Impaginato 157 Adv. Hort. Sci., 2018 32(2): 157-168 DOI: 10.13128/ahs-21876 Biochemical and physiological evaluations of common bermudagrass [Cynodon dactylon (L.) Pers.] Iranian accessions under cold stress M. Akbari, H. Salehi Department of Horticultural Science, School of Agriculture, Shiraz University, Shiraz, Iran. Key words: cold tolerance, Cynodon, Iranian accessions, physiological characters. Abstract: In this study, one foreign cultivar and forty-nine common bermuda- grass accessions were collected from 18 provinces of Iran. Turfgrasses were grown at four temperature regimes (24/17, 7.5/0, -7.5/-12 and -15/-15°C day/night cycles) in a factorial experiment based on the completely random- ized design with three replications. Physiological traits were evaluated to cate- gorize all accessions as either cold sensitive or tolerant using Hierarchical Clustering with Ward’s method in SPSS software. Our results revealed that cold-tolerant common bermudagrass accessions showed higher proline, pro- tein, antioxidant enzymes, color, visual quality and chlorophyll content and cold-sensitive accessions showed more severe cell membrane damage (EL) under cold stress conditions. Fall in temperature from 24°C severely decreased chlorophyll content, visual quality and color in all accessions. The highest antioxidant enzymes activity, chlorophyll content, color and visual quality at -7.5°C were observed in Taft, foreign cultivar, Naein, Malayear, Aligoudarz, Safashahr and Gorgan accessions. The increase in POD, SOD, CAT and APX activ- ity observed in this study led to protection against oxidative damage caused due to high ROS levels. The most cold-tolerant accessions at -15°C were Taft, Naein and Malayear. Great variations in freezing tolerance were observed between Iranian accessions of common bermudagrass. Further molecular stud- ies are needed to clarify better these findings. 1. Introduction Cold stress is the main serious problem that limits plant growth, agri- cultural productivity, survival, as well as geography of plant distribution. Common bermudagrass (Cynodon dactylon [L.] Pers.), from the grass (Poaceae) family, is a typical creeping grass grows in warmer parts of all continents between about 45 degrees north and 45 degrees south lati- tude (Harlan and de Wet, 1969; Anderson et al., 2003). This perennial, herbaceous, warm season, C4 grass is commonly known as ‘Chaiar’ or (*) Corresponding author: hsalehi@shirazu.ac.ir Citation: AKBARI M., SALEHI H., 2018 - Biochemical and physiological evaluations of common bermuda- grass [Cynodon dactylon (L.) Pers.] Iranian acces- sions under cold stress - Adv. Hort. Sci., 32(2): 157-168 Copyright: © 2018 Akbari M., Salehi H. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 10 October 2017 Accepted for publication 12 January 2018 AHS Advances in Horticultural Science Adv. Hort. Sci., 2018 32(2): 157-168 158 ‘Margh’ in Iran. Common bermudagrass’s high densi- ty, recuperative ability, high tolerance to drought, heat, salinity, wear, flood and most of soils cause the species to be extensively used in tropical and sub- tropical regions of Iran. This species is a major turf- grass for livestock herbage, golf courses, sport fields, public parks and soil conservation. Despite its good characters, C. dactylon has a considerable tendency to be damaged or killed by frost, especially in transi- tion zone (Munshaw et al., 2006). An important process in the winter perpetuity of common bermudagrass is acclimatization, which is an adapta- tion process to overcome the environmental stresses (Levitt, 1980). The most favorable temperature for root and shoot growth of cool-season turfgrass species varied from 10 to 18°C and 18 to 24°C, respectively. Warm season turfgrasses have C4 pho- tosynthetic pathway and are best adapted to warm climatic region of the world and grows well at tem- peratures between 24 to 29°C and 27 to 35°C for root and shoot growth, respectively (Beard, 1973). During cold stress, plants exhibit different mechanisms to develop their cold hardiness and increase their freez- ing tolerance (Zhu et al., 2004; Knight and Knight, 2012). Some of these processes includes changes in the concentration of amino acids, sugars, proteins, compatible solutes, certain hormones, and changes in the degree of fatty acid saturation level and antioxidant capacity that affect the freezing toler- ance (Karpinski et al., 2002; Munshaw et al., 2006; Zhang and Ervin, 2008). Genetic resources and wild plant species that genetically related to cultivated variety have gross value in plant breeding programs (Hajjar and Hodgkin, 2007). Today, many investigations have focused on the naturally occurring genetic differ- ences in stress tolerance of many plants such as Lolium perenne L., Brachypodium distachyon L. and Festuca arundinacea Schreb. (Luo et al., 2011; Hu et al., 2012; Salehi et al., 2013). For many years, improvement of warm season turfgrass quality and cold tolerance are the main goals in breeding pro- grams. Natural populations of bermudagrass should have considerable genetic variation for tolerance to environmental stresses. Since bermudagrass is cos- mopolite plant, its considerable genetic variation is predictable. There is great diversity among wild pop- ulations and cultivars of common bermudagrass for tolerance to freezing (Anderson et al., 2003) and other environmental stresses. In spite of that, there is little data about the cold tolerance of common bermudagrass, and many researches are being con- ducted to improve cold tolerance of this species (Zhang et al., 2011; Shi et al., 2015). The aims of the present research were to evaluate Iranian common bermudagrass accessions to find accessions with good freezing tolerance and examine physiological changes during cold stress. 2. Materials and Methods Forty-nine accessions of natural common bermudagrass were collected from 18 provinces of Iran with different climatic conditions ranging from Shiraz city with subtropical condition and Tabriz city with temperate climatic condition (Fig. 1), and one foreign cultivar ‘Blackjack’ was used as control. All accessions were collected originally from grasslands, roadside, seaside, and around orchards and agricul- ture fields, and transferred to the School of Agriculture, Shiraz University, under natural green- house condition (52°32’ E and 29°36’ N, elevation Fig. 1 - Map showing the sampling locations of common bermu- dagrass accessions from different regions of Iran. 1: Boroujerd, 2: Malayear, 3: Ghidar, 4: Zanjan, 5: Tabriz, 6: Sarein, 7: Talesh, 8: Anzali, 9: Nour, 10: Sari, 11: Gorgan, 12: Minoudasht, 13: Chenaran, 14: Mashhad, 15: Maiami, 16: Damghan, 17: Semnan, 18: Tehran, 19: Arak, 20: Malayear intersection, 21: Nahavand, 22: Firouzan, 23: Kamiaran, 24: Dehgolan, 25: Sanandaj Abidar, 26: Kermanshah Taghbostan, 27: Mahidasht, 28: Islamabad gharb, 29: Homail, 30: Ilam Saymareh bridge, 31: Holailan, 32: Poldokhtar, 33: Mamoulan, 34: Khoram abad, 35: Foreign cultivar, 36: Doroud Nahalestan, 37: Doroud Daneshjo park, 38: Doroud Siahvel, 39: Doroud Babahour, 40: Azna, 41: Aligoudarz, 42: Daran, 43: Naein, 44: Ardakan 1, 45: Ardakan 2, 46: Yazd, 47: Taft, 48: Abarkouh, 49: Safashahr, 50: Shiraz. Akbari and Salehi - Evaluation of common bermudagrass Iranian accession under cold stress 159 1810 m a.s.l.). Each accession was transplanted into 14 cm diameter pots filled with uniform mixture of 1:1:1 (v:v:v) of sand, loamy soil and decomposed manure. Turfgrasses were kept in natural green- house condition and were clipped to a height of 5 cm every 2 weeks. Low and freezing temperature treat- ments were conducted at 24/17, 7.5/0, -7.5/-12 and -15/-15°C day/night cycles and a 10 h light (300 µmol m-2 s-1) for 7 days, using a controlled-environment chamber. After each temperature regime, physiologi- cal traits including: superoxide dismutase (SOD) (Beauchamp and Fridovich, 1971), catalase (CAT) (Dhindsa et al., 1981), ascorbate peroxidase (APX) and peroxidase (POD) (Chance and Maehly, 1995) activities, proline (Bates et al., 1973), protein (Bradford, 1976), electrolyte leakage (Saadalla et al., 1990) and chlorophyll content (Saini et al., 2001) were measured. Turfgrass color and visual quality were rated visually after each treatment (Beard, 1973). To extract antioxidant enzymes, fresh leaf or stolon samples (0.5 g) were collected and ground to a fine powder in a mortar by adding liquid nitrogen and then homogenized with an ice cold enzyme extraction buffer containing 0.5% polyvinylpyrroli- done (PVP), 3 mM EDTA, and 0.1 M potassium phos- phate buffer (pH=7.5). The extracted samples were centrifuged for 10 min at 13500 rpm and 2-4°C and stored on ice until used. The resulting supernatant was used for enzyme analysis. SOD activity was determined according to the procedure used by Beauchamp and Fridovich (1971), CAT activity was determined as described by Dhindsa et al. (1981), ascorbate peroxidase and peroxidase activities were determined according to the method described by Chance and Maehly (1995). Proline was determined according to the method described by Bates et al. (1973). Using spectrophotometer (UV-120-20, Japan) at 520 nm wavelength, appropriate proline standards were included in calculation of its content in sam- ples. The protein content was quantified using Bradford method with bovine serum albumin (BSA) as standard. Electrical leakage measured, as described by Saadalla et al. (1990), using an electrical conductivity meter (Metrohm 644, Swiss) and calcu- lated with the following formula: Electrolyte leakage = EC1/EC2 × 100 Chlorophyll content was measured according to the method of Saini et al. (2001) using the following for- mula: mg Chl/g f.w. = [(20.2(OD 645 nm) + (8.02(OD 663 nm)] x V f.w. x 1000 where: OD is optical density, V is the final solution volume in ml, and f.w. is tissue fresh weight in mg. Turfgrass color and visual quality were measured after each treatment on a 1 to 9 scale where 1 was very poor quality turf, 6 was minimally acceptable turf, and 9 was exceptional turf quality (Beard, 1973). This study was conducted in a factorial experiment based on completely randomize design (CRD) with three replications. Factors were fifty accessions and four different concentrations of low and freezing temperatures (24/17, 7.5/0, and -7.5/-12 day/night cycles). In the case of treatment with -15/-15°C day/night cycles, only the seven most cold tolerant accessions were evaluated. Mean comparisons were performed using the least significant difference (LSD) at P= 0.05 probability level. Physiological traits were evaluated for accession clustering to determine cold sensitive or cold tolerant using Ward’s method of Hierarchical cluster analysis in SPSS software. 3. Results The Ward cluster analysis based on physiological traits before low temperature treatments (at 24/17°C day/night cycles) grouped the 49 accessions and the foreign cultivar into two major groups (Fig. 2). The first group contained 18 accessions with low antioxi- dant enzymes activity, proline, protein and chloro- phyll content including: Abidar Sanandaj, Boroujerd, Holailan, Malayear, Ghidar, Nour, Saymareh bridge, Anzali, Islamabad gharb, Tehran, Tagh bostan, Kermanshah, Homail, Maiami, Minodasht, Mashhad, Poldokhtar, Safashahr and Shiraz. The second group contained other accessions with more antioxidant enzymes activity, proline, protein and chlorophyll content. Two major groups were formed based on physiological characters after cold stress (at 7.5/0°C day/night cycles) (Fig. 3). The first group contained 36 accessions with low proline, protein, chlorophyll content and low antioxidant enzymes activity. Other accessions were in second group and had more pro- line, protein, chlorophyll content and more antioxi- dant enzymes activity included fourteen accessions: Arak, Doroud daneshjo park, Azna, Taft, Safashahr, Ardakan 2, Mahidasht, Mamoulan, Naein, Yazd, Chenaran, Semnan, Homail and Daran. The dendro- gram from physiological characters after cold stress (at -7.5/-12°C day/night cycles) grouped the 50 accessions in two main clusters (Fig. 4). The dendro- gram from physiological characters at -15/-15°C day/night cycles grouped the 7 most cold-tolerant genotypes into two main groups (Fig. 5). The first group contained 3 accessions with lower proline, pro- Adv. Hort. Sci., 2018 32(2): 157-168 160 tein, chlorophyll content and antioxidant enzymes activity than other elite accessions were: Aligoudarz, Gorgan and Safashahr. Other accessions were in sec- ond group and had more antioxidant enzymes activi- ty, proline, protein, chlorophyll, color and visual qual- ity, and the least EL included: foreign cultivar, Malayear, Naein and Taft. POD, CAT, SOD, APX, proline, protein, EL, chloro- phyll, color and visual quality were influenced by cold and freeze temperatures. With drop in temperature from 24 to 7.5°C, POD, CAT, SOD, and APX activities, and proline and protein content increased in all accessions; and from 7.5°C to -7.5°C significantly decreased the same parameters. The highest antioxi- dant enzymes activity, chlorophyll content, color and visual quality at -7.5°C were observed in Taft, foreign Fig. 2 - Dendrogram of the physiological relationships between 50 accessions of common bermudagrass and control cultivar before cold stress (at 24/17°C day/night cycles). Fig. 3 - Dendrogram of the physiological relationships between 50 accessions of common bermudagrass and control cultivar aftrer cold stress at 7.5/0°C day/night cycles. Akbari and Salehi - Evaluation of common bermudagrass Iranian accession under cold stress 161 cultivar, Naein, Malayear, Aligoudarz, Safashahr and Gorgan accessions. Peroxidase The results of peroxidase assay at 24°C showed that its maximum and minimum activity were belonged to Gorgan and Arak accessions, respectively (Table 1). The highest peroxidase activity at 7.5°C and -7.5°C was observed in Malayear accession. The maxi- mum peroxidase activity at -15°C was observed in Naein accession and the second rank was belonged to common bermudagrass accession collected from Taft. Catalase The results of catalase assay at 24°C showed that Fig. 4 - Dendrogram of the physiological relationships between 50 accessions of common bermudagrass and control cultivar after cold stress at -7.5/-12°C day/night cycles. Fig. 5 - Dendrogram of the physiological relationships between 7 cold-tolerant accessions of common bermudagrass after cold stress at -15/-15 °C day/night cycles. 162 Adv. Hort. Sci., 2018 32(2): 157-168 its maximum activity was belonged to Ghidar and Nour accessions, and its minimum activity was belonged to Abarkouh accession (Table 1). The high- est catalase activity at 7.5°C and -7.5°C was observed in Doroud daneshjo park and Taft accessions, respec- tively (Tables 2 and 3). The maximum catalase activi- ty at -15°C was observed in Taft accession and the second rank was belonged to common bermudagrass accession collected from Malayear (Table 4). Superoxide dismutase The results of SOD assay showed that there was difference between SOD activities of different acces- sions in all temperature regimes. The maximum and minimum superoxide dismutase activity at 24°C was belonged to Doroud nahalestan and Mamoulan Turfgrass visual quality and color based on a scale of 1-9, 1= brown/dead turf, 6= minimal acceptable turf, 9= ideal green, healthy turf. Table 1 - Amount of POD, CAT, SOD, APX, CHL, Proline, EL, protein, color and visual quality of different Iranian accessions and foreign common bermudagrass before cold stress (at 24/17°C day/night cycles) Location of accession POD (U mg−1 f.w.) CAT (U mg−1 f.w.) SOD (U mg−1 f.w.) APX U mg−1 f.w.) CHL Proline (µmol g−1 f.w.) EL (%) Protein (mg g−1 f.w.) Color VQ Doroud Nahalestan 13.26 36.11 120.50 81.25 4.06 13.20 11.90 15.20 7.2 7.1 Abarkouh 13.17 34.12 119.10 79.12 3.68 13.10 13.40 14.30 6.3 6.5 Abidar sanandaj 12.45 35.62 118.60 59.85 3.95 13.20 11.80 14.60 7.1 7.2 Aligoudarz 13.54 36.11 119.50 79.56 4.02 13.40 11.30 14.80 7.0 7.1 Anzali 13.36 34.25 116.40 69.55 3.78 12.80 12.60 14.90 6.2 7.3 Arak 12.11 35.25 119.70 75.25 3.92 13.10 12.70 14.90 7.0 7.1 Ardakan 1 12.26 36.11 116.80 72.12 3.97 12.70 12.30 14.30 7.1 7.2 Ardakan 2 13.21 35.17 117.50 81.21 4.02 13.10 12.50 14.80 7.0 7.0 Azna 13.12 36.24 118.60 80.12 3.96 13.50 11.30 14.90 7.6 8.1 Boroujerd 12.25 35.24 118.50 59.21 4.36 12.10 10.50 14.30 8.5 9.0 Chenaran 12.18 35.14 118.90 78.56 4.35 13.40 11.10 14.30 8.4 8.6 Damghan 12.17 36.24 117.50 77.56 3.81 12.50 13.70 13.80 6.0 6.2 Daran 13.18 35.59 117.30 74.21 4.09 12.90 11.80 13.50 7.0 7.2 Dehgolan 13.11 34.35 117.90 74.32 3.89 13.10 11.60 14.50 7.6 7.2 Doroud Babahour 13.14 36.12 116.50 79.65 3.94 13.40 11.20 15.20 7.8 8.0 Doroud Daneshjo park 13.21 35.59 117.40 84.21 4.41 13.50 10.80 15.30 8.2 8.5 Doroud Siahvel 13.54 36.15 114.50 85.17 4.51 13.30 10.40 15.10 8.5 9.0 Firouzan 12.45 35.14 118.50 77.89 3.89 13.20 11.30 14.30 7.0 7.0 Foreign cultivar 13.55 36.25 119.50 79.81 4.47 13.10 11.20 15.10 8.7 8.5 Ghidar 13.31 36.28 115.80 67.18 3.85 12.70 12.30 13.80 7.7 7.5 Gorgan 13.65 35.17 116.50 77.21 4.38 13.10 10.60 13.90 8.2 8.3 Holailan 12.58 35.18 115.50 58.52 4.12 12.40 12.50 14.50 7.2 7.4 Homail 13.26 35.26 117.80 68.74 3.96 12.90 12.40 14.70 7.6 7.4 Islamabad gharb 13.14 34.16 118.60 69.52 3.76 13.10 13.40 15.30 6.1 6.2 Kamiaran 13.89 34.23 117.60 75.12 4.02 13.40 11.50 14.50 7.2 7.3 Khorram abad 13.23 35.35 115.40 74.25 4.39 12.50 11.90 14.70 8.1 8.4 Mahidasht 13.45 36.21 118.30 72.32 4.32 13.50 10.60 15.10 8.6 9.0 Maiami 12.54 35.25 118.20 68.25 3.98 12.90 11.60 13.90 7.4 7.5 Malayear 13.12 36.12 119.40 63.14 4.18 13.20 11.10 15.20 7.3 8.0 Malayear intersection 13.24 36.25 118.60 81.32 4.32 13.40 11.20 14.50 8.0 8.2 Mamoulan 13.14 35.21 113.70 81.23 3.92 12.30 12.40 14.60 7.4 7.2 Mashhad 13.11 36.15 119.20 69.65 4.32 13.10 10.80 14.40 8.1 8.2 Minodasht 13.14 36.15 117.70 69.54 4.06 13.20 10.90 13.80 7.2 8.4 Naein 13.85 35.16 115.30 75.16 4.06 13.20 11.20 14.20 7.3 7.1 Nahavand 13.55 36.12 116.50 75.14 4.07 13.10 11.70 15.10 7.6 7.4 Nour 12.51 36.28 115.30 65.23 4.41 11.90 13.10 14.80 8.0 7.6 Poldokhtar 12.57 35.18 119.40 69.85 4.37 12.10 11.70 14.80 8.0 8.2 Safashahr 13.35 35.12 118.70 71.21 4.05 13.10 12.20 15.10 7.4 7.5 Sarein 13.15 35.15 119.20 81.25 4.81 13.20 10.90 15.30 9.3 8.4 Sari 13.12 35.21 117.50 78.54 3.96 12.50 11.20 14.60 7.8 8.2 Saymareh bridge 13.21 35.62 117.50 64.21 4.09 12.60 13.10 14.60 7.0 6.7 Semnan 13.32 35.27 116.50 76.5 3.35 12.40 14.70 13.80 5.3 4.2 Shiraz 12.56 35.52 119.10 70.25 3.89 12.80 12.50 14.80 7.1 7.2 Tabriz 12.74 34.18 120.10 75.65 4.38 12.90 11.10 14.60 8.2 7.6 Taft 13.54 36.18 118.60 82.35 4.49 13.20 11.20 14.90 8.3 8.1 Tagh bostan kermanshah 12.46 35.23 117.60 68.65 3.81 12.80 13.10 14.10 6.2 5.7 Talesh 12.18 36.14 117.50 74.32 4.85 13.50 10.80 14.70 9.0 8.1 Tehran 13.21 34.56 118.80 69.95 3.89 12.50 13.80 14.10 6.5 6.2 Yazd 12.96 35.11 116.40 81.24 3.95 12.80 12.60 14.50 7.2 7.5 Zanjan 12.25 35.62 117.20 81.25 3.96 13.40 11.40 15.10 7.5 8.0 LSD (5%) 0.458 0.264 1.860 0.650 0.508 0.320 1.520 0.458 1.452 1.286 163 Akbari and Salehi - Evaluation of common bermudagrass Iranian accession under cold stress accessions, respectively. The highest superoxide dis- mutase activity at 7.5°C and -7.5°C was observed in Doroud daneshjo park accession (Tables 2 and 3). The maximum superoxide dismutase activity at -15°C was observed in Taft accession and the second rank was belonged to common bermudagrass accession col- lected from Naein (Table 4). Ascorbate peroxidase The results of ascorbate peroxidase assay showed that there was difference between ascorbate peroxi- dase activity of different accessions in all tempera- ture regimes. The maximum and minimum ascorbate peroxidase activity at 24°C was belonged to Doroud siahvel and Holailan accessions, respectively. The Turfgrass visual quality and color based on a scale of 1-9, 1= brown/dead turf, 6= minimal acceptable turf, 9= ideal green, healthy turf. Table 2 - Amount of POD, CAT, SOD, APX, CHL, Proline, EL, protein, color and visual quality of different Iranian accessions and foreign common bermudagrass after cold stress (at 7.5/0°C day/night cycles) Location of accession POD (U mg−1 f.w.) CAT (U mg−1 f.w.) SOD (U mg−1 f.w.) APX (U mg−1 f.w.) CHL (mg g−1 f.w.) Proline (µmol g−1 f.w.) EL (%) Protein (mg g−1 f.w.) Color VQ Doroud Nahalestan 20.90 40.86 143.0 125.00 2.12 19.6 59.6 19.1 4.8 4.9 Abarkouh 21.73 42.29 136.0 108.57 2.28 21.9 57.8 21.8 5.7 5.6 Abidar sanandaj 25.64 40.86 132.5 59.29 2.17 20.8 59.3 19.0 5.0 5.1 Aligoudarz 22.18 42.75 138.5 148.57 3.11 26.4 52.5 21.2 7.0 7.1 Anzali 26.39 41.02 147.5 91.43 2.28 21.6 58.2 18.7 5.5 5.6 Arak 23.38 41.17 128.5 249.29 2.31 22.2 56.4 18.4 5.7 5.9 Ardakan 1 22.86 41.12 132.5 105.15 2.27 19.8 57.5 18.5 5.1 5.2 Ardakan 2 22.78 40.96 136.0 185.71 2.31 21.7 56.8 19.2 5.3 5.3 Azna 23.38 42.50 144.0 224.29 2.41 25.3 56.2 20.3 6.0 6.2 Boroujerd 15.19 41.48 140.0 91.43 2.24 21.3 57.4 19.8 5.2 5.3 Chenaran 24.36 43.16 136.0 156.43 2.31 21.9 57.5 18.9 5.8 5.9 Damghan 18.20 41.48 135.5 137.14 2.21 22.3 58.1 19.2 5.1 5.2 Daran 22.78 43.46 139.7 427.14 2.41 27.3 55.1 18.6 6.0 6.2 Dehgolan 23.53 44.69 137.0 42.14 2.34 21.9 58.2 18.6 5.7 5.9 Doroud Babahour 15.94 40.71 132.0 81.43 2.29 21.9 58.2 19.7 5.7 5.6 Doroud Daneshjo park 17.22 44.94 151.0 245.00 2.41 21.6 54.9 20.2 5.9 6.1 Doroud Siahvel 17.82 41.12 137.8 127.86 2.31 21.5 57.5 19.8 5.7 5.8 Firouzan 24.36 43.52 132.5 135.69 2.36 22.4 56.8 19.1 5.7 5.8 Foreign cultivar 22.11 42.24 137.5 122.86 3.28 27.9 51.1 21.5 7.5 7.5 Ghidar 20.08 42.44 138.5 132.71 2.26 21.5 58.5 18.7 5.4 5.5 Gorgan 24.14 41.99 129.5 135.00 2.35 27.3 56.5 18.8 6.4 6.5 Holailan 26.92 42.35 145.5 75.24 2.31 21.9 56.4 18.7 5.8 5.9 Homail 19.40 42.95 142.0 170.00 2.19 19.7 57.2 18.5 5.0 5.2 Islamabad gharb 21.82 41.85 141.4 120.24 2.15 19.5 57.5 18.8 5.0 5.1 Kamiaran 26.62 43.21 139.0 120.00 2.31 21.7 57.4 19.4 5.6 5.5 Khorram abad 24.59 42.55 130.0 85.71 2.35 21.8 57.5 18.7 5.7 5.8 Mahidasht 23.31 44.08 135.5 189.29 2.37 24.9 54.5 18.9 6.1 6.2 Maiami 22.41 41.37 136.0 132.72 2.35 23.2 55.5 18.5 6.0 6.1 Malayear 34.14 41.78 138.0 134.29 3.21 27.2 51.5 21.5 7.4 7.4 Malayear intersection 19.55 41.83 146.0 57.14 2.33 22.4 56.5 19.3 5.8 5.9 Mamoulan 22.78 43.98 139.5 191.43 2.39 24.3 55.5 19.1 6.0 6.1 Mashhad 22.56 42.95 136.0 62.86 2.28 21.5 57.2 19.1 5.7 5.8 Minodasht 20.38 42.09 147.0 140.00 2.25 21.5 57.9 19.1 5.2 5.4 Naein 23.08 42.29 139.0 191.43 3.26 27.5 51.2 18.7 7.5 7.6 Nahavand 24.74 41.22 135.5 110.71 2.26 21.8 57.2 20.8 5.5 5.4 Nour 24.66 42.90 133.5 89.14 2.21 20.7 57.1 18.6 5.0 5.3 Poldokhtar 22.33 40.81 139.2 121.43 2.37 22.5 54.5 18.6 6.0 6.2 Safashahr 21.95 41.53 132.5 212.86 2.38 27.1 53.5 21.3 6.4 6.3 Sarein 23.91 43.31 144.0 140.71 2.31 22.6 56.1 20.5 5.5 5.8 Sari 21.73 43.06 129.5 71.43 2.32 21.8 57.2 18.5 5.8 5.7 Saymareh bridge 25.86 40.96 129.5 75.00 2.34 19.8 57.8 19.4 5.7 5.1 Semnan 23.98 42.18 128.6 161.43 1.86 17.8 59.5 19.5 4.1 4.2 Shiraz 23.08 41.42 137.3 115.29 2.15 19.8 57.8 20.1 5.0 5.2 Tabriz 25.41 41.22 136.5 142.14 2.15 19.8 59.4 18.4 4.9 4.9 Taft 25.26 42.80 142.4 232.14 3.36 27.2 50.5 22.1 7.7 7.7 Tagh bostan kermanshah 21.23 41.46 134.5 81.74 2.14 26.4 59.8 17.1 5.0 4.9 Talesh 21.34 41.36 135.5 102.20 1.15 18.9 62.4 14.9 3.3 3.4 Tehran 19.85 41.27 143.5 140.71 2.24 21.4 57.3 18.7 5.2 5.2 Yazd 25.94 42.65 126.0 184.29 2.11 21.7 58.5 17.4 4.9 4.8 Zanjan 23.76 42.80 145.5 101.43 2.34 26.1 56.1 19.1 6.0 5.9 LSD (5%) 1.014 1.382 7.674 0.458 0.512 0.7279 4.086 1.825 0.988 1.027 164 Adv. Hort. Sci., 2018 32(2): 157-168 highest ascorbate peroxidase activity at 7.5°C and - 7.5°C was observed in Daran accession (Tables 2 and 3). The maximum ascorbate peroxidase at -15°C was observed in Taft accession and the second rank was belonged to common bermudagrass accession col- lected from Naein (Table 4). Total protein The results presented in Tables 2, 3 and 4 revealed that there was difference between total protein of different accessions in all temperature regimes. The maximum total protein at 24°C was belonged to Doroud daneshjo park, Sarein and Table 3 - Amount of POD, CAT, SOD, APX, CHL, Proline, EL, protein, color and visual quality of different Iranian accessions and foreign common bermudagrass after cold stress (at -7.5/-12°C day/night cycles) Location of accession POD (U mg−1 f.w.) CAT (U mg−1 f.w.) SOD (U mg−1 f.w.) APX (U mg−1 f.w.) CHL (mg g−1 f.w.) Proline (µmol g−1 f.w.) EL (%) Protein (mg g−1 f.w.) Color VQ Doroud Nahalestan 20.90 40.86 143.0 125.00 2.12 19.6 59.6 19.1 4.8 4.9 Abarkouh 21.73 42.29 136.0 108.57 2.28 21.9 57.8 21.8 5.7 5.6 Abidar sanandaj 25.64 40.86 132.5 59.29 2.17 20.8 59.3 19.0 5.0 5.1 Aligoudarz 22.18 42.75 138.5 148.57 3.11 26.4 52.5 21.2 7.0 7.1 Anzali 26.39 41.02 147.5 91.43 2.28 21.6 58.2 18.7 5.5 5.6 Arak 23.38 41.17 128.5 249.29 2.31 22.2 56.4 18.4 5.7 5.9 Ardakan 1 22.86 41.12 132.5 105.15 2.27 19.8 57.5 18.5 5.1 5.2 Ardakan 2 22.78 40.96 136.0 185.71 2.31 21.7 56.8 19.2 5.3 5.3 Azna 23.38 42.50 144.0 224.29 2.41 25.3 56.2 20.3 6.0 6.2 Boroujerd 15.19 41.48 140.0 91.43 2.24 21.3 57.4 19.8 5.2 5.3 Chenaran 24.36 43.16 136.0 156.43 2.31 21.9 57.5 18.9 5.8 5.9 Damghan 18.20 41.48 135.5 137.14 2.21 22.3 58.1 19.2 5.1 5.2 Daran 22.78 43.46 139.7 427.14 2.41 27.3 55.1 18.6 6.0 6.2 Dehgolan 23.53 44.69 137.0 42.14 2.34 21.9 58.2 18.6 5.7 5.9 Doroud Babahour 15.94 40.71 132.0 81.43 2.29 21.9 58.2 19.7 5.7 5.6 Doroud Daneshjo park 17.22 44.94 151.0 245.00 2.41 21.6 54.9 20.2 5.9 6.1 Doroud Siahvel 17.82 41.12 137.8 127.86 2.31 21.5 57.5 19.8 5.7 5.8 Firouzan 24.36 43.52 132.5 135.69 2.36 22.4 56.8 19.1 5.7 5.8 Foreign cultivar 22.11 42.24 137.5 122.86 3.28 27.9 51.1 21.5 7.5 7.5 Ghidar 20.08 42.44 138.5 132.71 2.26 21.5 58.5 18.7 5.4 5.5 Gorgan 24.14 41.99 129.5 135.00 2.35 27.3 56.5 18.8 6.4 6.5 Holailan 26.92 42.35 145.5 75.24 2.31 21.9 56.4 18.7 5.8 5.9 Homail 19.40 42.95 142.0 170.00 2.19 19.7 57.2 18.5 5.0 5.2 Islamabad gharb 21.82 41.85 141.4 120.24 2.15 19.5 57.5 18.8 5.0 5.1 Kamiaran 26.62 43.21 139.0 120.00 2.31 21.7 57.4 19.4 5.6 5.5 Khorram abad 24.59 42.55 130.0 85.71 2.35 21.8 57.5 18.7 5.7 5.8 Mahidasht 23.31 44.08 135.5 189.29 2.37 24.9 54.5 18.9 6.1 6.2 Maiami 22.41 41.37 136.0 132.72 2.35 23.2 55.5 18.5 6.0 6.1 Malayear 34.14 41.78 138.0 134.29 3.21 27.2 51.5 21.5 7.4 7.4 Malayear intersection 19.55 41.83 146.0 57.14 2.33 22.4 56.5 19.3 5.8 5.9 Mamoulan 22.78 43.98 139.5 191.43 2.39 24.3 55.5 19.1 6.0 6.1 Mashhad 22.56 42.95 136.0 62.86 2.28 21.5 57.2 19.1 5.7 5.8 Minodasht 20.38 42.09 147.0 140.00 2.25 21.5 57.9 19.1 5.2 5.4 Naein 23.08 42.29 139.0 191.43 3.26 27.5 51.2 18.7 7.5 7.6 Nahavand 24.74 41.22 135.5 110.71 2.26 21.8 57.2 20.8 5.5 5.4 Nour 24.66 42.90 133.5 89.14 2.21 20.7 57.1 18.6 5.0 5.3 Poldokhtar 22.33 40.81 139.2 121.43 2.37 22.5 54.5 18.6 6.0 6.2 Safashahr 21.95 41.53 132.5 212.86 2.38 27.1 53.5 21.3 6.4 6.3 Sarein 23.91 43.31 144.0 140.71 2.31 22.6 56.1 20.5 5.5 5.8 Sari 21.73 43.06 129.5 71.43 2.32 21.8 57.2 18.5 5.8 5.7 Saymareh bridge 25.86 40.96 129.5 75.00 2.34 19.8 57.8 19.4 5.7 5.1 Semnan 23.98 42.18 128.6 161.43 1.86 17.8 59.5 19.5 4.1 4.2 Shiraz 23.08 41.42 137.3 115.29 2.15 19.8 57.8 20.1 5.0 5.2 Tabriz 25.41 41.22 136.5 142.14 2.15 19.8 59.4 18.4 4.9 4.9 Taft 25.26 42.80 142.4 232.14 3.36 27.2 50.5 22.1 7.7 7.7 Tagh bostan kermanshah 21.23 41.46 134.5 81.74 2.14 26.4 59.8 17.1 5.0 4.9 Talesh 21.34 41.36 135.5 102.20 1.15 18.9 62.4 14.9 3.3 3.4 Tehran 19.85 41.27 143.5 140.71 2.24 21.4 57.3 18.7 5.2 5.2 Yazd 25.94 42.65 126.0 184.29 2.11 21.7 58.5 17.4 4.9 4.8 Zanjan 23.76 42.80 145.5 101.43 2.34 26.1 56.1 19.1 6.0 5.9 LSD (5%) 1.014 1.382 7.674 0.458 0.512 0.7279 4.086 1.825 0.988 1.027 Turfgrass visual quality and color based on a scale of 1-9, 1= brown/dead turf, 6= minimal acceptable turf, 9= ideal green, healthy turf. 165 Akbari and Salehi - Evaluation of common bermudagrass Iranian accession under cold stress Islamabad gharb accessions, and minimum total pro- tein was belonged to Daran accession. The highest total protein at 7.5°C and -7.5°C was observed in Taft accession (Tables 2 and 3). The maximum total pro- tein at -15°C was also observed in Taft accession and the second rank was belonged to common bermuda- grass accession collected from Malayear (Table 4). Proline Our results revealed that there was difference between proline content of different accessions in all temperature regimes. The maximum proline content at 24°C was belonged to Doroud daneshjo park, Azna, Mahidasht and Talesh accessions, and the mini- mum proline content was belonged to Nour acces- sion. The highest proline content at 7.5°C and -7.5°C was observed in foreign cultivar and Taft accession, respectively (Tables 2 and 3). The maximum proline content at -15°C was observed in Naein accession and the second rank belonged to common bermuda- grass accession collected from Taft (Table 4). Electrolyte leakage As shown in Tables 1, 2, 3 and 4, drop in tempera- ture severely increased EL in all accessions. The high- est EL at 7.5°C and -7.5°C was observed in Talesh and Abidar accessions, respectively, and the least was seen in Taft accession (Table 2 and 3). The minimum EL at -15°C was observed in Taft accession and the second rank was belonged to common bermudagrass accession collected from Naein (Table 4). Chlorophyll content, color and visual quality The highest chlorophyll content, color and visual quality were observed at 24°C and fall in tempera- ture under 24°C severely decreased these characters in all accessions (Tables 1, 2, 3 and 4). The highest chlorophyll content, color and visual quality at -7.5°C were observed in Taft, foreign cultivar, Naein, Malayear, Aligoudarz, Safashahr and Gorgan (Table 3). The maximum chlorophyll content, color and visu- al quality at -15°C were observed in Taft, Naein and Malayear accessions. 4. Discussion and Conclusions The 50 C. dactylon accessions were clustered into two major groups by Ward’s method on the basis of physiological characters at all temperature regimes. Accessions with high POD, CAT, SOD, APX, proline, protein, chlorophyll, color and visual quality, fall in same group. No complete relationships were found between the clustering of the common bermuda- grass accessions in dendrograms based on physiologi- cal characters and their geographical affiliations. These patterns of physiological variations within common bermudagrass accessions might be due to different genetic background because of various ploidy levels, cross pollination, genetic overlap, germplasm exchange and gene flow. Seven acces- sions collected from Taft, Naein, Malayear, foreign cultivar, Aligoudarz, Safashahr and Gorgan were the most cold-tolerant genotypes. Because, the highest antioxidant enzymes activity, chlorophyll content, color and visual quality at -7.5°C were observed in these accessions. Iran has a variable climate and we collected these species from different climatic regions of the country that shows its adaptation to wide ranges of climates. The results obtained from our physiological analysis demonstrated that the level of variation was great among Iranian C. dactylon accessions. The best color and visual quality in all common bermudagrass accessions were observed before cold stress. Reducing the temperature below 24°C severely decreased chlorophyll content, visual quality and color in all accessions. The results report- Table 4 - Amount of POD, CAT, SOD,APX, CHL, Proline, EL, protein, color and visual quality of the most cold-tolerant accessions of ber- mudagrass from Iran after cold stress (at -15/-15°C day/night cycles) In each column, means with the same letter are not significantly different according to Least Significant Difference (LSD) test at P= 0.05. Turfgrass visual quality and color based on a scale of 1-9, 1= brown/dead turf, 6= minimal acceptable turf, 9= ideal green, healthy turf. Location of accession POD (U mg−1 f.w.) CAT (U mg−1 f.w.) SOD (U mg−1 f.w.) APX (U mg−1 f.w.) CHL (mg g−1 f.w.) Proline (µmol g−1 f.w.) EL (%) Protein (mg g−1 f.w.) Color VQ Taft 10.12 a 31.35 a 96.00 a 71.45 c 1.85 a 16.350 a 78.1 d 14.20 a 6.20 a 6.30 a Naein 10.81 a 26.25 bc 93.86 a 85.39 a 1.74 a 16.41 a 82.3 c 13.70 a 6.10 a 6.00 ab Malayear 9.55 a 27.28 b 87.08 ab 74.65 c 1.36 e 14.21 ab 87.5 ab 13.90 a 5.36 b 5.30 ab Foreign cultivar 11.16 a 24.24 cd 76.18 bc 81.26 b 1.24 bc 13.82 ab 85.6 bc 12.80 a 5.10 b 5.10 bc Aligoudarz 10.13 a 23.36 d 71.22 c 67.25 d 0.94 cd 11.87 b 89.4 ab 11.85 a 4.30 c 4.30 c Gorgan 8.21 a 23.57 d 74.39 c 52.39 f 0.89 cd 11.68 b 91.2 a 12.10 a 4.20 c 4.50 cd Safashahr 9.17 a 22.11 d 69.00 c 57.48 e 0.75 d 11.89 b 90.8 a 12.80 a 4.10 c 4.10 d Adv. Hort. Sci., 2018 32(2): 157-168 166 ed by Esmaili and Salehi (2012) correspond to the results we obtained. The best temperature for growth and development of tropical grasses is rang- ing from 27 to 35°C (Beard, 1973). In warm-season turfgrasses, if temperature drops to 10-12.8°C their growth decrease considerably and enters dormancy at close to 0°C (Christians, 2004). McCarty (2001) suggested that a sudden air temperature decline to -5°C or a less rapid fall to below -12°C can cause dam- age to tropical turfgrasses. Photosynthetic apparatus directly affected by cold stress. In our study, the high- est POD, CAT, SOD and APX activity were observed at 7.5°C. As temperature diminished from 7.5 to -15°C, antioxidant enzymes activity decreased. This is in agreement with Manuchehri et al . (2014) for Cynodon dactylon (California origin). The study made by Zhang et al. (2006) on common bermudagrass ‘Riviera’ and ‘Princess-77’ showed an increase in the SOD activity during the first seven days of cold accli- mation and then after a decline was observed. Cold- acclimatized plants can tolerate freezing stress better than non-acclimated ones due to rapid development of metabolic defenses against freezing stress (Zhang et al., 2006). Overall, cold stress produces large amounts of ROS which causes oxidative injury to plants through vast destruction of proteins, carbohy- drates, lipids, cellular membranes, DNA and major decline of ATP reserve, and finally cell death (Dionne et al., 2001; Gill and Tuteja, 2010). Plants protect their cells from ROS damage by raising the activity of antioxidant enzymes like APX, SOD, CAT and POD (Apel and Hirt, 2004). Results of many studies indi- cate a positive correlation between freezing toler- ance in bermudagrasses and antioxidant enzymes activity (Zhang et al., 2006, Manuchehri et al., 2014). Over-production of ROS during chilling periods increase oxidative stress and can enhance the activity of antioxidant enzymes and stimulate synthesis of antioxidant metabolites (Karpinski et al., 2002). Our results regarding an increase in POD, SOD, CAT and APX activity found in this study is assumed to defense mechanism against oxidative damage caused by cold stress. The central role in the antioxidant defense system is perform by SOD via catalyzing O2 ·- into H2O2 and O2, while other antioxidant enzymes are also essential for breakdown of H2O2 through various pathways (Mittler, 2002; Apel and Hirt, 2004). CAT can produce O2 from H2O2. APX play a key role in ROS detoxification by conversion of H2O2 in to H2O. The balance between ROS production and elimination is vital mean for the protection of plant cells, and APX plays a major role in maintaining this balance (Asada, 1992; Lin et al., 2004). Karpinski et al. (2002) showed that tolerant plants have higher antioxidant enzyme activity that has dilatory effect on photooxidative injury during cold stress periods. It is postulated that under cold stress condition, cold tolerant plants increase the activity of their antioxidant enzymes that support active photosynthesis and development of carbohydrate reservation and other compounds such as protein and proline with protective functions. Our findings revealed that higher antioxidant enzymes activity can be attributed to better cold tol- erance in common bermudagrass. Decreasing the temperature increased EL, proline and protein con- tent. This negative correlation between temperature and EL can be due to cell membrane damage caused by cold temperatures. The EL method is commonly used to quantify the degree of cell membrane dam- age induced by cold temperatures and to assay the cold stress tolerance of turfgrasses (Shashikumar and Nus, 1993; Anderson and Taliaferro, 2002). Our results indicated that leaf EL increased during cold acclimatization. Cold acclimatization may induce ROSs production and oxidative stress, and may cause slight damage to cell membrane and later will cause an increase in EL. The production of free radicals under cold stress conditions may initiate the signal- ing pathways of plant metabolic defense responses, which may reduce cell membrane disruption and EL (Zhang and Ervin, 2008). One of the most important differences between cold tolerant and cold sensitive accessions may be greater development of defense responses to scavenging ROSs and lowering the EL in cold tolerant accessions. Cold acclimation increase proline content in all accessions, but we observed much higher proline content in cold tolerant acces- sions. This results for proline content in our research, is in accordance with the results presented by Munshaw et al. (2004), where they reported a signifi- cant increase of stolon proline concentration of the common bermudagrass ‘Princess-77’ during cold acclimation period. Other supporting results include those of Munshaw et al. (2006) and Zhang et al. (2006), where they found that higher proline content in bermudagrass cultivars during the winter can be related to greater freezing tolerance. Many studies have pointed to the cryoprotectiveness of proline and their function as osmolytes or compatible solutes (Koster and Leopold, 1988; Santarius, 1992; Karpinski et al., 2002). Proline is a cryoprotectant for chloroplast membranes of spinach (Spinacia oleracea L.) and plays a crucial role in plant protection against freezing stress (Santarius, 1992). Proline contributes Akbari and Salehi - Evaluation of common bermudagrass Iranian accession under cold stress 167 to the acclimatization of plants to cold stress by increasing osmotic potential (reducing the water potential) and decreasing the freezing point of cells. Protein content of all accessions was high during cold acclimation. Higher protein content was observed in cold-tolerant accessions compared to cold-sensi- tive ones. One of the studies that confirm our results also, is a study by Zhang et al. (2006), with their study on C. dactylon ‘Riviera’ as a cold tolerant cultivar with higher protein content, and ‘Princess-77’ as a cold sensitive cultivar with lower protein content follow- ing acclimatization. During cold acclimation plants increase their capacity for synthesis of novel proteins (Cloutier, 1983). This increase in protein content can be attributed to their determinant role in freezing tolerance of common bermudagrass. Our results showed rapid physiological alterations in common bermudagrass accessions in response to cold stress. Our results also revealed that cold-toler- ant common bermudagrass accessions showed high- er proline, protein, antioxidant enzymes, color, visual quality and chlorophyll content, and cold-sensitive accessions showed more severe cell membrane dam- age (EL) under cold stress conditions. We identified drastic natural variations in tolerance between com- mon bermudagrass accessions of Iran in response to freezing stress. Comparative study between bermudagrass accessions based on morphophysio- logical traits is one of the best method for its cold tol- erance improvement. According to our physiological investigations, accessions collected from Taft, Naein and Malayear were the most cold-tolerant genotypes compared to other accessions. Further molecular studies are in progress to clarify better these find- ings. This is the first report based on physiological characters of Iranian common bermudagrass acces- sions differences in cold tolerance and provides use- ful information for breeding programs. References ANDERSON J.A., TALIAFERRO C.M., 2002 - Freeze tolerance of seed-producing turf bermudagarsses. - Crop Sci., 42: 190-192. ANDERSON J.A., TALIAFERRO C.M., MARTIN D.L., 2003 - Longer exposure durations increase freeze damage to bermudagrasses. - Crop Sci., 43: 973-977. APEL K., HIRT H., 2004 - Reactive oxygen species: metabo- lism, oxidative stress, and signal transduction. - Ann. Rev. 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