Effect of Water Stress on Growth and Root Characteristics of Lentil

Lentil (Lens culinaris Medik) is an important “rabi” pulse crop in India which grows approximately 27% of the world’s annual production (FAO, 2010). Lentil plays a major role in the food and nutritional security of millions, particularly among low-income Asian families, because of high levels of protein, minerals and vitamins of their seed. The inclusion of lentil as a crop rotation benefits succeeding crops by improving soil fertility through biological nitrogen fixation and other rotational effects. Lentil predominantly grows under rainfed conditions and on residual soil moisture. In India national average seed yield of 619 kg ha^-1 which is much lower than the world average yield of 915 kg ha^-1 (FAO, 2010). The major constraints of this poor yield of lentil are cultivation of low yield potential cultivars and water scarcity. According to Saxena (2009), farmers usually sow lentil after rice (from late November to early December) and so it complies to complete their reproductive phase under soil moisture deficit conditions through exposing to mid-term or terminal drought stress. This leads to abortion of flowers and young pods and prevents seed filling and therefore, limiting economic yield (Das et al., 2014).
       
Additionally, the extent of yield reduction depends not only on the severity of the stress, but also on the stage of the plant development (Claasen and Shaw, 1970). Application of supplemental irrigation or moisture conservation technologies could alleviate mid-term or terminal drought in rainfed pulse cultivation (Bandyopadhyay et al., 2018). Keeping these in view, an attempt has been made to grow some promising varieties of lentil under four moisture regimes with the following objectives: (i) to investigate the effect of water stress on the physiology of four lentil cultivars; (ii) to study the roots under different moisture regimes and (iii) to evaluate the interaction between growth parameters and water stress.

Soil characterization and experimental setup
 
A pot experiment was carried out at the farm of Bidhan Chandra Krishi Viswavidyalaya, Nadia, West Bengal, India (22.99°N and 88.43°E), from 10 November 2018 to 15 February 2019 using  surface (0-0.15 m) soil of Aeric Haplaquept (Soil Survey Staff, 2006) (Table 1), collected from rice field. Two kg of soil were added to each 3 L pot, replicated thrice and the recommended dose of fertilizers for lentil crop was added to each pot. The four lentil varieties, namely Asha (V1), PL 06 (V2), Subrata (V3) and Ranjan (V4) were used. Lentil plants were provided with sprinkle water up to 20 days for proper establishment after which four different water regimes viz. 100 (I1), 75 (I2), 50 (I3) and 25% (I4) of 0.6 IW:CPE were maintained.
 

 
Chlorophyll content
 
Total chlorophyll as well as chlorophyll a and b concentrations (g fresh weight l^-1 ) were estimated at 35-40 (vegetative stage), 55-60 (initiation of flowering) and 75-85 days (pod formation) after the onset of the experiment following the method of Arnon (1949) using a Vis-spectrophotometer (Systronics, Hyderabad, India. Model-104).
 
Leaf area
 
The green leaves were separated and the area of it was measured in mm graph paper and the average was calculated. Total leaf area per plant was calculated by multiplying the leaf area with number of leaves per plant.
 
Relative leaf water content
 
Relative leaf water content (RLWC) was determined according to Barr and Weatherley (1962) using fresh, turgid and dry leaves as:
   
Root parameters
 
Root samplings were taken at the time of harvest by removing the soil from each pot. The roots with soil were soaked in water overnight and washed under gentle flow of water. The roots were then blotted by tissue paper and placed in a image analysis root scanner, “WinRHIZO”, fitted with computer to measure root length density, root volume, root diameter and root surface area (Himmelbauer et al., 2004).
 

 
Shoot dry biomass and seed yield
 
One plant from each pot in each replication was collected by cutting the whole plant at base portion at vegetative, flower initiation and pod formation periods. For dry biomass (g plant^-1), the collected plant samples were oven dried at 60°C for more than 48 h till constant weight. For seed yield the ripened pods were separated from plants and seeds were taken out by hand from pods and weighted (g plant^-1).
 
Statistical analysis
 
The experiment was done following a completely randomized design. All data obtained were subjected to a one-way ANOVA and the differences were compared by least significant difference (LSD) test. Comparisons of mean with P values <0.05 were considered significantly different.

Relative leaf water content
 
Relative leaf water content (RLWC) denotes the water status of plant decreased with the advancement of growth stages in all varieties. It maintained 54.3-89.2% at peak vegetative (40-45 DAS), 10-82.6% at flowering (60-65 DAS) and 0-66.1% during pod formation periods (80-85 DAS) (Table 2). The response of the cultivars for RLWC to different water treatments were observed different. Under I1 treatment, variety Asha showed more turgidity up to flowering period (82.6%) but variety Subrata got the maximum RLWC (69.1%) at pod formation. From peak vegetative stage, stress plots affected that signature with less RLWC and got severity during pod formation stage. Similar result was found by Bandyopadhyay et al., (2018). In mild stress condition (I2) Asha regulated its RLWC at 71.8% followed by Subrata (64.6%) and Ranjan (57.3%). But in stress and severe stress conditions, PL6 and Ranjan were unable to retain their turgidity to satisfactory level during flowering stage which resulted in dry out of plants during pod maturity stage. However, Asha and Subrata showed satisfactory RLWC of 61.8% and 41.4% at flowering and pod maturity periods under stress condition (I3). During severe stress (I4), only cultivar Asha somehow was able to retain its turgidity to 32.7% up to pod maturity period.
 

 
Chlorophyll content
 
Chlorophyll content, an important indicator of metabolic activity and sign of drought stress, was measured during peak vegetative, flowering and pod maturity periods. Generally, chlorophyll content of leaf increased from vegetative to flowering period and dropped towards pod maturity (Table 3). With the increase in stress intensity, chlorophyll content decreased. For no stress condition, Subrata got the maximum chlorophyll content during peak vegetative (1.83 g l^-1) and flowering stages (2.82 g l^-1). Asha experienced the maximum value for mild to severe stress situation during vegetative, flowering and pod formation periods. Severe stress affected PL 6 the most with 0.065 g l^-1 during vegetative to 0.101 g l^-1 at flowering period. In stress treatment Subrata and Asha stood well by showing 0.705 and 1.31 g l^-1 chlorophyll content, during flowering stage. In a nutshell, with increasing stress only Asha proved to produce chlorophyll content enough to withstand water scarcity than other varieties.
 

 
Leaf area
 
The result showed that under no stress condition leaf area (LA) was recorded higher for Ranjan and the least for PL 6 during peak vegetative stage. With the increase in the intensity of water scarcity, LA decreased rapidly for all the cultivars except Asha (Table 4). Drought stress caused little change in LA with the advancement of growth. The water stress significantly decreased the leaf area and the magnitude of reduction increased with the increase in soil moisture stress. Fig 1 shows a linear relationship between RLWC and LA indicating that the drought could reduce leaf water potential, rate of cell division and enlargement due to loss of turgor (Hussain et al., 2009). Under no stress treatment, maximum increase in LA from peak vegetative to flowering period was observed in Subrata (44.9%) followed by Asha (40.8 %). Under mild stress condition, Asha showed maximum LA (66%) whereas, PL6 resulted the highest under severe stress condition.
 

 



 
Root length density (RLD)
 
Root length density, an important parameter is closely related with soil moisture absorption (Krishnamurthy et al., 1999). Under different moisture regimes, RLD values of four varieties are presented in Fig 2. In general, RLD decreased with the increase in water scarcity for all the cultivars and ranged from 0.23 to 0.33, 0.10 to 0.16 and 0.07 to 0.11 cm cm^-3 under no stress, stress and severe stress conditions, respectively. From I1 to I2, the maximum rate of decrease in RLD was observed under PL 6, however,  the highest decrease of 63% from I2 to I3 was noticed under Asha variety. The highest RLD was observed in I1 for Asha (0.33 cm cm^-3) and the lowest in I4 under Subrata (0.07 cm cm^-3). An asymptotic relationship between the RLD and RLWC of lentil crop (Fig 3) indicating that plants can tolerate drought stress, because of expanded rooting system and better water uptake. In an experiment with chickpea, Lokhande et al., (2019) also found that the drought tolerant cultivars which retained higher root length and biomass, exploited the reduced water levels in a more efficient manner.
 

 

 
Root surface area and root volume
 
Root surface area and root volume are the water absorption sites, important for water stress condition (Hsiao and Xu, 2000). In this study, root surface area and root volume followed the same trend where I1 showed maximum values, followed by I2, I3 and I4, respectively (Fig 4 and 5). With the severity of water stress both root surface area and root volume were decreased and the rate of decrease was observed the highest from I2 to I3. Asha and Subrata produced better root volume and surface area than other varieties. In I1 regime, Asha and Subrata showed the highest whereas Ranjan being the least for both the parameters.
 

 

 
Average root diameter
 
Root diameter determines the strength of penetration through soil and influences net ion influx. Under no stress treatment average root diameter were maximum for all the varieties (Fig 6) with the highest with Subrata (0.68 mm). Plants with higher root diameter have more growth potential as it has direct relation with water absorption (Bao et al., 2014).  From I1 to I2 and I2 to I3 root diameter decreased in case of Asha, Subrata and Ranjan. An increase of average root diameter from I3 to I4 for all the varieties except Subrata was observed and this may be due to more pressure needed to penetrate by enlarging root diameter. Ranjan (0.64 mm) and PL 6 (0.10 mm) were noticed to have maximum average root diameter among other lentil varieties for I2 and I3, respectively. In severe stress condition the highest value of average root diameter was observed under Ranjan (0.08 mm).
 

It can be concluded that under drought stress condition shoot and root growth were significantly affected. Physiological traits like relative leaf water content, leaf chlorophyll content and leaf area were affected most during pod formation stage and were severe under PL 06 and Ranjan varieties. Root growth decreased significantly with increase in stress and was more sensitive than shoot biomass. Root diameter was increased under maximum water stress condition. Asha was found the best survival under sever water stress. The grain yield was controlled by stress and variety Subrata and Asha produced the highest yield.