Accepted Manuscript Title: Salinity tolerance among a large range of bermudagrasses (Cynodon spp.) relative to other halophytic and non-halophytic perennial C4 grasses Authors: Thinh Van Tran, Shu Fukai, Hayley E. Giles, Christopher J. Lambrides PII: S0098-8472(17)30248-4 DOI: https://doi.org/10.1016/j.envexpbot.2017.10.011 Reference: EEB 3308 To appear in: Environmental and Experimental Botany Received date: 11-9-2017 Revised date: 16-10-2017 Accepted date: 16-10-2017 Please cite this article as: Van Tran, Thinh, Fukai, Shu, Giles, Hayley E., Lambrides, Christopher J., Salinity tolerance among a large range of bermudagrasses (Cynodon spp.) relative to other halophytic and non- halophytic perennial C4 grasses.Environmental and Experimental Botany https://doi.org/10.1016/j.envexpbot.2017.10.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Salinity tolerance among a large range of bermudagrasses (Cynodon spp.) relative to other halophytic and non-halophytic perennial C4 grasses Thinh Van Tran1,2, Shu Fukai1, Hayley E. Giles1, Christopher J. Lambrides1,3 1 The University of Queensland, School of Agriculture and Food Sciences, Qld 4072, Australia. 2 Nong Lam University - Ho Chi Minh City, Vietnam. 3 Corresponding author. Email: [email protected] Highlights: ► A wide range of genetic variation for salt tolerance in bermudagrasses was identified. ► The best salt tolerance could be selected from saline environments. ► There was no correlation between drought resistance and salt tolerance although some drought resistant grasses did have excellent salt tolerance. ► Canopy temperature differential could be used as an effective attribute to select for salt tolerance in bermudagrasses. ► Using salinity levels above 20 dS m-1 for 8 weeks appears to be an effective method for detecting large variation for salt tolerance in bermudagrasses Abstract The increasing demand on potable water has resulted in a greater reliance on poorer quality water, including saline sources, for maintaining forage and turfgrasses in agricultural and urban landscapes. Consequently, it will be crucial to identify grasses that can tolerate saline irrigation water. This study aimed to determine salinity tolerance among a large range of bermudagrasses relative to other perennial C4 grasses and test the relationship between salt tolerance and drought resistance. We report the salinity tolerance of 70 genotypes of mostly Australian bermudagrass ecotypes that were compared to halophytic cultivars of seashore paspalum (Paspalum vaginatum Swartz) and a non- halophytic cultivar of Queensland blue couch (Digitaria didactyla Willd) using a flood and drain sand -1 culture system with salt treatments 1 to 40 dS m . For the first time for C4 grasses, salt tolerance was determined by comparing total biomass of the grasses with and without salt treatment. Large genetic variation in salinity tolerance was identified and six bermudagrasses collected from saline habitats had salinity tolerance equal to that of seashore paspalum under the salinity treatments used in this study. There was no correlation between salt tolerance and drought resistance phenotypes determined from our previous research. Canopy temperature differential during salt stress was negatively correlated (r = -0.71 to -0.91, P < 0.001) to salt tolerance and has potential to be used for screening 1 bermudagrasses for salt tolerance using flood and drain sand culture. Salinity levels above 20 dS m-1 for 8 weeks appeared to be effective for detecting large variation for salt tolerance in bermudagrass. Highlights: ► A wide range of genetic variation for salt tolerance in bermudagrasses was identified. ► The best salt tolerance could be selected from saline environments. ► There was no correlation between drought resistance and salt tolerance although some drought resistant grasses did have excellent salt tolerance. ► Canopy temperature differential could be used as an effective attribute to select for salt tolerance in bermudagrasses. ► Using salinity levels above 20 dS m-1 for 8 weeks appears to be an effective method for detecting large variation for salt tolerance in bermudagrasses. Keywords: Salt tolerance, drought resistance, canopy temperature differential. Abbreviations: CTD, canopy temperature differential; GC, green cover; CY, clipping yield; CCY, cumulative clipping yield; VB, verdure biomass; RB, root biomass; RTB, relative total biomass. 2 1. Introduction The Poaceae, represented by over 7500 species, shows an extreme range in salinity tolerance, from salt-sensitive to extremely salt-tolerant halophytic types (Richards, 1954; Gould and Shaw, 1983; Maas, 1986). Cynodon grasses, commonly known as green couch grasses in Australia and bermudagrasses elsewhere in the world, are widely used as turf and forage in tropical and subtropical regions. Recently, we assembled over 1000 Cynodon accessions from a range of soil types and climatic zones across Australia and subsets of this collection were evaluated with a focus on drought resistance (Kearns et al., 2009; Zhou et al., 2009; Lambrides et al., 2013; Zhou et al., 2013a, 2013b; Zhou et al., 2014), regrowth and sod strength (Zhou et al., 2015a; Tran et al., 2017) and wear tolerance (Zhou et al., 2015b). However, there have been no studies of salinity tolerance within this collection or other large collections of bermudagrass ecotypes with most studies focusing on fewer commercial releases (Marcum and Pessarakli, 2006; Bauer et al., 2009; Chen et al., 2014a, 2014b). In many regions of the world, fresh water shortage has resulted in restrictions on the use of potable water resources for irrigation of agricultural and urban landscapes. Secondary water sources are thus increasingly being used to irrigate large turf facilities as an attractive alternative to decrease operating budgets for managers. Large savings can be achieved by irrigating grasses with effluent water (recycled, non-potable, reclaimed or wastewater) that can cost 80% less than the fresh water equivalent (Huck et al., 2000). However, a great concern when using effluent water for irrigation is the quality of the water such as the amounts and types of dissolved salts included, resulting in the accumulation of high ion concentrations in normal soils. In this situation, identification and cultivation of salt-tolerant grasses offers a powerful approach for stabilising and vegetating soil profiles where accumulated salts from saline irrigation can be flushed with seasonal rainfall. Salinity and drought are the most damaging abiotic stresses affecting plants growth. Plant responds to salt and water stress in a similar way. High salinity severely reduces water uptake, leading to reduced growth, along with many changes identical to those caused by drought (Munns, 2002). Our previous studies indicated that a group of bermudagrasses collected from Australian Mediterranean environments were drought resistant and characterized by large rhizomes that could be a potential source of nutrients, water and carbohydrates when these grasses grow under saline conditions. The objectives of this study were to (i) determine genotypic variation for salinity tolerance among a large group of bermudagrasses recovered from saline environments relative to other perennial C4 grasses and (ii) test the relationship between salt tolerance and drought resistance. It was hypothesised that bermudagrasses collected from saline environments would be exposed to selection pressure for salt 3 tolerance leading to adaptation to saline conditions and that drought resistant bermudagrasses would be relatively tolerant to saline conditions. 2. Materials and methods 2.1. Experiment 1 This experiment was conducted in a semi-controlled glasshouse at Redlands Research Station, Cleveland, Australia (27.31oS, 153.15oE) in a flood and drain sand culture system. During the experiment, means of minimum temperature, maximum temperature, relative humidity and vapour pressure deficit (VPD) were 16oC, 25oC, 72% and 0.68 kPa, respectively (Fig. A1a). Twelve bermudagrass genotypes (Cynodon spp.) including 11 ecotypes and one commercial cultivar Wintergreen were chosen to represent a range of different levels of drought resistance based on previous research (Zhou et al., 2013b; Zhou et al., 2014). Genotypes were planted as vegetative sod in plastic pots (10.5 cm square 12.5 cm deep, 1.38 L capacity) filled with a coarse sand. The potted vegetative material was grown for 2 months using a 92 L basal nutrient solution containing 5.1 g of Flowfeed EX7, 10.2 g of KNO3, 51.1 g of Ca(NO3)2 and 12.8 g of MgSO4.7H2O [containing (µM): 8689 nitrogen (N), 58 phosphorus (P), 1345 potassium (K), 3383 calcium (Ca), 572 magnesium (Mg), 649 sulfur (S), 0.71 manganese (Mn), 0.09 copper (Cu), 1.29 iron (Fe), 0.01 molybdenum (Mo), 1.54 boron (B) and 0.25 zinc (Zn)]. During this 2-month period the grasses were clipped weekly to 25 mm. Three hundred and eighty-four pots were arranged in a split-plot design, with different salinity treatments allocated to main plots, and genotypes allocated to sub-plots. There were two duplicates of each genotype in each treatment and two biological replications. After establishment, salinity treatments were applied to the root system of the plants by applying solutions of sodium chloride (NaCl), with calcium added to each treatment to avoid sodium-induced calcium deficiency. The calcium activity ratio (CAR) of solution was maintained at ≥ 0.035 (Kopittke and Menzies, 2005). Salinity treatments were increased by 1.5 dS m-1 daily until the desired level was obtained at 1.1, 2.4, 4.8, 7.3, 11.5, 14.4, 17.3 and 20.1 dS m-1 (hereafter referred to as 1, 2, 5, 7, 12, 14, 17 and 20 dS m-1, respectively) corresponding to 0, 16, 44, 72, 95, 122, 148 and 175 mM NaCl, respectively.