application were determined to be statistically significant (p > 0.05) on anthocyanin level. It was observed that the anthocya- 94 A. Koc, G. Balci, Y. Erturk, H. Keles, N. Bakoglu, S. Ercisli Fig. 3: Root dry weights of strawberry ‘San Andreas’ plants under salt (0, 30 and 60 mM/L NaCl) stress and applications (control, my- corrhiza, bacteria and myc+bac) for the periods of 0, 15, 30, 45 and 60 days. Fig. 4: Proline in the leaf tissues of strawberry cv. ‘San Andreas’ plants under salt (0, 30 and 60 mM/L NaCl) stress and applications (control, mycorrhiza, bacteria and myc+bac) for the periods of 0, 15, 30, 45 and 60 days. nin content increased in line with the increasing salt concentration. Anthocyanin content increased in plants subjected to bacterial ap- plication. On the other hand, the 60th day uprooting was determined to be the uprooting with the highest content of anthocyanin (Fig. 5). In terms of salt × application interaction, it was determined that the anthocyanin content was high at 30 mM/L NaCl concentration with control and bacteria applications and at 60 mM/L NaCl concentrati- on with control, mycorrhiza and bacteria applications. Anthocyanin content was determined to be rather low at both salt concentrations with the application of mycorrhiza and bacteria (Tab. 2). Concerning the interaction between uprooting days, salt concentrations and im- plemented applications, high levels of anthocyanin were determined in plants subjected to bacteria application made on the plants uproo- ted on the 60th day with 30 mM/L NaCl concentration, and on those that were subjected to control, mycorrhiza and bacteria applications made on the plants uprooted on the 60th day with 60 mM/L NaCl concentration (Tab. 4). KEUTGEN and PAWELZIK (2009) reported that salt stress increased the contents of anthocyanins in fruits of both cvs and the highest increase of 94 % occurred in cv. ‘Elsanta’ at 40 mmol NaCl/l. Membrane permeability is an important indicator that points out to membrane stability. A low level of membrane permeability is impor- tant for the robustness of the membrane. In general terms, the salt applied on the plants causes an increase in membrane permeability thus disrupts membrane stability and becomes a significant factor damaging the plant. In studies conducted on strawberry, it was de- termined that application of salt increases membrane permeability (KAYA et al., 2003). In addition, subjecting plants under salt stress to bacteria application caused a decrease in membrane permeability and thus helped in the maintenance of membrane stability. In our study, the effects of salt concentrations, applications, uprooting days, the interaction between salt and application, and the interaction between day, salt and application were determined to be statistically signifi- cant (p < 0.05) on membrane permeability. Membrane permeability increased at applications with 30 mM/L and 60 mM/L NaCl concen- trations. Among the applications, it was determined that the mem- brane permeability of the control application increased and in other mycorrhiza, bacteria and myc+bac applications it was lower. It was observed that membrane permeability increased in the uprootings of the 15th, 45th and 60th days (Fig. 6). In salt × application interaction, membrane permeability was determined to be high in plants subjec- ted to control application at 30 mM/L and 60 mM/L NaCl concen- trations. The lowest interaction value was measured with bacteria and myc+bac applications at 0 mM/L NaCl concentration (Tab. 2). Concerning the interaction between uprooting days, salt concentra- tions and applications, it was determined that the control application Arbuscular mycorrhizae and plant growth promoting rhizobacteria affects strawberry (Fragaria × ananassa Duch.) growth under salt stress 95 was statistically different (p < 0.05) at 60 mM/L NaCl concentration on the plants uprooted on the 45th day (Tab. 4). Conclusions Our study demonstrates that under saline conditions, proline, antho- cyanins and membrane permeability increase in strawberry leaves, while LSA decreases. The mycorrhiza and bacteria applications car- ried out during planting caused an increase in the leaf area of plants under salt stress. Bacteria application was determined to be the pro- minent application that helps the plant to tolerate the negative effects of salt stress by increasing proline and anthocyanin levels. Acknowledgements We would like to thank to Dr. Ramazan Çakmakçi for providing the bacteria and to Bioglobal company for providing mychorriza, and to Bozok University Scientific Reseach Projects Division for providing financial support to my project. References AL-SHORAFA, W., MAHADEEN, A., AL-ABSI, K., 2014: Evaluation for salt stress tolerance in two strawberry cultivars. Am. J. Agric. Biol. Sci. 9, 334-341. ALKAN TORUN, A., AKA KAÇAR, Y., BICEN, B., ERDEM, N., SERCE, S., 2014: In vitro screening of octoploid Fragaria chiloensis and Fraga- ria virginiana genotypes against iron deficiency. Turk. J. Agric. For. 38, 169-174. AUGÉ, R.M., 2001: Water relations, drought and VA mycorrhizal symbiosis. Mycorrhiza 11, 3-42. AZIZ, A., MARTIN-TONGUY, J., LARHER, F., 1999: Salt stres, induced proline accumulation and changes in tyramine and polyamine levels are linked to ionic adjustment in tomato leaf discs. Plant Sci. 145, 83-91. BATES, L., WALDREN, R.P., TEARE, I.D., 1973: Rapid determination of free proline for water-stress studies. Plant and Soil. 39, 205-207. CAKMAKCI, R., DONMEZ, M.F., ERTURK, Y., ERAT, M., HAZNEDAR, A., SEKBAN, R., 2010: Diversity and metabolic potential of culturable bacte- ria from the rhizosphere of Turkish tea grown in acidic soils. Plant Soil. 332, 299-318. Fig. 6: Membrane permeability in the leaf tissues of strawberry cv. ‘San Andreas’ plants under salt (0, 30 and 60 mM/L NaCl) stress and applications (control, mycorrhiza, bacteria and myc+bac) for the periods of 0, 15, 30, 45 and 60 days. Fig. 5: Anthocyanins in the leaf tissues of strawberry cv. ‘San Andreas’ plants under salt (0, 30 and 60 mM/L NaCl) stress and applications (control, mycor- rhiza, bacteria and myc+bac) for the periods of 0, 15, 30, 45 and 60 days. 96 A. Koc, G. Balci, Y. Erturk, H. Keles, N. Bakoglu, S. Ercisli Tab. 4: Proline, anthocyanins and membrane permeability in the leaf tissues of strawberry cv. ‘San Andreas’ plants under salt (0, 30 and 60 mM/L NaCl) stress and applications (control, mycorrhiza, bacteria and myc+bac) for the periods of 0, 15, 30, 45 and 60 days. Salt Applications 0 15 30 45 60 Proline (μmol/100 mg) 0 mM/lt NaCl Control 1,536 b-e 0,746 m-t 0,315 w-z 0,157 z 0,156 z Mycorrhiza 1,209 f-i 0,725 n-t 0,188 yz 0,323 v-z 0,345 u-z Bacteria 1,105 h-k 0,775 l-s 0,538 r-x 0,276 x-z 0,442 t-z Myc+Bac 1,165 g-j 0,696 n-t 0,160 z 0,355 u-z 0,327 v-z 30 mM/lt NaCl Control 1,534 b-e 0,897 j-p 0,462 t-z 0,842 k-r 0,439 t-z Mycorrhiza 1,252 e-h 1,024 h-m 0,523 s-x 0,563 r-x 0,515 s-x Bacteria 1,232 f-h 1,442 d-g 0,634 o-v 1,636 b-d 0,748 m-t Myc+Bac 1,159 g-j 0,969 h-n 0,341 u-z 0,492 s-y 0,462 t-z 60 mM/lt NaCl Control 1,920 a 1,045 h-l 0,598 p-w 0,873 j-q 0,680 n-t Mycorrhiza 1,450 d-g 1,418 d-g 0,484 s-y 0,646 o-u 0,668 n-t Bacteria 1,494 c-f 1,814 ab 0,915 i-o 1,951 a 1,124 h-k Myc+Bac 1,768 a-c 1,062 j-l 0,605 p-w 0,555 r-x 0,573 q-x Anthocyanins 0 mM/lt NaCl Control 11,470 e-k 7,643 o-u 3,557 y- β 8,627 l-s 10,523 g-m Mycorrhiza 7,457 o-v 4,970 v-β 2,487 β 9,970 i-o 13,100 c-g Bacteria 8,627 l-s 5,753 t-y 2,873 z β 11,003 e-m 15,417 bc Myc+Bac 7,890 n-t 5,257 u-z 2,633 β 8,547 l-s 12,537 d-h 30 mM/lt NaCl Control 12,657 d-h 8,437 m-s 3,217 y- β 12,587 d-h 13,167 c-f Mycorrhiza 10,720 f-m 7,147 p-v 3,577 y- β 13,427 c-e 13,397 c-e Bacteria 10,457 h-n 6,970 q-w 3,487 y- β 13,530 c-e 17,827 a Myc+Bac 9,327 k-q 6,217 s-x 3,110 z β 11,097 e-l 14,607 cd 60 mM/lt NaCl Control 14,610 cd 9,743 j-p 3,870 x- β 14,400 cd 18,860 a Mycorrhiza 13,460 c-e 8,970 k-r 4,487 w- β 14,183 cd 17,630 ab Bacteria 12,110 d-j 14,740 cd 5,370 t-z 15,533 bc 19,963 a Myc+Bac 9,360 k-q 6,577 r-w 3,120 z β 12,347 d-i 15,277 bc Membrane permeability 0 mM/lt NaCl Control 8,918 b-g 9,265 b-f 9,150 b-f 9,378 b-f 9,375 b-f Mycorrhiza 7,913 hi 9,113 b-g 8,633 d-h 9,222 b-f 9,548 b-d Bacteria 8,808 c-h 9,070 b-g 8,396 f-h 7,964 hi 8,455 e-h Myc+Bac 8,819 c-h 9,088 b-g 7,244 i 8,146 gh 9,184 b-f 30 mM/lt NaCl Control 9,369 b-f 9,813 bc 9,421 b-e 9,626 b-d 9,584 b-d Mycorrhiza 9,081 b-g 9,679 bc 9,114 b-g 9,397 b-f 9,476 b-e Bacteria 9,202 b-f 9,436 b-e 9,575 b-d 9,467 b-e 9,509 b-d Myc+Bac 8,971 b-g 9,272 b-f 9,129 b-g 9,455 b-e 9,484 b-d 60 mM/lt NaCl Control 9,295 b-f 9,898 b 9,556 b-d 11,012 a 9,811 bc Mycorrhiza 9,062 b-g 9,347 b-f 9,481 b-d 9,457 b-e 9,674 bc Bacteria 9,136 b-f 9,280 b-f 9,442 b-e 9,456 b-e 9,544 b-d Myc+Bac 9,004 b-g 9,551 b-d 9,608 b-d 9,279 b-f 9,655 b-d *Values within by the same letter are not significantly different at P < 0.05 by Duncan CHALKER-SCOTT, L., 1999: Environmental significance of anthocyanins in plant stress responses. 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