Growth and Physiological Responses of Diverse Perennial Ryegrass Accessions to Increasing Salinity

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1 Growth and Physiological Responses of Diverse Perennial Ryegrass Accessions to Increasing Salinity TURFGRASS SCIENCE Yiwei Jiang 1, Jinchi Tang 2, Xiaoqing Yu 1 and James J. Camberato 1. 1 Department of Agronomy, Purdue University. 2 Guangdong Academy of Agricultural Science, Guangzhou, China. Summary: Ten diverse accessions of perennial ryegrass (Lolium perenne L.) were grown in sand culture and exposed to a half-hoagland solution amended with 0 (control), 50-, 100-, 150-, 200-, and 300 mm NaCl. Across all accessions, decreased plant height, K + concentration and K + /Na + and increased concentrations of fructan and Na+ were observed at 50 mm NaCl, while decreased leaf fresh and dry weight (, leaf water content (LWC), chlorophyll fluorescence (Fv/Fm), and increased water-soluble carbohydrate concentration (WSC) occurred at 150 mm NaCl. The maximum separations of salinity tolerance of accessions occurred at 200 to 300 mm NaCl. The results indicated that DW, LWC, Fv/Fm and Na + could be associated with variability in tolerance of diverse perennial ryegrasses to high salinity stress. Salinity is a major abiotic stress limiting plant growth and productivity. Salinity affects plant growth and development generally through osmotic stress limiting water uptake and the excessive uptake of ions, particularly Na + and Cl - that ultimately interfere with various metabolic processes (Munns and Tester, 2008). Salinized plants may suffer from metabolic toxicity, nutrient deficiencies and imbalances, membrane dysfunction, and antioxidative stress, which damage tissue and induce early senescence (Essah et al., 2003). Effects of salinity on plant growth vary highly among plant species and/or within a species. In perennial turf or forage grass species, seashore paspalum (Paspalum vaginatum Sw.) had superior shoot dry weight under salinity stress compared to several other warm-season turfgrass species including Japanese lawn grass (Zoysia japonica Steud.), manila grass (Zoysia matrella Additional index words: Carbohydrate, ion accumulation, Lolium perenne, salt stress Jiang, Y., J. Tang, X. Yu and J. Camberato Growth and Physiological Responses of Diverse Perennial Ryegrass Accessions to Increasing Salinity Annu. Rep. - Purdue Univ. Turfgrass Sci. Progr. p L.), hybridbermuda grass (Cynodon dactylon x. Cynodon transvaalensis.) and serangoon grass (Digitaria didactyla Willd.), although inhibition of growth occurred in all species exposed to salinity (Uddin et al., 2012). Turfgrasses are increasingly subjected to salinity stresses in many areas due to the accelerated salinization of agricultural lands and increasing demand on effluent water use for irrigating turfgrass landscapes (Carrow and Duncan, 1998). Perennial ryegrass (Lolium perenne L.) is a popular cool-season grass species cultivated in temperate climates. Originating in Europe, temperate Asia, and North Africa, it is commonly used as a turf and forage grass around the world. Perennial ryegrass has been ranked as moderate in salinity tolerance for commercial cultivars, tolerating soil ECe (saturated paste extract) ranging from 4 to 8 ds m 1 (Harivandi et al., 1992). Due to wide geographical distributions of perennial ryegrass, significant natural variation in growth and wholeplant physiological responses to salinity stress are expected in diverse ecotypes within this species. However, growth and physiological responses of diverse perennial ryegrasses to increasing levels of salinity stress as well as traits associated with genetic variability in salinity tolerance are not yet fully understood in perennial ryegrass accessions varying in origins. Therefore, the objectives of this study were to investigate growth

2 response, carbohydrate and ion accumulation of diverse perennial ryegrass accessions exposed to increasing salinity and to determine phenotypic traits associated with variability of salinity tolerance. The results will aid in determining natural variations in salinity tolerance of perennial ryegrasses and in providing a mechanistic formation for creating perennial grass materials that are more tolerant to salt-affected soils and water. Materials and Methods Ten accessions of perennial ryegrass from various origins were used in the experiment including 2 wild, 3 cultivars, 2 cultivated, and 3 uncertain materials, according to USDA National Plant Germplasm System (USDA-NAGS) classification (Table 1). Grasses were grown in plastic pots (10- cm diameter, 9-cm deep) containing a sandy-loam soil with a ph of 6.9 in a greenhouse at Purdue University, West Lafayette, IN, USA. Propagation of pots containing approximately 550 g sand were sprigged with 9 to 10 tillers on March 18, 2011 and grown in a greenhouse for 24 d prior to salinity treatment. Grasses were cut to 5.0-cm high once a week. The average air temperatures were 22ºC/20ºC (day/night) and the average photosynthetically active radiation (PAR) was approximately 350 µmol m -2 s -1, with a 10-h light period of both natural and artificial light during the period of growth and treatments. Plants were well-watered and fertilized every other day with a half-hoagland solution (Hoagland and Arnon, 1950) at a ph of 6.6 and an electrical conductivity (EC) of 1.5 ds m -1 ( 16 mm NaCl). Prior to initiation of the salinity treatments, all plants were cut to a height of 5-6 cm (the height of the smallest accession) so that later measurements would be made on tissue produced after the imposition of salinity stress. The control treatment received fresh water irrigation with a half-hoagland nutrient solution, and salinity treatments were watered with a half-hoagland solution amended with NaCl. The NaCl solution was added to the pots through soil drenching without contact of the plants. Final NaCl concentrations were 0 (control), 50, 100, 150, 200, and 300 mm (approximately 1.5, 4.2, 8.4, 12.6, 16.8, and 25.2 ds m -1, respectively). To avoid salinity shock in the plants, these concentrations were attained gradually by increasing the NaCl concentration of the irrigation water 25 mm each day until the final concentration was reached (except for 300 mm, which was increased with 50 mm from 200 mm to 300 mm). Salinity treatments lasted 20 days. To avoid salt accumulation in the sand media, irrigation was applied manually to each pot until free drainage occurred. The lack of salt accumulation in the pots was confirmed by measuring the electrical conductivity of the leachate (VWR Traceable Digital Conductivity Meter, VWR Inc., Chicago, IL). Table 1. Accession number (PI), origin, and collection status of perennial ryegrasses used in this experiment PI Origin Status Algeria Uncertain a Morocco Uncertain a Greece Uncertain a Yugoslavia Wild a Australia Cultivated a United Kingdom Cultivar a Netherlands Cultivated a France Wild a New Zealand Cultivar BrightStar SLT USA Cultivar a indicates core collection accession according to USDA classification

3 Plant height (HT), leaf fresh weight (FW), leaf dry weight (, leaf water content (LWC), leaf Na + and K + concentration, chlorophyll fluorescence (Fv/Fm), fructan and water-soluble carbohydrate (WSC) concentrations were measured as indicators of growth and physiological traits for all accessions. Plant height was measured from the soil surface to the top of the uppermost leaf blade. The chlorophyll fluorescence was measured using a Portable Modulated Chlorophyll Fluorometer (OS-30P, OPTI-Sciences, Hudson, NH, USA). All the leaves for each pot were collected and fresh weight (FW) was determined immediately. Dry weight ( was measured after drying at 80 C in an oven for 3 days. Leaf water content (LWC) was calculated as [(FW /FW] 100. The water-soluble carbohydrate (WSC) and fructan were extracted from 200 mg of leaf dry tissues with 1 ml double distilled water. The WSC and fructan contents were measured using the anthrone method (Koehler, 1952), with some modifications. Leaf Na + concentration were determined using an AA-6800 Shimadzu Atomic Absorption Spectrophotometer (Shimadzu Inc., Columbia, MD, USA) and K + concentration using a Digital Flame Analyzer (Cole-Parmer Instrument Inc., Chicago, IL, USA). The experiment was arranged in a split plot design, with salinity for the main plot and accession for the subplot. Each accession was randomly assigned within each treatment and each treatment was replicated three times. Statistical analysis was performed with Statistical Analysis System (SAS) (SAS Institute Inc., 2004). Plant height decreased 10%, 13%, 22%, 27% and 29% with 50-, 100-, 150-, 200- and 300 mm NaCl, respectively, compared to the non-salinity control (Table 2). The FW, DW, LWC and Fv/Fm were unaffected by NaCl 100 mm, but were progressively reduced at 150 mm to 300 mm NaCl. For example, DW significantly decreased 30%, 36% and 48% at 150-, 200- and 300 mm NaCl, respectively; compared to the non-salinity control. Water-soluble carbohydrate concentration was unaffected by NaCl level up to 150 mm but increased 53%, 85% and 94% at 150-, 200- and 300 mm NaCl, respectively. Fructan increased from 28% to 1.8-fold, Na + increased from 2.8- to 12.5-fold, and K + decreased from 17% to 39% from 50 mm and 300 mm NaCl, while reductions in K + / Na + were 36%, 52%, 1.1-, 1.3- and 1.9 fold for 50-, 100-, 150-, 200- and 300 mm NaCl, respectively; compared to the control. Accessions differed in overall salinity tolerance based on visual observation of the senescence of older leaves exposed to increasing salinity (Fig. 1). The degree of senescence of older leaf became apparently with increasing salinity concentration. Salinity injury mainly occurred at 150 mm NaCl in some accessions. PI and BrightStar SLT had better salinity tolerance followed by PI with more green tissues and little leaf senescence, while PI and PI were highly sensitive to high salinity with severe injury. Other five accessions ranked in the middle and showed moderate salinity injury. Results and Discussion Table 2. Effects of 20 d of salinity treatments on plant height (HT), leaf fresh weight (FW), dry weight (, water content (LWC), chlorophyll fluorescence (Fv/Fm), concentrations of water-soluble carbohydrate (WSC), fructan, leaf Na +, K + and ratio of K + /Na + across 10 perennial ryegrass accessions NaCl (mm) HT (cm) FW (g) DW (g) LWC (%) Fv/Fm WSC Fructan Na + K + K + /Na a 16.9 b 2.24 a 1.82 a 0.44 a 0.40 a 80.2 a 79.6 a 0.81 ab 0.81 ab 28.5 c 34.6 bc 17.7 d 25.9 c 3.20 d 12.2 c 38.9 a 32.3 b 12.7 a 2.74 b b 1.81 a 0.40 a 79.2 a 0.82 a 36.2 c 22.7 c 14.9 c 28.0 c 1.92 b c 1.25 b 0.31 b 76.5 ab 0.80 b 43.4 b 35.3 b 29.5 b 25.3 d 0.88 c d 1.06 b 0.28 bc 74.6 b 0.80 c 52.7 ab 42.7 a 33.0 b 23.1 e 0.76 c d 0.69 c 0.23 c 68.6 c 0.79 c 55.3 a 49.4 a 43.1 a 23.7 e 0.53 c Means followed by the same letter within a column for a given trait were not significantly different at P < 0.05.

4 Tolerant Intermediate Sensitive (mm) Figure 1. Differential responses of perennial ryegrasses to increasing NaCl concentrations Traits of DW, LWC, Fv/Fm, and Na + and accession of PI (sensitive), PI (tolerant, fast growing) and BrightStar SLT (tolerant, slow growing) were selected for assessing the patterns of variations between accessions and increasing salinity. Under the non-salinity control, there were some separations in three accessions based on selected traits; however, differences in accessions apparently became larger under salinity stress (Fig. 2). Generally, the maximum separations in accessions occurred at 150 mm NaCl for DW (Fig. 2A), at 200 mm or 300 mm NaCl for LWC (Fig. 2B), Fv/Fm (Fig. 2C), and Na + (Fig. 2D), respectively. Compared to the control, percentage deceases in DW, LWC, Fv/Fm and increase in Na + were also larger for accession PI with increasing NaCl levels, particularly compared to PI The results suggest that traits of DW, LWC, Fv/ Fm and Na + accounted for larger variations across accessions and could be more associated with variability in high salinity tolerance, severing appropriate parameters for assessing salinity tolerance in perennial ryegrasses. Ultimately, salinity concentration, duration of stress, species and genotypes and growing conditions can all impact selection of the salinity tolerant germplasm of perennial grasses. Acknowledgements This project is supported by the Midwest Regional Turfgrass Foundation of Purdue University and O.J. Noer Research Foundation References Carrow, R.N. and R.R. Duncan Salt-affected turfgrass sites: assessment and management. John Wiley & Sons, Inc., New York, USA. Essah, P.A., R.J. Davenport, and M. Tester Sodium influx and accumulation in Arabidopsis thaliana. Plant Physiol. 133: Harivandi, M.A., J.D. Butler, and L. Wu Salinity and turfgrass culture. In: D.V. Waddington, R.N. Carrow, and R.C. Shearman (Ed), Turfgrass. Agronomy Monograph 32. pp Amer. Soc. Agron. Madison, WI, USA. Hoagland, D.R., and D.I. Arnon The water-culture method for growing plants without soil. Univ. Calif. Agri. Exp. Station. Berkley Circ: 347. Koehler, L.H Differentiation of carbohydrates by anthrone reaction rate and color intensity. Anal. Chem. 24: Munns, R., and M. Tester Mechanisms of salinity tolerance. Annu. Rev. Plant Biol. 59: SAS Institute Inc., SAS Procedures Guide, Release 9.1 Edn. SAS Institute Inc., Cary, NC, USA Uddin, M.K., A.S. Juraimi, M.R. Ismail, M.A. Hossain, R. Othman, and A.A. Rahim Physiological and growth responses of six turfgrass species relative to salinity tolerance. The Sci. World J.:

5 A B C Fv/Fm LWC (%) DW (g) D Na (mm) Figure 2. The variation of dry weight ( (A), leaf water content (LWC) (B) and chlorophyll fluorescence (Fv/Fm) (C) in accession PI231595, PI and BrightStar SLT (Bstar SLT) exposed to 0, 50, 100, 150, 200, and 300 mm NaCl, respectively. Values above x axis within a salinity column indicate a least significant difference (LSD) between accessions within a salinity level. Numbers on the right side represent accessions of perennial ryegrass, and values outside the accession designation represent LSD between accessions across salinity levels.