ARCHIVES OF AGRONOMY AND SOIL SCIENCE, 2016 VOL. 62, NO. 6, 769–780 http://dx.doi.org/10.1080/03650340.2015.1094182

Screening different crested wheatgrass ( cristatum (L.) Gaertner.) accessions for drought stress tolerance Hassan Bayata, Hossein Nematia, Ali Tehranifara and Ali Gazanchianb aDepartment of Horticultural Science, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran; bDepartment of Genetic and Physiology, Agricultural and Natural Resources Research Center of Khorassan Razavi, Mashhad, Iran

ABSTRACT ARTICLE HISTORY A pot experiment was conducted in a greenhouse to evaluate the effect Received 10 May 2015 of drought stress on growth and turf quality of 24 accessions of crested Accepted 10 September wheatgrass ( (L.) Gaertner.) and ‘Rembrandt’ tall 2015 fescue, and to find the most drought-tolerant accession(s) of crested KEYWORDS wheatgrass. The grasses were treated in well-watered or exposed to Cluster analysis; correlation; drought stress by withholding water for 20 days. Visual turf quality of growth; low-maintenance; drought-stressed had no significant differences with well- turf quality; wheatgrass watered plants until day 8 of drought and 1727 P10 had the highest turf quality at day 20 of drought. Cluster analysis classified the acces- sions and Rembrandt into five clusters comprised of drought tolerant (I), intermediate (II, IV and V) and susceptible (III) in response to soil drying. Turf quality, green tissue, relative water content and electrolyte leakage of cluster I (1727 M, 208 M, 4056, 2854 and 1727 P1) were 1.8-, 2.0-, 1.6- and 0.6-fold of cluster III (Rembrandt) under drought stress conditions, respectively. Genetic diversity of cluster I could be used for breeding programmes and introduction of the drought-tolerant accessions to turfgrass breeders for drought tolerance varietal devel- opment programme.

Introduction Water is becoming more and more rare resource in arid and semi-arid regions of Iran that is characterized by little rainfall, high solar radiation and high temperatures in the summer (Madani

Downloaded by [Dokuz Eylul University ] at 21:15 06 May 2016 2014). In recent years, the normal seasonal droughts that have occurred in most regions of Iran have caused local and state government to enact water conservation ordinances. Urbanization and increases in population, however, are seriously threatening sustainable natural resources. At pre- sent, non-renewable groundwater resources are being depleted to an alarming extent. On the other hand, turf grass has been extensively planted in urban green spaces in recent years and demand for water for the irrigation of turfgrass will also increase substantially. Under severe drought conditions, water restrictions may be imposed on turfgrass areas. One strategy to mitigate irrigation demands for turfgrass is to identify and develop and that maintain better quality with less water (Hanks et al. 2005; Robins & Waldron 2007). Extensive landscape areas that have been planted to high-maintenance turf could be replaced with low-maintenance turf species or cultivars (Wu & Hari-vandi 1988; Watkins et al. 2011). Low-maintenance turf is a relative term describing areas that receive reduced or no inputs of irrigation, fertilizer, herbicides andmowing,andcanwithstandweedinvasion(Meyer

CONTACT Hossein Nemati [email protected] © 2015 Taylor & Francis 770 H.BAYATETAL.

1989; Dernoeden et al. 1994) and thus help conserve natural resources and reduce pollutants. Conservation of water is a primary goal for low-maintenance turfgrass development and management in arid and semi-arid areas (Hanks et al. 2005). Moreover, maintenance costs of turfgrass in many areas could be reduced by planting low-maintenance turfgrasses that need limited irrigation and mowing (Feldhake et al. 1983;Watkinsetal.2011). One of the main limitations to the use of low-input turf is the availability of appropriate plant material. Common cool season turfgrasses cultivars are genetically improved for turf quality, resistance to plant pests and diseases and perform well in areas with sufficient water. However, many regions of Iran are in arid and semi-arid areas with less water where these species may have a lot of problems. Crested wheatgrass (Agropyron cristatum (L.) Gaertner.) is a potential source of turf germ- plasm for arid and semi-arid regions. The genus originated from central Asia, including parts of Iran, Turkey, Afghanistan, Russia and China (Dewey & Asay 1975) and includes 150 species, of which more than 20 species are distributed in different areas in Iran (Mozaffarian 1996). Crested wheatgrass is a persistent, long-lived perennial and characteristics include excellent seed production, seedling vigour, drought and cold resistance. It withstands weed competition, tolerates insect depredation, is easily established and is adapted to a wide variety of soils. Although crested is normally bunch-type growth habit, accessions with varying degrees of rhizome development have been identified from Iran and Turkey (Asay & Jensen 1996;Asay et al. 1999). Plant response to drought stress involves changes in various morphological and physiological factors (Nilsen & Orcutt 1996; Shinozaki & Yamaguchi-Shinozaki 1997; Sanchez-Blanco et al. 2009). Water stress reduced dry matter production of grasses by reduction of leaf area, height of plant and tillering (Gazanchian et al. 2007; Pessarakli & Kopec 2008). Drought stress increased electrolyte leakage (EL) of cool season grass and resistant cultivars exhibited better membrane stability than susceptible ones (Jiang & Huang 2001; Jinrong et al. 2008). Drought stress has also adverse effects on grass quality and persistence, particularly for cool season turfgrasses that require a relatively large amount of water for maintaining growth (Bian & Jiang 2009; Sanchez-Blanco et al. 2009). Genetic differences among cultivars in drought tolerance traits could be used by turf breeders as selection in breeding programmes for improving drought tolerance. The objectives of this study were to find the most drought-tolerant accession(s) of crested wheatgrass for optimum growth and turf quality under drought stress, and introduction of the tolerant accession(s) to turfgrass breeder for broad cultural practices.

Downloaded by [Dokuz Eylul University ] at 21:15 06 May 2016 Materials and methods Plant materials and growth conditions Seeds of 24 accessions of crested wheatgrass (A. cristatum (L.) Gaertner.) native to Iran were collected from different geographical regions by the Rangelands and Forestry Research Institute (Table 1) and planted in the greenhouse of Department of Horticultural Science in Ferdowsi University of Mashhad, Mashhad, Iran, in 2014. ‘Rembrandt’ tall fescue (Festuca arundinacea Schreb.) was used as a control turf to compare with the accessions. The grass seeds were sown in plastic pots (18 cm diameter and 21 cm length) filled with sandy loam soil (50% fine-loamy soil and 50% sand) with pH = 7.0. The soil had 18.2 g water per 100 g dry soil at field capacity. Prior to the drought treatments, plants were watered three times per week to maintain soil moisture at field capacity and grasses were cut every two weeks with scissors and maintained at 9 cm height. The greenhouse environmental conditions were 22/18°C (day/night), relative humidity of 50–60%, 14 h photoperiod and a photosynthetically active radiation of 900 µmol m −2 s−1. ARCHIVES OF AGRONOMY AND SOIL SCIENCE 771

Table 1. Collection site and turf quality (1–9 visual scale, 9 indicating the best turf quality) of 24 crested wheatgrass accessions native to Iran and ‘Rembrandt’ tall fescue under well-watered (C) and drought stress conditions (D). Day 0 Day 4 Day 8 Day 12 Day 16 Day 20 Accession Collection site C D C D C D C D C D C D 3029 Bojnord 6.33 6.66 6.00 6.66 6.33 6.66 6.33 4.66 6.33 2.33 6.66 1.66 1727 P10 Golestan 6.66 6.66 7.00 6.66 6.66 6.66 6.66 5.00 6.33 4.00 6.66 3.00 1727 M Golestan 4.66 4.33 4.66 4.33 5.00 4.33 4.33 4.46 4.66 4.10 5.00 2.66 208 M Esfahan 6.03 6.43 6.20 6.43 6.16 6.43 6.50 5.30 6.20 3.00 6.16 2.66 4056 Chadegan 6.00 6.66 6.33 6.66 6.66 6.66 6.50 5.00 6.33 2.66 6.00 2.00 2854 Arak 6.33 6.65 6.33 6.65 6.33 6.65 6.33 5.20 6.33 3.00 6.33 1.33 1727 P12 Golestan 4.76 4.43 4.76 4.46 5.06 4.43 4.76 4.20 4.76 2.33 4.76 1.66 1550 Bojnord 5.10 4.56 5.10 4.56 5.10 4.56 5.10 4.10 5.10 1.66 5.03 1.00 619 S Esfahan 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 2.33 5.00 2.00 208 P2 Esfahan 5.33 4.86 5.33 4.86 5.33 4.86 5.33 5.13 5.33 2.66 5.33 2.33 4049 Kerman 6.33 6.16 6.33 6.10 6.33 6.16 6.33 5.33 6.33 1.00 6.33 1.00 208 P13 Esfahan 6.00 6.00 6.00 6.00 6.00 6.00 6.33 5.10 6.00 2.00 6.00 1.00 619 M Esfahan 6.33 6.43 6.00 6.43 6.33 6.43 6.33 5.06 6.33 2.00 6.33 1.00 4056 M Chadegan 6.26 6.00 6.26 6.00 6.26 6.00 6.50 5.10 6.26 1.50 6.26 1.00 210 M Hamand 4.90 5.03 4.90 5.03 5.06 5.03 5.06 4.00 4.90 2.33 5.06 2.33 619 Esfahan 6.00 6.00 6.00 6.00 6.00 6.00 6.00 5.16 6.00 1.00 6.00 1.00 619 P13 Esfahan 5.96 6.10 6.13 6.10 5.96 6.10 5.96 5.06 5.96 1.00 5.83 1.00 208 MH Hamedan 4.83 5.03 4.83 5.03 4.83 5.03 4.83 4.23 4.83 1.33 4.83 1.00 1727 P1 Golestan 5.10 5.10 5.10 5.10 5.10 5.10 5.10 5.00 5.10 3.00 5.10 3.00 1727 Golestan 5.40 5.20 5.40 5.20 5.40 5.20 5.40 4.16 5.40 2.00 5.40 1.00 209 M Esfahan 5.23 5.10 5.23 5.10 5.23 5.10 5.23 4.03 5.23 1.00 5.23 1.00 4050 Lorestan 6.10 6.10 6.10 6.10 6.10 6.10 6.00 5.33 6.00 1.00 6.10 1.00 4049 M Kerman 6.00 6.00 6.00 6.00 6.33 6.00 6.00 5.40 6.00 1.00 6.00 1.00 1551 Bojnord 4.33 4.40 4.76 4.40 4.33 4.40 4.33 4.00 4.66 1.00 4.33 1.00 Rembrandt 7.00 6.66 7.00 6.65 7.00 4.33 7.00 1.20 7.00 1.00 7.00 1.00 LSD (0.05) 0.71 0.69 0.71 0.55 0.92 0.79 LSD, least significant difference.

Treatments and experimental design After 120 days of growing, the grasses were exposed to two soil water treatments: (1) well-watered (control): plants were watered every other day to soil reaching field capacity; and (2) drought stress: irrigation was withheld for 20 days. The experiment was conducted as a completely randomized design with three replicates.

Measurements Downloaded by [Dokuz Eylul University ] at 21:15 06 May 2016 Evapotranspiration (ET) was calculated by weighing the pots at 10:00 every 4-day intervals. Turf quality was visually rated on a scale of 1–9, with a rating of 1 being a completely desiccated brown turf canopy and a rating of 9 representing healthy plants with dark green, turgid leaf blades and a dense turf canopy (Turgeon 1996). A rating of 6 was considered the minimal acceptable turf quality level. Green tissue ratings were evaluated based on the amount of green tissue remaining (where 1 point is no green tissue remaining and 9 points is all green tissue) (Watkins et al. 2007; Karcher et al. 2008). Relative water content (RWC) was calculated according to Barrs and Weatherley (1962) using the formula: [(FW − DW)/(TW − DW)] * 100, where FW is leaf fresh weight, TW is leaf turgid weight and DW is leaf dry weight after oven-drying leaf samples for 72 h at 100°C. Turgid weight was determined as weight of fully turgid leaves after soaking leaves in distilled water in the refrigerator for 24 h. Electrolyte leakage was evaluated by Barranco et al. (2005) method. Also, 0.5 g fresh weight of the leaves were placed in Erlenmeyer flask containing 20 mL distilled water and incubated for 24 h in a shaker at 23°C under continuous light. The initial electrolyte conductivity (EC1) of each sample was measured to obtain an indirect indication of the amount of ion released at each treatment. 772 H.BAYATETAL.

Then, the samples were placed in an autoclave at 121°C for 20 min and a second reading (EC2) was recorded after cooling the solution to room temperature. The EL was calculated as EC1/EC2 and expressed as per cent. At the end of the experiment period (20 days), the shoot length and the maximum root length were measured for each pot. Plant material from six pots for each accession were washed, shoots and roots separated, oven dried (78°C for 48 h) and weighed.

Statistical analysis Analysis of variance for all the variables was carried out using the JMP8 software (SAS Campus, Cary, NC, USA), and LSD test at 5% level was used for mean separation. Ward’s(1963) method based on Euclidean distance was used to grouped all 24 accessions and ‘Rembrandt’ tall fescue by the Minitab software.

Results Evapotranspiration There were significant differences among the accessions for their ET under drought stress (Figure 1). At day 12 of drought, 208 P13 had the lowest ET (20.01 g day−1) and total ET water losses ranged from 698.33 g day−1 (210 M) to 764.33 g day−1 (208 MH) at day 20 of drought (Figure 1).

Turf quality and green tissue ratings Turf quality under drought stress as determined by visual turf quality is presented in Table 1.Turf quality was maintained at >6 throughout the treatment period in 12 accessions under well-watered conditions and in the others were <6. Under drought stress, turf quality of drought-stressed plant had no significant differences with well-watered plants until day 8. At days 12–20 of drought, turf quality exhibited a steady decline and all accessions had no acceptable turf quality <6. Acceptable turf quality was only maintained until day 4 for Rembrandt and decreased up to 1.2 at day 12 of drought (Table 1). As with turf quality ratings, green tissue ratings declined more slowly under drought stress (Table 2). 1727 P1 had the highest green tissue at day 20 of drought. All accessions showed significantly higher green tissue than Rembrandt throughout the drought treatment period (Table 2).

Downloaded by [Dokuz Eylul University ] at 21:15 06 May 2016 Growth parameters Growth parameters under well-watered and drought stress conditions are shown in Tables 3 and 4. Drought stress stopped shoot vertical growth of all accessions and Rembrandt at days 16 and 8, respectively. At day 20 of drought, shoot length decreased by 20% in comparison to control (Table 3). Soil drying had significant reduction in RL and 619 M (23.00 cm) had the longest root system among all accessions (Table 4). 1727 M (0.88) and 619 M (1.80) had the lowest and greatest root to shoot length ratio under well-watered conditions, respectively (Table 4). Drought stress increased root to shoot length ratio by 24% in comparison to control. Significant differences among accessions were measured for root, shoot and total dry weight under well-watered and drought-stressed conditions (Table 4). Drought stress decreased root, shoot and total dry weight by 4%, 10% and 7% in comparison to control plants (Table 4). Interestingly, the highest root dry weight loss was observed in Rembrandt (44.5%) compared to control (Table 4). Drought stress increased root to shoot dry weight ratio of all the accessions by 11% in comparison to control. The highest increase in root to shoot dry weight ratio was observed for 1727 P10 (48.8%) (Table 4). ARCHIVES OF AGRONOMY AND SOIL SCIENCE 773 Downloaded by [Dokuz Eylul University ] at 21:15 06 May 2016

Figure 1. Evapotranspiration of 24 crested wheatgrass accessions native to Iran and ‘Rembrandt’ tall fescue as affected by soil drying for 20 days. Bars indicate LSD values for treatment comparisons within a given day of treatment for each cultivar (P ≤ 0.05).

Relative water content and electrolyte leakage Leaf RWCs of all the accessions significantly decreased with drought stress (Table 4). RWCs declined from 74% in well-watered plants to 32% in drought-stressed plants. Under drought stress, RWC were significantly higher in the leaves of 208 M (46.37%), 2854 (46.33%), 1727 P1 (45.55%), 1727 M (45.00%) and 4056 (43.66%), indicating their higher drought tolerance (Table 4). Drought stress significantly increased EL (2.3 times in comparison to control plants) in the leaves of the crested wheatgrass accessions. Electrolyte leakages were significantly lower in 208 M (42.88%), 1727 M (45.07%), 2854 (48.41%), 4056 (48.44%), and 1727 P1 (51.06%) (Table 4). 774 H.BAYATETAL.

Table 2. Green tissue rating (1–9 visual scale, 9 indicating the best green tissue rating) of 24 crested wheatgrass accessions native to Iran and ‘Rembrandt’ tall fescue under well-watered (C) and drought stress (D) conditions. Day 0 Day 4 Day 8 Day 12 Day 16 Day 20 Accessions CDCDCDCDCDCD 3029 9.00 8.66 9.00 8.66 9.00 8.66 9.00 8.66 9.00 3.63 9.00 3.00 1727 P10 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 8.10 9.00 7.20 1727 M 8.00 8.00 8.00 8.00 8.00 8.00 8.00 8.00 8.00 7.20 8.00 5.10 208 M 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 5.85 9.00 4.66 4056 9.00 9.00 9.00 9.00 9.00 9.00 8.83 9.00 8.66 4.20 8.66 2.7 2854 8.33 9.00 8.33 9.00 8.33 9.00 8.33 8.33 8.33 4.20 8.33 2.13 1727 P12 8.00 8.00 8.00 8.00 8.00 8.00 8.00 8.00 8.00 4.20 8.00 2.56 1550 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 2.73 9.00 1.26 619 S 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 5.10 9.00 4.95 208 P2 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 8.33 5.55 8.33 4.95 4049 8.66 8.00 8.66 8.00 8.66 8.00 8.66 7.65 8.66 1.26 8.66 1.00 208 P13 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 4.50 9.00 2.16 619 M 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 3.30 9.00 2.73 4056 M 8.00 8.00 8.00 8.00 8.00 8.00 8.33 2.30 8.33 2.30 8.33 2.30 210 M 8.10 8.00 8.16 8.00 8.16 8.00 8.00 8.00 8.00 4.50 8.00 3.46 619 9.00 9.00 9.00 9.00 9.00 9.00 8.66 9.00 8.66 2.16 8.66 2.16 619 P13 8.66 8.00 8.66 8.00 8.33 8.00 8.66 8.00 8.66 3.90 8.66 3.60 208 MH 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 8.00 4.20 8.00 4.20 1727 P1 9.00 9.00 8.50 8.00 8.50 8.00 8.50 8.00 8.50 7.65 8.50 7.50 1727 8.00 9.00 8.16 9.00 8.00 9.00 8.00 9.00 8.00 4.80 8.00 3.60 209 M 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 1.50 9.00 1.00 4050 9.00 8.33 9.00 8.33 8.66 8.33 8.66 8.33 8.66 1.56 8.66 1.00 4049 M 8.00 9.00 8.00 9.00 8.00 8.00 8.00 8.66 8.00 1.00 8.00 1.00 1551 9.00 9.00 9.00 9.00 8.66 8.00 8.66 8.00 8.66 1.00 8.66 1.00 Rembrandt 9.00 9.00 9.00 9.00 9.00 5.80 9.00 1.80 9.00 1.00 9.00 1.00 LSD (0.05) 0.30 0.32 0.38 0.48 1.86 1.69

Table 3. Vertical shoot growth of 24 crested wheatgrass accessions native to Iran and ‘Rembrandt’ tall fescue under well- watered (C) and drought stress (D) conditions. Day 0 (cm) Day 4 (cm) Day 8 (cm) Day 12 (cm) Day 16 (cm) Day 20 (cm) Accessions CDCDCDCDCDCD 3029 9.00 9.00 10.66 11.33 12.66 12.00 14.33 14.33 15.50 14.33 16.50 14.33 1727 P10 9.00 9.00 12.25 12.50 16.00 15.00 17.75 15.66 18.50 15.66 19.50 15.66 1727 M 9.00 9.00 11.00 10.66 13.50 12.33 15.50 13.16 17.50 13.16 19.25 13.16 208 M 9.00 9.00 11.00 10.33 13.33 11.33 14.00 11.33 14.66 11.33 15.50 11.33 4056 9.00 9.00 11.75 10.16 14.25 11.33 14.50 11.33 14.50 11.33 15.00 11.33 2854 9.00 9.00 12.00 11.00 15.00 12.83 15.00 12.83 16.00 12.83 16.75 12.83 1727 P12 9.00 9.00 11.50 12.00 15.66 14.50 15.66 14.66 15.66 14.66 15.93 14.66 1550 9.00 9.00 11.16 10.83 15.75 13.33 16.00 13.33 16.50 13.33 17.50 13.33

Downloaded by [Dokuz Eylul University ] at 21:15 06 May 2016 619 S 9.00 9.00 10.33 10.50 12.00 11.66 12.33 11.66 12.66 11.66 13.00 11.66 208 P2 9.00 9.00 11.83 12.00 14.00 13.00 14.66 13.00 15.33 13.00 16.00 13.00 4049 9.00 9.00 9.83 10.00 10.83 10.33 11.16 10.33 11.66 10.33 12.16 10.33 208 P13 9.00 9.00 12.00 11.25 13.50 12.50 14.00 12.50 14.66 12.50 15.33 12.50 619 M 9.00 9.00 12.00 10.83 13.33 13.00 13.33 13.00 13.33 13.00 13.33 13.00 439056 M 9.00 9.00 12.00 11.00 13.50 12.00 15.00 12.00 16.00 12.00 17.00 12.00 210 M 9.00 9.00 11.00 12.16 12.00 14.00 13.25 14.00 14.25 14.00 15.00 14.00 619 9.00 9.00 13.33 13.00 15.00 14.33 17.16 14.33 18.33 14.33 19.33 14.33 619 P13 9.00 9.00 11.83 13.50 14.33 15.00 15.16 15.00 16.16 15.00 17.33 15.00 208 MH 9.00 9.00 15.00 14.00 16.00 14.33 17.00 15.00 18.33 15.00 19.00 15.00 1727 P1 9.00 9.00 13.33 12.66 15.00 13.33 16.00 13.33 16.33 13.33 16.66 13.33 1727 9.00 9.00 11.66 11.33 12.50 12.00 14.16 12.00 15.00 12.00 16.00 12.00 209 M 9.00 9.00 10.00 10.50 12.00 11.33 13.00 11.33 14.00 11.33 15.00 11.33 4050 9.00 9.00 13.00 10.83 14.25 11.83 15.50 11.83 16.50 11.83 17.50 11.83 4049 M 9.00 9.00 14.00 13.50 15.00 14.25 16.00 14.25 17.00 14.25 18.00 14.25 1551 9.00 9.00 12.50 12.00 13.50 12.83 15.00 13.16 16.50 13.16 18.00 13.16 Rembrandt 9.00 9.00 9.33 9.66 11.00 9.66 11.83 9.66 12.33 9.66 13.00 9.66 LSD (0.05) - 1.71 2.12 2.15 2.12 2.40 ARCHIVES OF AGRONOMY AND SOIL SCIENCE 775

Table 4. Root length, root to shoot length ratio, root dry weight, shoot dry weight, root to shoot dry weight ratio, total dry weight, relative water content and electrolyte leakage of 24 crested wheatgrass accessions native to Iran and ‘Rembrandt’ tall fescue under well-watered (C) and drought stress (D) conditions. Root/ shoot Root dry Shoot dry Root/shoot Total dry Relative Root length length weight weight dry weight weight water Electrolyte (cm) ratio (g pot −1) (g pot −1) ratio (g pot −1) content (%) leakage (%) Accessions CDCDCDCDCDCDCDCD 3029 21.00 20.10 1.29 1.54 4.39 4.34 2.92 2.75 1.49 1.57 7.32 7.09 74.33 27.33 20.33 79.68 1727 P10 21.00 19.00 1.17 1.23 3.58 2.85 3.85 1.44 1.32 1.96 6.68 4.29 70.00 31.66 23.14 79.28 1727 M 17.33 14.50 0.88 1.08 2.19 2.10 1.66 1.57 1.33 1.28 3.86 3.67 65.66 45.00 28.55 45.07 208 M 19.33 16.00 1.25 1.33 3.78 3.42 2.08 1.92 1.81 1.78 5.86 5.34 78.66 46.37 22.44 42.88 4056 19.16 21.00 1.33 1.75 3.46 3.76 2.57 2.35 1.37 1.59 6.03 6.10 79.66 43.66 21.40 48.44 2854 20.00 18.50 1.19 1.42 3.83 3.88 2.44 2.43 1.56 1.58 6.27 6.31 79.33 46.33 19.57 48.41 1727 P12 19.33 15.50 1.21 1.00 2.76 2.69 2.00 1.80 1.38 1.61 4.76 4.49 71.00 23.33 24.90 81.46 1550 21.33 21.00 1.17 1.75 2.97 3.93 2.45 2.35 1.22 1.68 5.42 6.28 70.00 25.00 20.11 85.55 619 S 20.50 20.00 1.58 1.74 3.02 2.69 2.54 1.70 1.19 1.62 5.56 4.39 75.00 30.00 20.07 81.82 208 P2 21.66 20.00 1.36 1.53 3.92 3.00 3.20 2.47 1.22 1.22 7.12 5.47 78.66 35.00 17.60 79.50 4049 21.66 20.00 1.78 2.00 3.41 2.91 3.44 2.38 0.99 1.22 6.86 5.29 76.00 22.36 19.11 77.38 208 P13 22.00 20.50 1.44 1.81 3.34 3.05 3.31 2.49 1.00 1.23 6.65 5.55 68.00 27.33 26.55 79.40 619 M 24.00 23.00 1.80 2.09 4.09 4.33 2.72 2.66 1.45 1.62 6.92 6.99 70.66 34.33 19.42 79.01 4056 M 22.33 20.00 1.29 1.53 3.83 5.14 2.89 2.85 1.33 1.80 6.73 7.99 72.00 31.00 24.50 76.92 210 M 20.00 21.00 1.42 1.50 3.59 3.94 2.90 2.93 1.24 1.33 6.49 6.87 68.33 25.33 29.90 82.88 619 22.00 24.00 1.13 1.67 3.17 3.63 3.33 3.09 0.98 1.22 6.50 6.73 74.66 33.33 23.85 77.26 619 P13 20.66 20.00 1.20 1.75 3.83 2.99 2.93 3.72 1.36 0.83 6.77 6.71 72.33 27.66 23.66 80.93 208 MH 19.50 21.00 1.10 1.50 3.84 3.96 3.09 2.89 1.24 1.37 6.94 6.85 77.33 32.23 19.66 86.01 1727 P1 20.00 21.50 1.25 1.44 2.95 2.62 2.04 1.80 1.44 1.45 4.99 4.42 79.31 45.55 22.40 51.06 1727 19.50 20.00 1.26 1.81 2.85 2.95 2.23 2.69 1.30 1.09 5.08 5.65 72.65 37.27 21.45 60.81 209 M 21.66 22.00 1.33 1.99 3.25 3.62 3.10 3.16 1.06 1.14 6.30 6.78 74.16 28.94 19.29 78.30 4050 20.00 21.50 1.14 1.78 3.44 3.92 3.03 3.49 1.12 1.12 6.47 7.41 72.66 31.66 24.83 80.36 4049 M 20.83 22.50 1.22 1.58 2.71 2.41 3.95 2.04 0.71 1.23 6.66 4.45 78.33 28.00 17.31 77.29 1551 21.33 21.00 1.24 1.55 2.97 3.93 2.45 2.35 1.22 1.68 5.42 6.28 70.33 26.66 20.11 85.55 Rembrandt 21.66 21.50 1.67 2.15 9.57 5.31 5.05 5.59 1.39 0.95 16.41 10.90 81.66 22.33 15.32 84.87 LSD (0.05) 2.13 0.51 1.02 0.87 0.45 1.55 4.31 4.47

Correlation Correlation coefficients among studied traits are shown in Table 5 under well-water (upper of diagonal) and drought stress (lower of diagonal) conditions. Under drought stress condition, negative correlations occurred between turf quality and root length (r =−0.63**) and between turf quality and total dry weight (r =−0.56**) (Table 5). Significant correlation occurred between root to shoot dry weight and turf quality (r = 0.55**) and between root to shoot dry weight and Downloaded by [Dokuz Eylul University ] at 21:15 06 May 2016 green tissue (r = 0.45**) under drought stress (Table 5).

Cluster analysis On the basis of the results of cluster analysis, the accessions were assigned to five clusters (Figure 2). Cluster I had five accessions, namely 1727 M, 208 M, 4056, 2854 and 1727 P1 and had the highest RWC (45.80%), root to shoot dry weight (1.69), turf quality (3.15) and green tissue (6.23) and lowest electrolyte leakage (47.18%) and shoot length (12.40 cm) among all accessions under drought stress (Table 6). There were six accession in cluster II and had middle averages for most traits. Cluster III (Rembrandt) had the highest root (5.31 g pot−1), shoot (5.60 g pot−1) and total (10.91 g pot−1) dry weight, while root to shoot dry weight (0.95) was the lowest (Table 6). Cluster IV included six accessions and had the longest root length (21.67 cm). Cluster V with seven accessions including the largest group obtained the longest shoot length (13.55 cm). Downloaded by [Dokuz Eylul University ] at 21:15 06 May 2016 776 H.BAYATETAL.

Table 5. Correlation coefficients among 11 measured traits of 24 crested wheatgrass accessions native to Iran and ‘Rembrandt’ tall fescue under well water (above diagonal) and drought stress (below the diagonal). Turf Green Shoot Root Root to shoot Root dry Shoot dry Root to shoot dry Total dry Relative water Electrolyte Correlation quality tissue length length length ratio weight weight weight ratio weight content leakage Turf quality 0.456* –0.256ns 0.416* 0.394* 0.579** 0.566** –0.011ns 0.590** 0.397* –0.308ns Green tissue 0.893** –0.338ns 0.517** 0.418* 0.325ns 0.265ns 0.085ns 0.299ns 0.060ns –0.279ns Shoot length 0.205ns 0.339ns –0.284ns –0.873** –0.402** –0.260ns –0.003ns –0.377ns –0.226ns 0.297ns Root length –0.632** –0.523** –0.039ns 0.617** 0.307ns 0.413* –0.254ns 0.374ns –0.004ns –0.384* Root to shoot length ratio –0.644** –0.602** –0.569 ** 0.701** 0.444* 0.399* –0.067ns 0.456* 0.211ns –0.399* Root dry weight –0.387* –0.504* –0.285ns 0.382ns 0.420* 0.848** –0.008ns 0.973** 0.380* –0.427* Shoot dry weight –0.629** –0.599** –0.333ns 0.411* 0.624** 0.674** –0.443* 0.944** 0.277ns –0.432* Root to shoot dry weight ratio 0.530** 0.455** 0.190ns –0.182ns –0.461* –0.009ns –0.672** –0.213ns 0.259ns 0.062ns Total dry weight –0.561** –0.605** –0.339ns 0.434* 0.575** 0.907** 0.923** –0.388* 0.359ns –0.454* Relative water content 0.623** 0.583** –0.061ns –0.298ns –0.346ns –0.226ns –0.415* 0.359ns –0.355ns –0.692** Electrolyte leakage –0.609** –0.501* 0.225ns 0.460* 0.340ns 0.294ns 0.402* –0.287ns 0.383ns –0.903** Note: *, ** and ns indicate significance at p < 0.05, p < 0.01 levels and non-significance, respectively. ARCHIVES OF AGRONOMY AND SOIL SCIENCE 777

Figure 2. Ward ’s cluster analysis classifications of 24 crested wheatgrass accessions native to Iran and ‘Rembrandt’ tall fescue under drought stress conditions.

Table 6. Means of traits used in the identification of five clusters formed from 24 crested wheatgrass accessions native to Iran and ‘Rembrandt’ tall fescue under drought stress conditions.

Root/ Root/ Relative Turf Green Root Shoot shoot Root dry Shoot dry shoot dry Total dry water Electrolyte Cluster quality tissue length length length weight weight weight weight content leakage number (1–9) (1–9) (cm) (cm) ratio (g pot −1) (g pot −1) ratio (g pot −1) (%) (%) 1 3.15† 6.23† 18.30 12.40 1.40 3.16 1.91 1.69 5.06 45.80 47.18 2 2.56 5.38 19.17 13.25 1.52 2.87 2.10 1.46 4.98 30.77 77.05 3 1.00 1.00 21.50 9.67 2.15 5.31 5.60 0.95 10.91 22.33 84.87 4 1.00 1.91 21.67 12.58 1.79 3.25 2.98 1.13 6.23 28.66 78.99 5 1.74 3.10 21.01 13.55 1.63 4.23 2.68 1.58 6.91 28.84 82.24 Note: † Means of turf quality and green tissue were obtained at day 20 of drought stress. Downloaded by [Dokuz Eylul University ] at 21:15 06 May 2016 Discussion This study documented differences among crested wheatgrass accessions regarding their ability to resist drought. The accessions were collected from different ecological regions of Iran and they have different adaptability to stresses, including drought due to their genetic differences (Hays et al. 1991; Beard & Sifers 1997; Gazanchian et al. 2006; Zandi Esfahan & Azarnivand 2013). The accessions showed high genetic diversity for turf quality, green tissue rating, RWC, EL, ET water loss and growth parameters like shoot length, root and shoot dry weight and root to shoot length ratio. Cluster analysis has been used to group accessions in genetic diversity studies (Garcia et al. 1997; Bayuelo-Jimenez et al. 2002; Mohammadi & Prasanna 2003). In the present study, the accessions and Rembrandt were classified into five clusters comprised of drought tolerant (I), intermediate (II, IV and V) and susceptible (III) clusters in response to soil drying. Turf quality declined with drought stress for all accessions of crested wheatgrass. The results confirm the report of Fu et al. (2004), Bian and Jiang (2009) and Sanchez-Blanco et al. (2009), where 778 H.BAYATETAL.

they found water stress decreased turf quality. Drought stress also decreased green tissue rating which varied among accessions. Cluster I had the highest turf quality and green tissue under drought stress. Green tissue rating provides a good assessment of overall turfgrass drought resistance (Carrow & Duncan 2003). The physiological and morphological mechanisms for the improved stress tolerance were associated with RWC, ET, EL, root growth in our study, as discussed subsequently. There were significant differences among the accessions for their ET under drought stress. The ability to maintain low ET rates has long been considered a trait for water conservation and drought resistant (Kirkham 1983; Bacon et al. 1998; Alves & Setter 2000). The results showed that drought stress significantly decreased leaf RWC of all accessions. These results are similar to those found for other cool season turfgrass species (Huang et al. 1998; Jiang & Huang 2000; Huang & Fu 2001; Xu & Huang 2001; Mohsenzadeh et al. 2006). RWC is a widely used parameter to determine the level of internal water status and resistant cultivars maintain high RWC than sensitive ones (Ritchie et al. 1990). The results showed that cluster I had the highest RWC. In the present study, higher RWC was correlated positively with higher turf quality (r = 0.633**) and green tissue (r = 0.583**), revealing that maintenance of leaf water status is essential for continuation of physiological and biochemical functioning. Lower EL was associated with reduced water content in the leaves. In this study, water stress increased EL and damaged cell membrane stability. The lowest EL was obtained from cluster I under drought stress that negatively correlated with higher turf quality (r = −0.609**) and green tissue (r = −0.501*), indicating its potential use for screening drought tolerance of plant accessions. In fact, the resistant accessions exhibited better membrane stability than susceptible ones under drought conditions, as demonstrated by the lower EL (Jiang & Huang 2001; Jinrong et al. 2008). The results showed that dry weight of shoots and roots, shoot and root length decreased with drought, which were in agreement with the results of Sanchez-Blanco et al. (2009). Several authors have reported a reduction in the dry matter yield of grasses caused by water stress (Gerakis et al. 1975;Mott&McComb1975; Pessarakli & Kopec 2008). The highest root to shoot dry weight ratio (1.69) and root length (21.67 cm) were obtained from clusters I and IV, respectively. Longer root system had greater volume and surface areas of roots in contact with soil to facilitate water and nutrient uptake under drought stress. The root system has been chosen as a selection trait in breeding programmes to improve drought tolerance (Qian & Fry 1997;Balasubramaniyametal.2015). Reduction of dry weight of shoots and roots under drought stress is evidently due to a decline in net assimilation caused by decreased water potentials in the leaves (De Puit & Caldwell 1975; Farooq et al. 2009; Junjittakarn et al. 2014). Declining effects of drought stress on shoot length may be related to the impression of drought on meristematic cells of shoot and interfere with the process of cell division and Downloaded by [Dokuz Eylul University ] at 21:15 06 May 2016 elongation. It seems that cell elongation rather than cell division has been affected by drought because of dehydration and potential negative influence on cells water absorption (Pessarakli & Kopec 2008).

Conclusions In conclusion, the present results demonstrated genetic variation in drought tolerance in crested wheatgrass accessions. The results showed that crested wheatgrass accessions were better able to survive drought stress due to mechanisms such as maintaining higher RWC, root to shoot length ratio, root and shoot dry weight ratio, root elongation and lower EL under drought stress. Cluster I had five accessions (1727 M, 208 M, 4056, 2854 and 1727 P1) and performed better than other clusters under drought stress conditions. The highest RWC, root to shoot dry weight ratio, turf quality and green tissue and the optimum shoot vertical growth were obtained from cluster I under drought stress, and these genetic diversity could be used for future turf breeding programmes. ARCHIVES OF AGRONOMY AND SOIL SCIENCE 779

Acknowledgement

The authors wish to acknowledge Gene Bank of the Rangelands and Forestry Research Institute of Iran for providing seeds.

Disclosure statement

No potential conflict of interest was reported by the authors.

Funding

This work was supported by the Ferdowsi University of Mashhad, Iran [No. 28735].

References

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