Evaluation of salt stress tolerance of chilli (Capsicum annuum L.) genotypes at seedling stage

Salinity is increasing problem and is one of the main threats to crop productivity worldwide. Understanding the adverse impact of salt stress (NaCl) at early growth stages is essential for evaluation of salt stress tolerance in plants. A trial was performed during 2017 following completely randomized design (CRD) factorial with three replicates. Three chilli genotypes varieties were received 5 salt stress levels. The canal irigation water having Electrical conductivity (EC) of 0.5 dSm -1 was kept as control. The Salinity levels included 1, 3, 5 and 7 dS m -1 , respectively. It was noted that all the studied traits were considerably influenced by salinity (P<0.05) while chilli genotypes showed similarity (P>0.05) for germination, leaves plant -1 and seedling vigour index and significant (P<0.05) differences for other traits. The control (non-saline) showed better performance with 84.4% germination, 5.64 leaves plant -1 , 905.4 seedling vigour index, 10.7 cm shoot length, 0.7 g fresh root biomass weight, 13.3 cm length of the root, 4.8 g fresh biomass of the shoot 42.5% electrolyte leakage. Salinity at 1.0 dS m -1 revealed a slight reduction in germination (80.5%) 5.12 leaves plant -1 , 843.2 seedling vigour index 10.4 cm shoot length, 0.6 g fresh root biomass weight 13.0 cm length of the root, 4.5 g fresh biomass of the shoot and 48.4% electrolyte leakage. Further increase in salinity showed simultaneous negative impact on all the observed parameters. There was a negligible difference in studied traits when Sky lane-4 was compared with Ghotki. Overall variety Pusa Jawala was most sensitive to salt stress.


Introduction
Chilli, (Capsicum annum L.) is a member of Solanaceae family grown for its fruit and is popular for its aromatic and pleasant flavor and pungency. The use of chillies is common for culinary purposes, beverage and pharmaceutical industries. There is diverse utility of chilli as spice, culinary supplement, condiment, vegetable and medicine besides its importance as commercial crop. [1]. Salinty is one of the main threats to crop productivity globally [2]. Available reports reveal that more than 7% of the surface of the earth is occupied by salt affected soils and because of salt stress 20% decrease in yield of cultivated plants has been observed globally [3]. It is well documented that cultivated plants tolerate salt stress to a specifed limit, however, after that threshold level, salt stress significantly induced reduction in yield. The salinity affects plants in different ways, like the first instant impact of salinity for plants is an osmotic effect through which plants loose their internal water balance from their cells [4]. The second hazard of salinity is an ionic effect, where plants face problems of ionic toxicity, especially toxicity of Na + and Clions [5]. The third negative impact of salinity for plants is the nutrient imbalance, where plants uptake toxic elements like Na+ rather than nutrient elements particularly K + [6]. Plants use several mechanisms to adopt these influences of salinity. Generally, plants synthesize organic acids (proline, glycinebetane, etc) as osmo-protectant the Seed germination is the most vulnerable to salt stress in comparision with other growth stages of plants [7,8]. At this stage most vegetable crops exhibit sensitivity even to low concentration of salts to about 75 mM NaCl [9]. Salinity reduces seed germination percentage and impairs nodule formation and the growth and yield is decreased and survival of plant is jeopardized at later stages. The seed germination is adversely affected by salt stress through osmotic effects and ionic toxicity [10]. The past studies relevant to saline water irrigation showed that the saline water effect is proportional to salinity level and crop species. The seed germination in several crops including chillies is highly sensitive to salinity [11]. While the seedling growth at early stage is also face severe adverse effects of salinity, but some plants species and varieties may have ability to tolerate salinity at this stage [12]. The inhibitory effects of NaCl on germination are well established [13], it causes reduction in germination rate considerably [14], and consequently caused decline in plant performance in terms of growth, development and yield [15]. The application of phytohormones can mitigate the negative effects of salinity. The concentration of the salts at higher rates reduces the water potential in medium which hinders water acquision and thus reduce seed germination [16]. The speed of germination and the final seed germination percentage determine the ability of the seed to tolerate stresses like salt stress. The rate of seed germination and days taken to seedling emergence in chilli, cabbage, spinach, sugar beet and cauliflower were adversely affected by increasing the salinity levels that salinity considerably affected the Na content of plant traits including roots, shoots and fruits of pepper and tomato varieties without any effect on crop yields. Evaluating salinity tolerance of plants has been found very successful [17]. Several plant species and varieties have been evaluated and reported as salt-tolerant and sensitive to salinity [18]. To the knowledge of researchers very little information is available on salinity tolerance of condiment plant species, including chillies. In Pakistan chillies are cultivated on large scale however; this crop is facing several problems, including Aflotoxin, waterlogging and salinity. The crop species, cultivars and growing conditions have been documented to affect the response of plants to salinity and associated nutrient acquisition [19]. In the light of above mentioned facts, a pot trial was performed to assess the salt stress tolerance of chilli genotypes at seedling stage.

Materials and Methods
A pot trial was performed during 2017 following completely randomized design (CRD) factorial with three replicates. Three chilli genotypes viz, Ghotki, Skylane 4 and Pusa Jawala received 5 salt stress levels. Three pots represented each replication; and in each pot seven seeds were sown. The earthen pots were filled with a growing medium that included canal sediment, well rooten FYM and garden soil at the ratio of (1:1:1). In this research, the seeds of three Chilli genotypes including Ghotki, Skylane 4 and Pusa Jawala were sown in pots. The sodium chloride (NaCl) was used to maintain EC levels in accordance with the treatment levels. Five different NaCL levels including control (canal water having EC 0.05 dSm -1 ) and 1, 3, 5 and 7 dSm -1 were applied. Plants were irrigated with saline water on alternate days and all other cultural operations in the pots were performed manually. After one and half month (45 days), the data were taken for plant traits including leaves plant -1 , seedling vigour index, shoot length, fresh biomass of shoot, root length, fresh biomass of root and electrolyte leakage of leaf. Procedures for recording the observations Observations recording methodology Seed germination percentage The germination percentage of the seed was done by applying the formula suggested by [20]. GP = Σn/ N ×100 Where n is seeds that were germinated and N is total seeds that were sown.

Shoot and root related traits
The measuring scale was used to assess the root and the length of the shoot while fresh root and shoot biomass were measured with electrical balance.

Seedling vigour index
Seedling Vigor Index (SVI) was measured by using the formula given by [21].
(SVI) = [shoot length (cm) × germination percentage] Electrolyte leakage of leaf The leaf Electrolyte leakage % was assessed by applying the formula given by [22]. The random leaf sample, weighing 0.5 g was used for measurement of electrolyte leakage. The 25 ml deionized water was used to rinse the leaf samples before incubation for 3 hours at room temperature. The conductivity of the bathing solution was calculated by electrical conductivity (EC) meter after incubation that was considered as (value A). The petal discs were boiled with bathing solution for 15 minutes to lyses all cells. After cooling at room temperature, the conductivity of the bathing solution was again calculated that was considered as (value B). The electrolyte leakage was measured in percent (%) value by using the following formula. Electrolyte leakage of leaf % = (Value A/Value B) x100 Statistical analysis The data were analyzed using Statistix-8.1 computer software [23]. The least significant difference (LSD) test at P≤ 0.05 probability level was performed to compare the results and performance of the treatments.

Results and Discussion Germination (%)
The data given in (Table 1) indicate the impact of salt stress levels on seed germination of chilli genotypes and comparison was made with control (Canal irrigation water having EC 0.5 dS m -1 ). The analysis of variance (ANOVA) described significant (P<0.05) influence of salinity levels on germination. Among salinity levels, chilli genotypes sown in media of 1.0 dS m -1 resulted in maximum germination (80.5%) against 84.4 percent germination in control. A decline in germination was recorded with increased salt stress levels. It was noted that with each unit increase in salinity, the germination was adversely affected when compared to control. The genotypes effect showed that chilli genotypes Sky lane-4 and Ghotki resisted to salinity effect with higher germination of 70.5 and 69.5%, respectively; while lowest germination was found in Pusa Jawala (65.2%). The germination was markedly higher (86.6%) in variety Sky lane-4 when treated with with control (canal water). Similarity in germination (P>0.05) was recorded between control and 1.0 dS m -1 salt stress level; while linear decrease in germination with increasing salinity was recorded in rest of the treatments. The highest concentration of NaCl might have created external osmotic potential by which caused obstacles in acquisition of the optimum quantity of the water for better germination of the seed [24]. These findings are also endorsed by [25], who also got the same results on pepper plants.

Seedling vigour index
The data shown in (Table 3), reflects the impact of salinity levels on seedling vigour index of chilli genotypes over control. The ANOVA revealed that the effect of salinity on seedling vigour index was significant (P<0.05), while genotypes did not vary significantly in seedling vigour index. It was noted that among salinity levels, seedling vigour index was maximum

Shoot length (cm)
The results given in (Table 4) The length of the shoot was adversely affected by increasing salinity level as well as genotypes also responded differently to salinity levels, in salinity influenced significantly shoots and roots of the plants for their size and fresh weight. [28]. has reported that because of toxicity of ions (Na + and Clions) caused by salt stress, the plant performance in terms of growth, and yield reduced significantly. Moreover, this also led to soil and plant osmotic imbalances.

Fresh shoot biomass (g)
The data shown in (Table 5), indicated the salinity impact on fresh shoot biomass of different genotypes of chilli over control. The ANOVA demonstrated that salt stress showed highly negative impact on fresh shoot biomass. The genotypes also differed in fresh shoot biomass considerably. The data show that among treatments (salinity levels), genotypes sown in media of 1.0 dS m -1 showed highest shoot biomass (4.5g) against 4.8 g fresh shoot biomass in control. The fresh shoot biomass reduced to 3.3 g and 2.2 g when the genotypes were sown in media of 3.0 and 5.0 dS m -1 , respectively. The lowest values pertaining to fresh shoot biomass was determined under highest salt stress level. In genotypes, the fresh shoot biomass was higher in chilli genotypes Sky lane-4 while Pusa Jawala showed minimum fresh shoot biomass. In the present investigation the reduced shoot biomass under higher salinity level might be due to the negative influence of NaCL on growth of shoot. This also reflects that less fresh biomass under high salt stress might have been associated with dcreased acquisition of water by plants at early stage [29], also found that seedling height, leaves and branching, fresh and dry biomass of shoots and roots were declined under salt stress.

Root length (cm)
The data given in (Table 6), represent the salinity effect on root length of various chilli genotypes over control. The ANOVA displayed that the varied salinity levels had negative impact on root length; and chilli genotypes also differed significantly for root length at various salt stress levels. The plants grown under the influence of canal water produced longer roots (13.3 cm). The root length declined to 11.4 cm and 9.2 cm when the chillies were sown in media of 3.0 and 5.0 dS m -1 , respectively; while the lowest root length (8.1 cm) was recorded under highest salt stress level. The root length was negatively influenced by increased salinity level; and varieties also had significant difference for this trait under various salinity levels. Among genotypes, the root length was higher in chilli cultivars Sky lane-4 (11.3 cm) as compared to Ghotki (11.1 cm); while Pusa Jawala showed least root length (10.7 cm). The salt stress levels of 1dSm -1 and control showed nonsignificant (P>0.05) response for root length. Root length is one of the important parameters that largely determines the success of the plants under diverse environmental conditions especially salt stress growth conditions. In the present investigation the highest salt concentration showed poor growth of the roots largely due to ion toxicity if Na and chloride salts. This might have created hinderances in normal metabolic processes of plants consequently plants with poor growth of the roots were produced. [30], have also described the highly adverse effect of salinity on canola cultivars.

Fresh root biomass (g)
The data shown in (Table 7), disclose the salinity influence on fresh root biomass of various genotypes of chilli as compared to control. The ANOVA exhibited that the increasing salt stress had detrimental effect on fresh root biomass; and chilli genotypes also showed significantly different response to salinity levels for fresh root biomass. The results indicated that among treatments (salinity levels), genotypes sown in media of 1.0 dS m -1 showed maximum root biomass (0.6 g) against 0.7 g fresh root biomass in control. The fresh root biomass decreased to 0.5 g and 0.4 g when the salt stress levels increased to 3.0 and 5.0 dS m -1 , respectively. The minimum fresh root biomass (0.4 g) was recorded under highest salt stress of 7 dSm -1 . The fresh root biomass was adversely influenced by increasing salinity level and genotypes also responded differently to salinity. Among varieties, the fresh root biomass was higher in chilli cultivars Sky lane-4 (0.6 g) as compared to Ghotki (0.5); while Pusa Jawala showed lowest root biomass. For root biomass, the differences were not-significant between control and 1.0 dS m -1 . These results refelected that plant mechanism regarding to salt stress is not well developed at early stages of chilli plants [31]. It has been well documented that salt stress causes adverse effect on uptake of vital nutrients elements such as phosphorus (P) and potassium (K) that could lead to poor growth of young seedlings [32].

Electrolyte leakage of leaf (%)
The Data in regards to electrolyte leakage in different genotypes of chilli as affected by salinity levels are given in ( in Pusa Jawala × 7.0 dS m -1 ; while minimum electrolyte leakage of 40.8% was determined in interaction of Sky lane-4 × 1.0 dS m -1 . The differences were insignificant statistically (P>0.05) in electrolyte leakage between Pusa Jawala and Ghotki. In the current study, the highest electrolyte leakage of the leaf at elevated salinity was possibly due to the inability of the leaf to reorganize cellular membranes promptly and efficiently. Moreover, plants failed to avoid dehydration of water that showed more electrolyte leakage of leaf in the water [33, 34], mentioned that increasing salinity level not only adversely affects the agronomical performance of the crop, but the physiological aspects are also influenced simultaneously in negative direction.

Conclusion
It was concluded that increase in salinity showed severe adverse effects on the seed germination and early seedling growth of chilli genotypes. The effect on germination and seedling growth was negligible upto 1.0 dS m -1 salinity. Moreover, the threshold level for chilliies was recorded 1.0 dS m -1 in the current study. The increasing salt stress beyond this level showed severe adverse effect on germination and seedling growth. Among chilli varieties, Sky lane 4 and Ghotki showed tolerance to salinity; while Pusa Jawala did not prove its tolerance to salinity.