Growth and Physiological Responses of
Diverse Perennial Ryegrass Accessions to
Increasing Salinity
TURFGRASS S CIENCE
Yiwei Jiang1, Jinchi Tang2, Xiaoqing Yu1 and James J. Camberato1. 1Department 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 (DW),
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 Clthat 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. 2013. Growth and
Physiological Responses of Diverse Perennial Ryegrass
Accessions to Increasing Salinity. 2012 Annu. Rep. - Purdue
Univ. Turfgrass Sci. Progr. p. 7-11.
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
Purdue Turfgrass Science Program 2012 Annual Report
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 (10cm 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
231587
Algeria
Uncertain
231595 a
Morocco
Uncertain
a
231597
Greece
Uncertain
Yugoslavia
Wild
251141 a
275660 a
Australia
Cultivated
303011 a
United Kingdom Cultivar
Netherlands
Cultivated
303022 a
418727 a
France
Wild
a
New Zealand
Cultivar
462339
BrightStar SLT USA
Cultivar
a
indicates core collection accession according to USDA classification
Purdue Turfgrass Science Program 2012 Annual Report
Plant height (HT), leaf fresh weight (FW), leaf dry
weight (DW), 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 (DW) was
measured after drying at 80 °C in an oven for 3 days.
Leaf water content (LWC) was calculated as [(FW –
DW)/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).
Results and Discussion
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. PI275660 and BrightStar SLT
had better salinity tolerance followed by PI462339
with more green tissues and little leaf senescence,
while PI231595 and PI251141 were highly
sensitive to high salinity with severe injury. Other
five accessions ranked in the middle and showed
moderate salinity injury.
Table 2. Effects of 20 d of salinity treatments on plant height (HT), leaf fresh weight (FW), dry weight (DW), 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
WSC
Fructan
Na+
K+
NaCl
HT (cm)
FW (g) DW (g) LWC (%) Fv/Fm
(mg g-1
(mg g-1
(mg g-1
(mg g-1
K+/Na+
(mM)
DW)
DW)
DW)
DW)
0
18.7 a
2.24 a
0.44 a
80.2 a
0.81 ab 28.5 c
17.7 d
3.20 d
38.9 a
12.7 a
50
1.82 a
0.40 a
79.6 a
0.81 ab 34.6 bc
25.9 c
12.2 c
32.3 b
2.74 b
16.9 b
100
16.2 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
150
14.7 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
200
13.6 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
300
13.2 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.
Purdue Turfgrass Science Program 2012 Annual Report
Tolerant
Intermediate
Sensitive
0
50
100
150
200
300 (mM)
Figure1.DifferentialresponsesofperennialryegrassestoincreasingNaClconcentrations
Traits of DW, LWC, Fv/Fm, and Na+ and accession
of PI231595 (sensitive), PI275660 (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 PI231595 with increasing
NaCl levels, particularly compared to PI275660.
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
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Purdue Turfgrass Science Program 2012 Annual Report
DW (g)
A
0.19
0.24
0.23
0.13
0.17
0.17
7.9
10.7
4.2
6.3
14.9
10.5
0.02
0.03
0.02
0.03
0.14
0.05
6.1
4.7
12.8
11.6
7.1
LWC (%)
B
Fv/Fm
C
Na+ (mg g-1 DW)
D
2.8
050100150200300(mM)
Figure2.Thevariationofdryweight(DW)(A),leafwatercontent(LWC)(B)andchlorophyll
fluorescence(Fv/Fm)(C)inaccessionPI231595,PI275660andBrightStarSLT(Bstar‐SLT)
exposedto0‐,50‐,100‐,150‐,200‐,and300mMNaCl,respectively.Valuesabovex‐axiswithina
salinitycolumnindicatealeastsignificantdifference(LSD)betweenaccessionswithinasalinity
level.Numbersontherightsiderepresentaccessionsofperennialryegrass,andvaluesoutsidethe
accessiondesignationrepresentLSDbetweenaccessionsacrosssalinitylevels.