AGRICULTURE AND BIOLOGY JOURNAL OF NORTH AMERICA ISSN Print: 2151-7517, ISSN Online: 2151-7525, doi:10.5251/abjna.2015.6.1.34.46

© 2015, ScienceHuβ, http://www.scihub.org/ABJNA

Interactive effects of potassium and sodium on the growth and nodulating capacity of pea (Pisum sativum L) var: Merveile de Kelvedon under salt

stress

Chougui Saida *, Said Hadjar *, Belgat Houria ** Boudour Leila *, Gharoucha Hocine *, Baka Moubarek.*

*Faculty of science, Department of biology and ecology, Constantine University 1, Algeria.

** Faculty of science, Department of biology, Sétif University 1, Algeria.

E-mail – saida.chougui@yahoo.fr

ABSTRACT

The objective of the research is to evaluate the interactive effects of salinity and potassium salt on the growth and nodulating capacity. The experiment was conducted during the development of pea seedlings (Pisium sativum) var: Merveile Kelvedon, the test was conducted in a randomized complete block (split plot) with three levels of NaCl (S0: 0 , S1:50 , S2: 150) mMol, and two levels of potassium salt: potassium nitrate (KNO3) (N1: 20, N2: 40) mMol, and potassium acetate (CH3COOK) (A1:20 , A2: 40) mMol with four replicates grown in pots, under controlled conditions ,the work was executed on 48 expérimentales units. The results are shown that treatment salts decreased significantly pigment chlorophyll (chla, Chlb, Chlt), leaf area (La), the number of nodules (Nnd ), root length and stem (Rl , Rs) and the dry weight of leaves and roots (Dwl, Dwr) with increasing levels of salinity, the content of potassium (K+) and the selectivity coefficient (K / Na) of leaves and roots and the content of total nitrogen (TN) in the leaves is reduced with a high salinity concentration compared to the control, whereas the content of proline (Pro) and sugar (Sr) in the leaves and the content of sodium (Na +) in the leaves and roots have been accumulated considerably when salinity levels are increased. The application of the two potassium salts in the soil potassium nitrate (KNO3) or potassium acetate (CH3COOK)) is removed deleterious effects of salinity .In comparing these two potassium salts we result, the level of potassium acetate (CH3COOK) (A2) increased the number of nodules (Nnd), total nitrogen (NT) in the leaves and the potassium content (K+) in the shoots and roots, but potassium nitrate (KNO3) (N2) increased sodium content (Na +) in the roots and the leaves compared to the other treatment one, we can conclude that the potassium acetate (CH3COOK) is a good fertilizer than potassium nitrate (KNO3) in saline conditions. Keywords: Salinity / Pisum sativum / Potassium acetate / Potassium nitrate / Salinity.

INTRODUCTION

Legumes have been suggested as appropriate crops for the enhancement of bioproductivity and the reclamation of marginal lands, because these plants not only yield nutritious fodder protein –rich seeds, but they also enrich soil nitrogen in symbiotic association with Rhizobium (El Nady and Belal, 2005 ; Hassan, 2009). Pea plant (Pisum sativum L) is one of the major crops in the arid and semi-arid region, especially in the Mediterranean bassin , where symbiotic N2 fixation contributes to its adaptations (Noreen et al., 2007), unfortunately soil salinity is also common in arid and semi-arid region and N2fixation can be extremely

sensitive to salinity stress (Serraj and Drevon, 2013) . Laguerre et al., 2001. found that inoculation with rhizobial strains increased shoot , dry weight after eight weeks by (21.4 and 29)% , compared to uninoculated plants under salts –stress .Rhizobial strains are very sensitive to soil environmental factors like high salt drought , pH , and temperature stresses , which affect their nitrogen fixation capacity and hence the productivity of legumes (Nogales et al., 2002).Approximately 40% of the worlds land surface is estimated to have potential salinity problems and most of these areas are confined to the Mediterranean region (Hatice et al., 2010).Saline conditions may limit the symbiosis by (a) affecting survival and proliferation

Agric. Biol. J. N. Am., 2015, 6(1): 34-46

35

of Rhizobium spp in the soil , and rhizosphere (b) inhibiting the infection process (c) directly affecting root nodule function (d) reducing plant growth (Sohrabi et al., 2008 ;Fatma et al., 2011 ; Sharma et al., 2013) , the presence of high sodium chloride (NaCl) concentration has been reported to cause a reduction in the number of rhizobia in legumes inoculants (El-Sayad and Belal, 2013). Lopez et al., (2008); Baghbani et al., (2012) showed that the saline conditions reduced significantly the total numbers of nodes and active nodes of nitrogen-fixation and reduced K+concentration, in the roots and shoots of alfalfa cultivars. Increased treatment of NaCl increases Na+ and Cl- and decrease in Ca2+, Mg2+and K+ levels in number of plants (Kham et al., 2003). There is a negative relationship between Na+ and K+concentration in roots and leaves , the selective uptake of K+as opposed to Na+is considered to be one of the important physiological mechanisms contributing to salt tolerance in many plant species (Ghazi and Al-Karaki, 2000 ; Heidari and Jamshid, 2010), thus under saline and sodic conditions K+ fertilization management may need to be modified because of K+competition with other cations and especially Na+ in the plant and to the effects of salinity on K+reactions in soils (Badr and Shafei, 2002 ; Cherel, 2004 ). K+has been also considered often to play a role in osmotic stress and salt toxicity, K+ uptake occurs through different transport mechanisms depending on the external K+ concentration (Rubiol and Botella, 2004). The specific symptoms of sodium toxicity include high tissue sodium concentration and low K+/Na+ratio (Tejera et al., 2005 ; Yanshou et al., 2009) .Plant species adjust to high salt concentration by lowering tissue osmotic potential with the accumulation of inorganic as well as organic solutes (Hameda, 2011) .The accumulation of some organic solutes has been considered as an adaptation of plants against osmotic stress (Karimi et al., 2005 ;Gama et al., 2007) , Amino acids such as proline , asparatine and γ-amino-butyric acid can play an important role in the osmotic adjustment of the plant under saline conditions (Gama et al., 2007 ;Hameda 2011) ,other organic compounds that accumulate in response to stress such as sugars apparently play a role in the development of salt tolerance (Hisschnyder and Schmidhalter, 2000 ; Neera and Ranju, 2004) . Accordingly the objectives of this study were to study the interactive between K+ nutrient and water salinity and their effects growth and nodulating capacity of pea ( Pisum sativum L ) var: Merveile Kelvedon under salt stress and test the ability to reduce damage to crops by applying different levels of potassium salts:

potassium nitrate (KNO3) and potassium acetate (CH3COOK).Thus we investigated in relation to changes in growth media .We were also interested in knowing whether salt stress resistance mechanisms might develop in pea ( Pisum sativum L ) var: Merveile Kelvedon seedling living under salinity and how that response may influenced . MATERIALS AND METHODS

Plant materiel: Pea plant (Pisum sativum L) var: Merveile Kelvendo cultivar was obtained from the department of biology and ecology/ faculty of Sciences University 1 / Constantine / Algeria.

Experiment design and location: Pea plant (Pisum sativum L) var: Merveile Kelvedon seeds were sterilized for 20 min using 3% sodium hypochlorite, and then they were 3-5 times rinsed with distilled water. After sterilization, they were transferred into 9 cm sterile Petri dishes on filter paper and then were wetted with 7ml distilled water before imbibitions during 24h. After that, two seeds were sown in plastic pots (20cm height and 70cm diameter) contained soil and peat moss (2:1) at depth of 1cm. Pots were kept in laboratory (temperature 26°C, light 5000lux for 12h photoperiod and relative humidity of 51.70%). They were treated separately with two potassium salts in the soil potassium nitrate KNO3 (in two levels N1:20, N2:40) mMol and potassium acetate (CH3COOK) (A1:20, A2: 40) mMol for two weeks. After full germination, the number of plants was reduced to one seedling per pot. Salinity stress induction was done when the fourth leaf was completely expanded, and the symbiosis was well established. 17 days after planting, the plants were subjected to salt stress by adding NaCl to the growth medium (S0:0, S1:50, S2:150) mMol (standing for 4 replications of each treatment). The plants were harvested after 6 weeks of culture in the soil.

Growth analysis: At the end of the experimental period, the roots were separated from the soil by washing culture medium from the roots with a flowing water stream. The lengths of stems and roots were measured. The number of nodules for each plant at different levels of NaCl under different potassium salt concentration levels was counted. The fresh weight of the plant (root and leaves) was immediately calculated on the electronic scale. They were then washed with distilled water blotted dry and kept in oven at 70C ° to constant weight to measure the dry weight. Leafs area was determined with a D-image analysis system (LTD, England).

Agric. Biol. J. N. Am., 2015, 6(1): 34-46

36

Biochemical analysis :

Pigments analysis: Chlorophyll a and b were extracted and measured according to Bidel et al., 2007. One plant per replicate for each isolate at different NaCl concentrations and different levels of potassium salt was used for chlorophyll determination. 1g leaf sample was ground in 2ml of 85% acetone using pestle and mortar. The absorbance was measured with a UV/Visible spectrophotometer and chlorophyll concentrations were calculated using the following equations:

Chla / 12.25(A663.2) – 2.79(A646.8) = mg/g MF Chlb/21.5(A646.8) - 5.10 (A663.2) = mg/g MF ChlT /7.15(A663.2) +18.71(A646.8) = mg/g MF

Proline Determination: Proline in the leaves was determinate according to the method described by Khedv et al., (2003). For each isolate at different NaCl concentrations and different levels of potassium salt approximately 0.5g of fresh leaf material was homogenized in 10ml of 3% aqueous sulfo-salicydic acid, filtered through wattman’s n°2 paper and 2ml of solution was mixed with 2ml of ninhydrin acid and 2 ml of glacial acetic acid in a test tube. The mixture was placed in a water bath for 1h at 100°C. The reaction mixture was extracted with 4ml toluene and cooled at room temperature. The absorbance was measured at 520nm with UV/visible spectrophotometer using toluene as a blank .The proline concentration was determined from a standard curve.

Carbohydrate content: Carbohydrate content in the leaves was determined according to Dubois et al., 1956. The absorbance was measured at 485nm with UV/visible spectrophotometer. The soluble carbohydrate concentration was determined using a calibration curve obtained by plotting the rate of intensity that increased according to D-glucose concentrations.

Nutrient analysis

Na+/K+ Analysis: The roots and leaves of pea plant (Pisum sativum L) var: Merveile de Kelvedon was prepared for nutrient analyses according to the method described by Kaçar, 1972. The dried samples were crushed into powder using mortar and pestle and transferred into individual Erlenmeyer flasks. To the powder in each flask were added 6ml of nitric acid + perchloric acid solutions. The samples were digested

for 30min in a water bath at 4°C, and then heated at 150-180°C until the extracts were reduced to 1ml. this residue was dissolved in distilled water and made up to 100 ml in standard flasks. The samples for Na+ and K+ were analyzed using flame photometer (FP Jeaway UK).

Nitrogen Content: Nitrogen content of plant leaves was determined using the semi-micro Kjeldahl method. Samples of 0.2 dry shoots were digested by sulfuric acid (H2SO4). Distillation was carried out with 40% NaOH and ammonia was received in 4% boric acid solution. The distillates were then titrated with 0.02 HCl using the mixed methyl red bromocresol green indicator. Nitrogen percentage was calculated on dry-weight basis according to the methods of (El-Nady and Belal, 2005).

Statistical analysis: All statistical analysis was performed using SPSS software version 13 based on the one way ANOVA (for P<0.05) result a Duncan test for mean comparison was performed for a 95% confutation level to test for significant differences among treatments.

RESULTS AND DISCUSSION

Growth analysis

0

5

10

15

20

25

Ls Lr Dwl Dwr Nnd La

the

sal

init

y le

vels

( m

MO

L/l)

The morphological parameters

S0 S1 S2

The effect of salinity levels on the morphological traits

Fig : 1The effect of salinity levels on the morphological traits Length stem (Ls), length roots(Lr) ,dry weight of leaf(Dwl) , dry weight of roots (Dwr) ,number of nodules(Nnd) ,leaf area (La)

The effect of potassium levels in the form of acetate and

nitrate on the morphological traits

Agric. Biol. J. N. Am., 2015, 6(1): 34-46

37

Results presented in Table.1 showed that higher levels of salinity decrease all parameters of growth ( Sl , Rl , Dwl ,Dwr , La, Nnd ), throughout the experiment it was found that the general trend of the treatment reflect a gradual decrease in this parameters of growth with the increase of salt concentration compared with the plants of the control experiment (Fig.1) , these results have been confirmed by the results of Gama et al., (2007) on beans (phaseolus vulgaris ) they mention that there is a decrease in growth when treated with 50 and 100Mm of sodium chloride . The decrease of parameters of growth may due to the accumulation of sodium chloride in the cell walls and cytoplasm (Brolain et al., 2001). Previous studies have shown that the reasons for the inhibition of growth under salt stress are (1) the inhibition of the extension and leaf development (2) the thinnest layer of epidermal tissue mesophilic (3) the reduction of CO2 absorption rate (4) difficulties of stomatal regulation (5)

reducing the number of nodes (6) reduction in dry weight of organs (leaves, stems and roots) (7) inhibition of lateral roots ( Parida et al., 2004 ;Mathur et al., 2006 ;Rui et al., 2009; Memon et al., 2010) .This study revealed a significant difference between the pea cultivar and the effects of NaCl on growth based on two sources of K+ , KNO3 and CH3COOK, that were used in this study to assess whether these salts influence on the different concentrations of NaCl through the growth parameters, as indicated in Table.1 for this study included the dry weight of plant (leaves and roots), the number of nodes and the leaf area. The use of KNO3 source influence on some parameters while CH3COOK significantly affected almost all growth parameters, except the dry weight of the root and leaves that have remained unchangeable. CH3COOK (A2: 40 mmol) increased the length of the roots and stems approximately 52.45%, 58.08%, respectively, compared to the control, however CH3COOK caused an increase of leaf area approximately 64.35% in A2 (40 mmol) from KNO3 about 45.21% N2 (40 mmol). CH3COOK the source is more effective in A1 and A2 on the development of pea in particular on the number of nodes of the roots which have increased up to 71.15% relative to control. While the source KNO3 reduced the number of nodes above under N2 concentration (40 mmol) about 52.61% compared to the control (Fig. 2). Significant correlation coefficient were found between all parameters of growth and the potassium and sodium contents in the leafs and roots, when seedling growth under the supplementary potassium acetate CH3COOK and potassium nitrate KNO3 at salinity conditions ,the highest correlation observed between K(l)/Rl, r=0.994, Na (l)/ Sl, r =0.961 when plant treated with KNO3 and K(l) /La, r = 0.995, Na (l) /Sl, r = 0.941 when they treated with CH3COOK(Table. 2 -3).

0

5

10

15

20

25

Ls Lr

Dw

l

Dw

r

Nn

d

La

po

tass

ium

leve

ls m

MO

L/L

the morphological parameters

C

A1

A2

Fig :2The effect of potassium levels in the form of acetate and nitrate on the morphological traits Length stem (Ls), length roots(Lr) ,dry weight of leaf(Dwl) , dry weight of roots (Dwr) ,number of nodules(Nnd) ,leaf area (La)

Agric. Biol. J. N. Am., 2015, 6(1): 34-46

38

Table. 1. The effect of the interaction between Na+and K+ on morpho-physiological and biochemical parameters in pea (Pisum sativum) var: Merveille de kelvdon

Means followed by the same letter are not significantly different within rows and column according to Duncan’s test (P≤0.05).

(A1/ 20mMol/L, A2:40mMol/L CH3COOK) ;( (N1/ 20mMol/L, N2:40mMol/L KNO3)

S0 S1 S2

CH3COOK KNO3 CH3COOK KNO3 CH3COOK KNO3

variables A1 A2 N1 N2 A1 A2 N1 N2 A1 A2 N1 N2

Sl (cm) 19.45c 25.25a 18.75d 22.95b 14.2c 16.5a 13.37d 15.5b 10.65c 13.52a 9.52d 12.62b

Rl (cm) 13.52c 16.42a 12.7d 15.12b 9.45b 11.44a 9.12b 11.18a 6.72b 9.05a 5.82b 8.95a

Dwl (g) 0.70c 1.28a 0.58d 1.01b 0.20c 0.48a 0.10c 0.39b 0.070b 0.098a 0.062b 0.080a

Dwr (g) 0.40c 0.60a 0.3d 0.5b 0.091c 0.20a 0.081d 0.12b 0.041c 0.080a 0.036d 0.073b

Nnd 23b 34a 15d 8.5d 13b 18.5a 11.5d 10d 6b 11.25a 4.75d 3d

La (cm2) 3.97b 4.98a 3.70b 4.40a 2.75b 3.02a 2.61b 2.96a 1.81b 2.35a 1.72b 2.03a

Chla (µg/100mg /Mf) 2.65d 4.75a 2.37c 3.69b 1.85b 2.03a 1.69c 1.99a 1.13d 1.52b 1.04d 1.39b

Chlb (µg/100mg /Mf) 1.90c 3.03a 1.75c 2.71b 1.48c 1.77a 1.39d 1.69b 0.92c 1.31a 0.80d 1.22b

Chlt (µg/100mg /Mf) 4.55c 7.02a 4.14d 6.38b 3.33c 3.80a 3.08d 3.68b 2.05c 2.83a 1.84d 2.61b

Pro (µg/100mg /Mf) 1.90a 1.86a 1.94a 1.88a 2.51a 1.94a 2.72a 2.17b 4.74a 4.16b 4.82a 4.26b

Sr (µg/100mg /Mf) 2.15a 2.10a 2.18a 2.11a 3.92a 3.28b 4.02a 3.37b 5.65a 4.91b 4.80a 5.09b

Na+(r)% 1.28a 1.01b 1.33a 1.10b 1.61a 1.38b 1.68a 1.40b 2.25a 1.81b 2.30a 1.9b

Na+(l)% 1.19a 0.95b 1.23a 1.03b 1.60a 1.27b 1.65a 1.31b 1.89a 1.60b 1.96a 1.71b

K+(r)% 1.95b 2.38a 1.83b 2.29a 1.26b 1.48a 1.21b 1.43a 0.85c 1.03a 0.83c 0.95b

K+(l)% 2.36b 2.97a 2.20b 2.81a 1.58b 1.84a 1.53b 1.76a 0.89b 1.06a 0.81b 1.01a

K+/ Na+ (r) 1.52b 2.35a 1.37b 2.10a 0.78b 1.07a 0.72b 1.02a 0.34b 0.58a 0.31b 0.55a

K+/ Na+ (l) 1.98c 3.12a 1.78d 2.72b 1.03b 1.44a 0.92b 1.34a 0.62b 0.80a 0.59b 0.71a

TN(l)% 2.53c 3.01a 2.30d 2.78b 1.68c 2.06a 1.47d 1.79b 0.97c 1.11a 0.80d 0.98b

Agric. Biol. J. N. Am., 2015, 6(1): 34-46

39

Biochemical analysis

The treatment effect of salinity on chlorophyll (a, chlorophyll b and total) is shown in Table 1,.the levels of NaCl are trying to reduce the chlorophyll content, and salt stress causes yellowing leaves which eventually led to significant damage to chlorophyll pigments (Al-Khajari et al., 2002) Similar results have been reported for other legumes (Delgado et al., 1994), the inhibitory effects of salts on the chlorophyll content could be due to a suppression of specific enzymes responsible synthesis green pigments (Neera and Ranjus, 2004) .The reduction of chlorophyll can be attributed to the increased activity of the enzyme chlorophyllase (Parida et al., 2004) An effect that depended on biological processes and stages of plant development and also the concentration of salts (Fig.3) .The two potassium salts (CH3COOK, KNO3) eliminates the deleterious effects of salinity on chlorophyll pigment content (a, b and total chlorophyll). Comparing these two potassium salts we observe ,that potassium acetate (CH3COOK) increased significantly by 72.5%, 66.39%, 79.43% and KNO3 by 46.87% , 53.27%, 49.64% (chlorophyll a, b and total chlorophyll content) respectively compared to the control, has revealed a significant difference between the two treatments, and we observed that the CH3COOK treatment caused an increase in the

content pigment chlorophyll (a, b, and total chlorophyll) under A1 and A2 concentration which appears to exceed the treatment of KNO3 in levels N1 and N2( Fig :4). The concentration of organic solutes (proline and sugar) in the plant tissues were generally very low (Table.1) but the levels were significantly affected by salinity (Jampeetong and Brix, 2009). Also they found that concentration of this organic solutes (proline –sugar) were higher in the plants grown at 150mMol /L salinity compared with the control, proposed that it might be related to the mechanism of this tolerance plant salt stress (Parida et al., 2004; Garg and Singla, 2009). The Selective accumulation of intercellular organic solutes is a generalized physiological response to osmotic stress (Ashraf and Haris, 2004). They are involved in osmotic adjustment (Demiral and Turkan, 2005), and suggests that these osmolytes can be caused by proteolysis increase or decrease in protein synthesis (Gama et al., 2007, Kapoor and Srivastava 2010 Hameda, 2011) (Fig.3). The application of two potassium salts in the soil (KNO3 , CH3COOK) removed the deleterious effects of salinity compared to control .The organic solutes concentration (proline and sugar) varied with potassium salts treatment , under potassium acetate treatment both proline and sugar decreased

0

1

2

3

4

5

6

chla chlb chlT Pro Sr

The

sal

init

y le

vels

( m

Mo

l/ L

)

The biochemical parameters

S0 S1 S2

0

1

2

3

4

5

6

chla chlb chlT Pro Sr

the

po

tass

ium

leve

ls m

Mo

l/L

the biochemical parameters

C

A1

A2

N1

N2

The effect of salinity levels on the biochemical traits

Fig: 3.The effect of salinity levels on the biochemical traits chlorophyll a(Chla), chlorophyll b(Chlb), total chlorophyll (Chl T), proline (Pro), sugar (Sr)

Fig: 4. The effect of potassium levels in the form of acetate and nitrate on the biochemical traits chlorophyll a(Chla), chlorophyll b(Chlb), total chlorophyll (Chl T), proline (Pro), sugar (Sr)

The effect of potassium levels in the form of acetate and nitrate on the biochemical traits

Agric. Biol. J. N. Am., 2015, 6(1): 34-46

40

significantly by 31.52, 22.92 , in A2 respectively, and under potassium nitrate treatment this organic solutes decreased significantly by 28.42, 20.89 in N2 levels respectively compared to control we revealed no significant variations between the two salts treatment , the decrease in organic solutes under two potassium salts it can be stated that the ability of plants to retain K+ at high Na+ concentration of the external solution may be involved in reducing the damage associated with excessive Na+ concentration in plant (Heidari and Jamshid, 2002).Cherel, (2004) reported potassium plays an important role in balancing membrane potential and turgor activating enzymes , regulating osmotic pressure , stoma movement and tropisms (Fig.4) . Significant correlation coefficient were found between all parameters of biochemical content ( Chla , Chlb , ChT , Sr , Pro ) and the potassium and sodium contents in the leafs and roots when seedling growth under the supplementary potassium acetate CH3COOK and potassium nitrate KNO3 at salinity conditions (Table.1) the highest correlation observed between K(l)/Chlb, Na(l) / Sr, r=0.971when seedling growth under CH3COOK levels and K(l)/Chlb r= 0.978, Na(l) / Sr, r=0.966 when seedling treated by KNO3 levels under salt stress condition (Table .2-3) .

Agric. Biol. J. N. Am., 2015, 6(1): 34-46

41

Table.2.Matrix of correlations coefficients between the variables analyzed in pea (Pisum sativum) var: Merveille de kelvdon (leaves, roots, stems) treated with acetate of potassium (CH3COOK) under saline conditions

ARIABLES Sl Rl Dwr Dwl La Nnd Sr Chla Chlb Chlt Pro Na+(r) K+(r) K+/ Na+ (r)

Na+(l) K+(l) K+/ Na+ (l)

TN(l)

Sl 1

Rl 0.968** 1

Dwr 0.862* 0.957** 1

Dwl 0.862* 0.958** 0.990** 1

La 0.973** 0.985** 0.931** 0.921** 1

Nnd 0.979** 0.976** 0.910** 0.913** 0.980** 1

Sr -0.877*

-0.816*

-0.680*

-0.684*

-0.878*

-0.853*

1

Chla 0.876* 0.961** 0.967** 0.979** 0.919** 0.892* -0.679*

1

Chlb 0.931** 0.988** 0.967** 0.979** 0.956** 0.948** -0.743*

0.988** 1

Chlt 0.900** 0.974** 0.970** 0.982** 0.936** 0.916** -0.706*

0.998** 0.996** 1

Pro -0.862*

-0.817*

-0.696*

-0.701*

-0.875*

-0.844*

0.997** -0.691*

-0.747*

-0715* 1

Na+(r) -0.935**

-0.875*

-0.749*

-0.754*

-0.922*

-.0937**

0.966** -0.728*

-0.910*

-0.762*

0.957** 1

K+(r) 0.982** 0.968** 0.894* 0.888* 0.990** 0.991** -0.895*

0.875* 0.930** 0.899* -0.885*

-0.954**

1

K+/ Na+ (r) 0.947** 0.948** 0.885* 0.892* 0.970** 0.980** -0.913**

0.854* 0.912** 0.879* -0.913**

-0.966**

0.981** 1

Na+(l) -0.941**

-0.861*

-0.718*

-0.716*

-0.915**

-0.918**

0.971** -0.709*

-0.789*

-0.742*

0.953** 0.990** -0.948**

-0.938**

1

K+(l) 0.962** 0991** 0.959** 0.949** 0.995** 0.981** -0.833*

0.941** 0.971** 0.955** -0.832*

-0.891*

0.982** 0.961** -0.879*

1

K+/ Na+ (l) 0.948** 0.979** 0.945** 0.944** 0.991** 0.978** -0.870*

0.923** 0.958** 0.939** -0.874*

-0.918**

0.981** 0.983** -0.895*

0.991** 1

TN(l) 0.953** 0.983** 0.943** 0.955** 0.982** 0.976** -0.861*

0.939** 0.971** 0.954** -0.866*

-0.908**

0.975** 0.977** -0.887*

0.984** 0.991** 1

(**highly significant; * significant; ns not significant).

Agric. Biol. J. N. Am., 2015, 6(1): 34-46

42

Nutrient analysis

The result of nutrient analysis indicated that salinity treatment increased significantly Na+ concentration and decreased K+content in all organs of pea plants (leafs and roots) mainly in 150mMol/L NaCl compared to control (Table.1), these decrease could be due to the antagonism of Na+on K+ transport into the xylem or the inhibition of uptake processes (Menzel and Kirkby, 2001), the depressive effect of salt on the decrease of total N in the leaves with a decrease to 34.5%. . Etelvina et al., (2005) found that the nitrogen content in Pisum sativum has been more greatly reduced with an increase in the water of saline irrigation, other results include those in support of Singh et al., 2003 with their study Vigna radiata L they found that exposure to NaCl salinity reduces the nitrogen content in the leafs and they proposed that salt stress can induce a disordered structure of nodules that could be the reason for the decline in the rate of N2 fixation by legumes subjected to salt stress or NaCl directly affects nitrogenase synthesized by Azotobacter (Bouhmouce al., 2005) (Fig :5) . To investigate the effect of both potassium salts in saline conditions. We notice that these two salts under salinity stress differ in their effects on the accumulation and nutrient absorption in all plant organs (leaves and roots). Tables.1, Figure.6,

shows in CH3COOK treatment caused a decrease in Na+ content (26.16%, 30.69%) and an increase in K+ concentration (39.28%, 40.51%) in A2 in the leaves and roots respectively compared to control. As against the treatments with KNO3 levels the concentration of Na+ and K+ in all plant parts (leaves and roots) was significantly different from the control supply, the Na+ decreased by 32.85%, 33, 62% in the leaves and roots respectively in N2, but K+content increased by 33.62%, 32.85% in the leaves and roots respectively compared to control, there is a different effect from A2, A1 and N2, N1 on the accumulation of Na+ and K+ in the leaves and roots under salt stress (Kaya and David, 2003 ).The two different K+ sources were greater than the control because the K+ reduced excess uptake of ions such as Na+in saline soils (Heidari and Jamshid, 2002), The decrease of the Na+ content can be attributed to the competition K+ and Na+ on the absorption sites of the plasma membrane that rejects the Na+ in the external solution (Al-Uquali, 2003). The K+ / Na+ ratio was significantly higher in CH3COOK potassium acetate (99%, 133.33%), compared to potassium nitrate KNO3 (84.88%, 114.03%) in the leaves and roots respectively compared to control.

0

0.5

1

1.5

2

2.5

3

Na+

(r )

Na+

(l)

K+(

r)

K+(

l)

K+/

Na+

(r)

K+/

Na+

(l)

TN(l

)

The

sal

init

y le

vels

(m

MO

L /

l )

The nutrients in the leaves and roots

S0 S1 S2

0

0.5

1

1.5

2

2.5

Na+

(r )

Na+

(l)

K+

r

K+(

l)

K+/

Na+

r

K+/

Na+

(l)

TN(l

)

The

po

tass

ium

leve

ls m

MO

L/l

the nutrients in the leaves and roots (%)

C

A1

A2

N1

N2

The effect of salinity levels on the nutrients in the leaves and roots

Fig: 5. The effect of salinity levels on the nutrients in the leaves and roots: sodium in the roots Na+(r), sodium in the leafs Na+ (l), potassium in the roots K+(r), potassium in the leafs K+ (l), total nitrogen (NT)

Fig: 6. The effect of potassium levels in the form of acetate

and nitrate on the nutrients in the leaves and roots: sodium

in the roots Na+(r), sodium in the leafs Na+ (l), potassium in

the roots K+(r), potassium in the leafs K+ (l), total nitrogen

(NT)

The effect of potassium levels in the form of acetate and

nitrate on the nutrients in the leaves and roots

Agric. Biol. J. N. Am., 2015, 6(1): 34-46

43

Table.3.Matrix of correlations coefficients between the variables analyzed in pea (Pisum sativum) var: Merveille de kelvdon (leaves, roots, stems) treated with potassium nitrate (KNO3) under saline conditions

VARIABLES Sl Rl Dwr Dwl La Nnd Sr Chla Chlb Chlt Pro Na+(r) K+(r) K+/ Na+ (r)

Na+(l) K+(l) K+/ Na+ (l)

TN(l)

Sl 1

Rl 0.969** 1

Dwr 0.841* 0.946** 1

Dwl 0.850* 0.942** 0.981** 1

La 0.967** 0.988** 0.909** 0.915** 1

Nnd 0.679* 0.654* 0.527ns 0.522ns 0.694* 1

Sr -0.910**

-0.866* -0.696* -0.719* -0.923**

-0.810* 1

Chla 0.905** 0.972** 0.975** 0.967** 0.951** 0.548* -0.788* 1

Chlb 0.931** 0.982** 0.968** 0.968** 0.958** 0.562ns -0.797* 0.994** 1

Chlt 0.918** 0.978** 0.974** 0.969** 0.956** 0.556ns -0.794* 0.999** 0.998** 1

Pro -0.900**

-0.862* -0.698* -0.723* -0.921**

-0.815* 0.998** -0.796* -0.796* 0.794* 1

Na+(r) -0.954**

-0.903**

-0.736* -0.775* -0.941**

-0.762* 0.964** -0.809* -0.842* -0.825* -0.956**

1

K+(r) 0.977** 0.980** 0.884* 0.894* 0.984** 0.680* -0.906**

0.919** 0.944** 0.932** -0.894* -0.955**

1

K+/ Na+ (r) 0.951** 0.960** 0.868* 0.904** 0.981** 0.719* -0.924**

0.907** 0.929** 0.918** -0.921**

-0.968* 0.980** 1

Na+(l) -0.961**

-0.897* -0.714* -0.741* -0.931**

-0.749* 0.966** -0.794* -0.827* -0.810* 0.953** 0.991** -0.952**

-0.943**

1

K+(l) 0.956** 0.994** 0.947** 0.941** 0.987** 0.624* -0.856* 0.967** 0.978** 0.973** -0.849* -0.900**

0.983** 0.962** -0.891**

1

K+/ Na+ (l) 0.949** 0.985** 0.930** 0.940** 0.993** 0.664* -0.889* 0.960** 0.970** 0.966** -0.887* -0.925**

0.981** 0.984** -0.904**

0.991** 1

TN(l) 0.948** 0.987** 0.940** 0.956** 0.989** 0.679* -0.887* 0.969** 0.977** 0.974** -0.887* -0.915**

0.974** 0.980** -0.896**

0.984** 0.994** 1

(**highly significant; * significant; ns not significant).

Agric. Biol. J. N. Am., 2015, 6(1): 34-46

44

The K+ / Na+ ratio was pronounced in relatively higher in the roots than in the leaves (Figure. 6, Table.1). The nitrogen content in the leaves varies with potassium sources, The CH3COOK caused an increase in the N content in the leaves of 47.7%, but KNO3 treatment increased only 32.14% compared to controlled levels in A2 and N2 respectively (Cengiz and Divid, 2013) .A high significant correlation was observed between potassium K (l) and K+ / Na+ ratio in leaves (r=0.991) and between the sodium content in the leaves Na(l) and the sodium content in the roots Na(r) (r=0.991,r= 0.990) when the seedlings have been treated with KNO3 and CH3COOK respectively in salt stress conditions (Table.2-3). CONCLUSIONS: In this study we can draw the following conclusion. • Both source of potassium have eliminated the deleterious effects of salinity on the pea plant (Pisum sativum L.) var: Merveile Kelvedon, there have effects on the increase of physio-morphological and biochemical traits of seedlings and they reduced the accumulation of proline and sugar content in leaves. • Potassium nitrate KNO3 was found to have less effect on the accumulation of Na+ in the leaves and roots of pea plants compared to potassium acetate CH3COOK. • Potassium acetate CH3 COOK (A2) has increased the number of nodules (Nnd), total nitrogen (NT) in the leaves and the content of potassium (K+) in the leaves and roots, compared with KNO3. • CH3COOK potassium acetate is a good fertilizer compared to potassium nitrate (KNO 3) for the development of pea (Pisum sativum L.) var: Merveile Kelvedon in saline conditions.

REFERENCES

1. Al-khanjari, S; Al-Kathiri, A; Esechie, H.A. (2002).Variation in chlorophyll meter reading nodulation and dry –matter yields of alfalfa (Medicago sativa) cultivars differing in salt tolerance. Crop .Res .24:350-356.

2. Al-Uquali .J.K. (2003). Potential of using drainage water of wheat production in Iraq .Emirates. J. Agric. Sci .15:36-43.

3. Ashraf .M and Haris, P.J.S. (2004). Potential biochemical indicators of salinity tolerance in plants .Plant.Sci .166: 3-16.

4. Badr, M.A and Shafei, A.M. (2002).Salt tolerance in two wheat varieties and its relation to potassium nutrition. Al azhar .J.Agric .Res .35:115-128.

5. Baghbani, A; Namdari, A; Kadkhodaie, A. (2012). Effects of salinity and nitrogen supply on nitrogen fixation nodules and nitrogen, sodium and potassium concentration of Alfalfa cultivars. J.Bas .Appl . Sci. Res. 2(10):9978-9984.

6. Bidel, L.P.R; Meyer, S; Goulas .Y; Cadot, Y and Cerovic, Z. G. (2007). Responses of epidermal phenolic compounds to light acclimation in vivo qualitative and quantitative assessment using chlorophyll fluorescence extraction spectra in leaves of three woody species. J. Photochem. Photobiol. 88: 163-179

7. Bolarin, M.C; Estan .M.T; Caro, M; Romero-amanda, R and Cuartero, J. (2001) .Relationship between tomato fruit growth and fruit osmotic potential under salinity. Plant. Sci .160:1153-1159.

8. Bouhmouce, L; Souad, M.B; Brhada, f. (2005). Influence of host cultivars and Rhizobium species on the growth and symbiotic performance of phaseolus vulgaris under salt stress. J Plant.physiol.162:1103-1113

9. Cengiz.K and Divid, H. (2013). Supplementary potassium nitrate improves salt tolerance in bell pepper plant .J. Plant Nutr. 26(7): 1367-1382.

10. Cherel, L. (2004). Regulation of K+ channel activities in plant from physiological to molecular aspects. J.Exp. Bot .55:337-351.

11. Cordovilla .M.P; Ligero.F; Liuch .C. (1994). The effect of salinity on Nfixation and assimilation in vicia faba. J.Exp. Bot. 279:1483-1498 .

12. Delgado .M.J; Ligero .F; Liuch .C. (1994). Effect of salt stress on growth and N2 fixation by pea, faba bean, Common bean and soybean. Plant Soil Biology and Biochemistry .26:371-376.

13. Demiral .T and Turkan .I. (2005). Comparative lipid peroxidation antioxidant defense systems and proline content in roots of two rice cultivars differing in salts tolerance. Env .Exp.Bot.53:247-257.

14. Dubois, M; Gilles, K; Hamilton, J.K; Rebers, P. A and Smith, F. (1956) .colorimetric method for determination of sugar and

Agric. Biol. J. N. Am., 2015, 6(1): 34-46

45

related substances. Analytical .Chemistry .28(3): 350-356.

15. El-Sayed, B ; Belal Hassan, M.M ; El Ramady, H.R. (2013).Phylogenetic and characterization of salt tolerant rhizobial strain nodulating faba bean plants .Afric .J .Biotech .12(27): 4324-4337.

16. El-Nady, M.F and Belal, E.B. (2005). Responses of faba bean (Vicia faba) plants to root nodule bacteria under salinity conditions. J.Agri. Res. Tanta. Univ. 49:245-253.

17. Etelviva, M; Almeida, P; and Calderia, G. (2005) .Effect of nitrogen on salt tolerance of Pisium sativum during vegetative growth. J. Plant. Nutrition. 168: 359-363.

18. Fatma, B; Sener, A; Ahmet, E. (2011).Growth and uptake of sodium and potassium in broad bean (Vicia faba) under salinity stress.Comm. Soil. Sc. Plant. Analy. 42(8): 945-961.

19. Gama, P.B; Inanaga, S; Tanka, K and Nakazawa, R. (2007). Physiological response of Common bean (phaseolus vulgaris) seedlings to salinity stress. Afric.J.Biotech .6(2): 79-88.

20. Garg, N; Singla, R. (2009).Variability in the response of chickpea cultivars to short –term salinity in terms of water retention capacity membrane permeability and osmoprotection .Turk.J.Agric.For. 33:57-63

21. Ghazi, N; Al-Karaki. (2000). Growth sodium and potassium uptake and translocation in salt stressed tomato .J.Plant. Nutr. 23(3)369-379.

22. Hameda, E.S.A.S. (2011). Influence of salinity (NaCl and Na2So4) treatments on growth development of broad bean (phaseolus vulgaris) plant .Amer.Euras.J.Agric. Envir .Sci. 10(4):600-610.

23. Hassan, M.M. (2009). Genetic studies on Rhyzobium and legumes symbiosis relationship. PhD .Thèses . Dep. Fac .Agri . Min. Egypt.

24. Hatice, Ö; Caner, K; Erdal, E. (2010).Effect of Rhizobium strains isolated from wild chickpea on the growth and symbiotic performance of chickpea (Cicer arietinum L.) under salt stress .Turk .J. Agric. For. 34: 361-371 .

25. Heidari, M and Jamshid, P. (2010). Interaction between salinity and potassium

on grain yield carbohydrate content and nutrient uptake in pearl millet .ARPN. J.Agric Biol .Sc.5 (6):39-46.

26. Hisschnyder, H.Y.H and Schmidhalter, U. (2000). Carbohydrate deposition and parhrioning in elongation leaves of wheat under saline soil conditions .Aust.J.Plant.Physiol.27:363-370.

27. Jampeetong .A and Brix.H. (2009) .Effect of NaCl salinity on growth, morphology, photosynthesis and proline accumulation of Salvinia natans .Aquatic .Bot .91:181-186.

28. KaÇar, B. (1972) .Bitki ΰe topraĝin kimyyasal. Analizleri.Ankara. Bizim. BÜro

.Basimeri. 113-261: ׀׀׀.

29. Kapoor, K; Srivastava, A. (2010).Assessment of salinity tolerance of Vinga mungo var. Pu-19 using ex vitro and in vitro .Metho.Asian. J. Biotech., 2 (2): 73–85

30. Karimi, J.M; Ghorbanli, H; Heidari, R.A; Khavari, N and Assareh, M.H. (2005). The effect of NaCl on growth, water relations, osmolytes and ion content in Kochia prostata. Biol. Planta .49:301-304.

31. Kaya, C; Erol, B and David, H. (2003). Response of salt –stressed strawberry plants to supplementary calcium nitrate and /or potassium nitrate. J.Plant. Nutr. 26(3) .543-560.

32. Kham, A.A; Rao, S.A and Moneilly, T. (2003). Assessment of salinity tolerance based upon seedling root growth response function in maize (Zea mays L.). Euphetica .131:81-89.

33. Kheder, A.A; Abbas, M.A; Abdelwahid, A.A; Quik, W.P and bdogadallah, G.M. (2003). Proline induces the expression of salts stress responsive proteins and may improve the adaptation of Pancratium maritum L to salt stress. J .Exp .Bot. 54: 2553-2562.

34. Laguerre, G; Mhamdi, R; Mohamed, E. (2001). Different species and symbiotic genotypes of field Rhysobia can nodulate phaseolus vulgaris in Tunisian soils. Microbial .Ecol .41:77-84.

35. Lopez, M; Herra-cervera, J; Iribarne, C; Tejra, N and Liuch, C. (2008). Growth and nitrogen fixation in luus japonicus and Medicago truncatula under salinity stress noodle carbon metabolism. J.Plant .Physiol. 165:641-650.

Agric. Biol. J. N. Am., 2015, 6(1): 34-46

46

36. Mathur, N; Singh, J; Bohra, S; Bohra, A; Vyas, A. (2006).Biomass production, productivity and physiological changes in moth bean genotypes at different salinity levels .Am. J. Plant Physiol., 1 (2: 210-213

37. Memon, S.A. X; Hou, L; Wang, J. (2010). Morphological analysis of salt stress response of pak Choi. EJEAFChe, 9 (1), pp. 248–254

38. Menzel .K; Kirkby .E.A. (2001). Principales of plant nutrition .5th Ed, Kluwer Academic .Publishers Dordrecht

39. Neera, G; Ranju, S. (2004) .photosynthesis nodule nitrogen and carbon fixation in the chickpea cultivars under salts stress. Braz.J.Plant.Physiol.16(3):137-146.

40. Nogales, J; Campos, R; Benabdelkhaled, H; Olivares, J; Liuch, C; Sanjuan, j. (2002). Rhizobium tropici genes involved in free-living salt tolerance are required for the establishment of efficient nitrogen fixing symbiosis with (phaseolus vulgaris).Mol.plant.Microb. Interact. 15:225-232.

41. Noreen, Z; Ashraf, M and Hassan, M.U. (2007). Inter-accessional variation for salt tolerance in pea (Pisum Sativum) at germination and seedling stage. Pak.J.Bot .39(6):275-285.

42. Parida .A.K; Das.A.B and Mittra.B. (2004). Effects of salt on growth ion accumulation photosynthesis and leaf anatomy of the mangrove Brugulera paviflora .Trees: Structure and function .18:167-174.

43. Rubiol, L and Botella, M.A. (2004). Regulation of K+ tansport in tomato roots by the TSSI locus implications in salt tolerance. Plant .Physiol .134:452-459.

44. Rui, S. L; Wei, C; Mu-xiang, J; Cheng-jun, W. Min, Y. Bo-ping. (2009) Leaf anatomical

changes of Burguiera gymnorrhiza seedlings under salt Stress.J. Trop. Subtrop. Bot., 17 (2): 169–175

45. Serraj, R; Drevon, .j.j. (2013). Effect of salinity and nitrogen source on growth and nitrogen fixation in Alfalfa. J.Plant. Nutr.21(9):1805-1818.

46. Sharma, S.R; Rao, N.K; Gokhal, T.S; Ismail, S. (2013). Isolation and characterization of salt tolerant rhizobia native to the desert soil of United Arab Emirates. Emir.J.Food.Agric .25(2) : 102-108.

47. Singh, R.P; Tripathi,R.D; Dabas, S; Rhizvi,S.M.; Ali,M.B; Sinha .S.K; Gupta , D.K; Mishra ,S; and Rai ,U.N .(2003) . Effect of lead on growth and nitrate assimilation of Vigna Radiata (L) Wilezek seedling in a salt affected environment. Chemosphere .70 :566-575.

48. Sohrabi, Y; Heidari, g and Esmailpoor, B. (2008). Effect of salinity on growth and yeild of desi and Kabuli chickpea cultivars. Pak.J.Bot .Sc. 11: 664-667.

49. Tejera, N.A; Campos, R; Sanjuan, J; Liuch, C. (2005). Effect of sodium chloride on growth nutrient accumulation and nitrogen fixation of Common bean plants in symbiosis with isogenic strains J.Plant. Nutr.28:1907-1921.

50. Yanshou, W.U; Yibing, H.U and Guohua, X.U. (2009). Interactive effects of potassium and sodium on root growth and expression of K/Na transporter genes in rice. Plant. Grow .Regul. 57(3):271-280.