Foliar application of urea and potassium chloride minimizes terminal moisture stress in lentil (Lens culinaris L.) crop

Experimental site and conditions
 
A field experiment was conducted at the Research Farm facilities of ICAR Research Complex for North Eastern Hill (NEH) Region, Tripura Centre, Lembucherra, Tripura (W), India, during winter seasons in the year 2017-18. The annual rainfall of the study site was 2200 mm. The soil of the experimental site was sandy loam type, acidic in reaction [pH (1.25-5.2)] and the baseline soil sample had 5.6 g kg^-1 soil organic carbon (SOC), 251.1 kg ha^-1 available nitrogen (N), 20.2 kg ha^-1 available phosphorus (P) and 170.1 kg ha^-1 available potassium (K).
 
Experimental design and crop management
 
The experiment trial was designed to test three levels of foliar spray of agro-chemicals (Urea and KCl) @ 2% either individually or in combination at three distinct application stages viz. 1) 50% flowering, 2) 50% podding and 3) at both 50% flowering + 50% podding compared to a control (no spray).  The experiment was laid out in a factorial completely randomized block design with three replications. Lentil var. Tripura Selection 1 was sown at 25 cm spacing under reduced tillage, two time power tilling only, condition in a gross plot size of 9.8 m × 2.3 m. A starter dose of 20 kg ha^-1 N, 27 kg ha^-1 P and 33 kg ha^-1 K were applied in furrows before sowing the seeds. The crop was raised with residual soil moisture and lifesaving irrigation was provided at the flowering stage for better growth.
 
Chlorophyll content
 
Leaf chlorophyll content was estimated by the method of Arnon (1949). Fresh leaf sample was finely cut and mixed well; 1 g of tissue was weighed and homogenized with the addition of 20 ml of 80 % acetone. The solution was centrifuged at 5000 r.p.m for 5 minutes and the supernatant was made upto 100 ml with 80 % acetone. The absorbance of the supernatant was recorded at 645 and 663 nm against the solvent (80 % acetone) blank. Chlorophyll content in the extract was calculated using the formulae:
Where,
A = Absorbance at specific wavelength.
V = Final volume of chlorophyll extract in 80 % acetone.
W = Fresh weight of tissue extracted.
 
Leaf relative water content (LRWC)
 
Data related to Leaf relative water content (LRWC) was recorded at 50% flowering and 50% pod formation stages separately. The third leaves from top (fully expanded youngest leaf) of two plants from each treatment were used to determine the LRWC. Immediately after sampling, leaves were sealed within a plastic bag and processed in the laboratory. Fresh weight (FW) was determined within two hours after leaves excision. Then turgid weight (TW) was obtained after soaking leaves in distilled water for 16-18 hr at room temperature. After soaking, leaves were blotted dry using tissue paper to calculate the TW. The samples were dried in an oven for about 72 hours at 65°C and measured to a constant weight. LRWC was calculated using the formula given by Lazcano-Ferrat and Lovatt (1999):  
Excised leaf water loss (ELWL)
 
Data for ELWL was recorded at both flowering and podding stages separately. The third leaf from top (fully expanded youngest leaf) of two plants for each treatment was used to determine ELWL. The leaves were placed in polythene bags and transported to the laboratory as quickly as possible in order to minimize water losses by evaporation. The leaves were weighed at three stages, immediately after sampling (fresh weight: FW), after placing at room temperature for 6 h (wilted weight: WW) and after drying in an oven for 24 h at 65°C and (dry weight: DW). ELWL was calculated by using formula given by Clarke (1987):
   
Plant sampling and yield measurement
 
The growth parameters (plant height, primary and secondary branches), yield attributes (pods/plant and seeds/pod) and seed yield of lentil were recorded at harvest. Number of pods were taken by counting the total number of pods of five tagged plants in each plot and then averaged was calculated. Number of seeds were taken by counting the total number of seed per pod of each of five tagged plants and then averaged was recorded. For biological yield, 4 square meter crop area was harvested from each plot. Then, crops were sun dried and biological yield was converted into kg per hectare. From 4 square meter crop area, seed yield was obtained and expressed into kg per hectare. The yield of lentil was estimated from the weight of sun-dried seeds (12% moisture content) obtained from each plot after threshing and cleaning.
 
Statistical analysis
 
The experimental data pertaining to each parameter of the study were subjected to statistical analysis by using the technique of analysis of variance and their significance was tested by “F” test (Gomez and Gomez, 1984). The standard error of means (SEm+) and least significant difference (LSD) at 5% probability were worked out to evaluate the differences between treatment means for each parameter studied.

A significant effect (p<0.05) of foliar spray application of agro-chemical on the measured physiological parameters viz. chlorophyll a, chlorophyll b and total chlorophyll content, relative water content and excised leaf water loss (ELWL) and yield, as well yield attributes was observed in the present study (Table 1, 2). Compared to the control, significant (p<0.05) increases in chlorophyll a, chlorophyll b and total chlorophyll content were noticed in leaves of lentil plant received either singular or combined application of 2% urea and 2% KCl suggesting the potential of agrochemicals in reducing the extent of chlorophyll loss under low moisture. In the present study, significant increase (p <0.05) in mean values of chlorophyll a (6.7 µg g^-1 fw, 10.3 µg g^-1 fw), chlorophyll b (5.74 µg g^-1fw, 2.94 µg g^-1fw) and total chlorophyll content (12.44 µg g^-1 fw , 13.24 µg g^-1 fw) at 50% flowering and 50% podding, respectively, were observed in leaves of treated plants either 2% KCl alone or in combination with 2% urea as compared to control and 2% urea. Earlier reports are also indicative of an efficient nutrient uptake and utilization under soil moisture stress in crops through application of nitrogen and potassium fertilizer (Waraich et al., 2011; Ge et al., 2012). Additional supply of key nutrients as either 2% KCl alone or in combination with 2% urea might have played an ameliorative function in this study. Interestingly, the effect was more pronounced in case of 2% KCl compared 2% urea, indicating its effectiveness in managing soil moisture stress. Further, 2% KCl had no significant difference (p < 0.05) with its combined application, suggesting that KCl application singularly will be an economically viable option in effectively controlling such stressor. Nitrogen supplied through urea contributes to the structural organization of chlorophyll, whereas potassium in KCl functions as an activator or coenzyme in chlorophyll biosynthesis (Abdel Mohatgally, 2014). Foliar application has already been proven as an effective means for improving nutrient uptake as well as its utilization (Fageria et al., 2009). Thus, foliar nutrition with either KCl or urea alone or in combination might have potentiated nutrient balance in our study, thereby, minimizing the stress effect. Decline in chemical activity of water and a loss of turgor in plant cells is often encountered as a result of drought induced osmotic stress (Zhang et al., 2013; Yýldýztugayet_al2014). These changes, may further lead to impairment of nitrate assimilation due to reduced activity of nitrate reductase (NRA) enzyme (Zhang et al., 2009). These, ultimately leads to reduction in plant growth and/or plant death as well as reduced LRWC, which is being recognized as an effective indicator of plant water status (Zhang et al., 2009). In this study, LRWC and ELWL were found to be the highest (83%) and the lowest (0.7%), respectively (Fig 1 and 3), in case of combined application as compared to other two treatments, although the values were comparable and statistically non-significant between 2% KCl alone or in combination with 2% urea. This observation indicates the effectiveness of the used agrochemicals in improving the water status of lentil crop. Positive response to several agro-chemicals are also reported in crops like oilseeds (Fanaei et al., 2009), mung bean (Nandwal et al., 1998), maize (Premachandra et al., 1991) and wheat (Pier and Berkowitz, 1987) under drought stress. We further observed higher LRWC under soil moisture deficit conditions using KCl application than the control (Fig 1). It is known that application of K contributes to improved cell turgor through osmotic adjustment (Maathuis and Sanders, 1996). Improvement in relative water contents, cell membrane stability, water use efficiency, leaf area index, chlorophyll contents, grain protein contents, plant height, number of primary branches, number of secondary branches, pods per plant, grains yield were also reported in legumes (Namvar et al., 2013) through application of urea.
 

 

 

 

 
Drought stress causes drastic reduction in leaf chlorophyll content, affecting the photosynthetic efficiency of plants, reducing dry matter accumulation and poor grain yield (Wang et al., 2018). In this study, a significant increase (p < 0.05) in plant height, number of branches per plant, number of pods per plant and biomass as well as seed yield with agrochemical application as compared to control was observed. This might be related to enhanced chlorophyll content on account of foliar application of the agrochemical; thus, enabling higher photosynthesis under stress conditions (Arabzadeh, 2013). Remarkable increase in plant height (37.7 cm), number of branches per plant (10.5), number of pods per plant (68.2), biomass yield (2439 kg ha^-1) and seed yield (995 kg ha^-1) were observed through combined application, followed by singular application of 2% KCl and 2% urea (Table 2). This was in agreement with many earlier studies in chickpea (Parimala et al., 2013), mungbean (Beg and Ahmad, 2012, Majeed et al., 2016), lentil (Nagraju, 2017) and cowpea (Choudhary and Yadav, 2011), wherein foliar application of KCl reportedly enhanced growth characteristics and seed yield. This may be attributed to the role of potassium in drought adaptations (Sardans et al., 2012, Grzebisz et al., 2013) linked to various biological processes such as cell osmoregulation, which helps in maintaining the cell turgor and expansion required for promoting root and shoot growth and regulate stomatal opening, thereby optimizing water-use efficiency (Egilla et al., 2005; Kanai et al., 2011, Majeed et al., 2016). Besides, potassium helps in maintaining the inner membrane of chloroplast and proton gradient of thylakoid membranes, which promote photosynthetic phosphorylation which is essential for sustaining photosynthesis during stressful condition, leading to higher dry matter production and seed yield (Hermanset_al2006; Yurtseven et al., 2005). Drought mitigation through foliar application of urea is reported in various crops (Asadullah et al., 2017; Gou et al., 2017). Nutrient uptake is highly reduced under water stress condition; therefore, drought imposed nutrient deficiency becomes severe during terminal growth stages (Gunes et al., 2006). Foliar application of nitrogen fertilizer, supplemented the nitrogen uptake deficiency which resulted the improvement of lentil yield performance under limited availability. This is due to the fact that nitrogen is vitally involved in various physiological processes such as protein biosynthesis, nucleic acid linked processes, protoplast formation, chlorophyll synthesis, leaf area, cell size and photosynthetic activity (Dordas and Sioulas, 2008; Waraich et al., 2011). Except for number of pods per plant and number of seeds per pod as well, no difference (p < 0.05) was observed between yield attributes and yield w.r.t 2% KCl and combined application of 2% urea + 2% KCl.

Time of foliar spray application significantly affected the chlorophyll content, leaf relative water content and excised leaf water loss (Fig 2 and 4). It was observed that foliar spray at both 50% flowering + 50% podding stage exhibited better response than either at 50% flowering or at 50% podding stage. The highest chlorophyll a, chlorophyll b and total chlorophyll content were recorded at 75 days after sowing: DAS (7.1 µg g^-1 fw, 6.16 µg g^-1 fw and 13.26 µg g^-1 fw). Similarly, at 90 DAS, maximum treatment response was observed at both 50% flowering + 50% podding with highest chlorophyll a (10.8 µg g^-1 fw), chlorophyll b (2.97 µg g^-1 fw) and total chlorophyll content (13.77 µg g^-1 fw). The highest LRWC and the lowest ELWL were recorded with a percentage value of 86% and 0.5%, respectively, in case of foliar spray at both 50% flowering + 50% podding as compared to application of foliar spray at flowering and podding alone. Timing of foliar application also significantly affects the yield attributes and yield of lentil. Foliar spray at both 50% flowering + 50% podding resulted a maximum increase in plant height (40.1 cm), number of branches per plant (12.2), number of pods per plant (74.9), number of seeds per pod (2), biomass yield (3617 kg ha^-1) and seed yield (1231 kg ha^-1) followed by application at both 50% flowering + 50% podding stage. Our result corresponds the findings of Palta et al., (2005) and Zeidan (2003) wherein foliar application of urea at 50% flowering resulted in increase of yield and seed protein. Foliar spray of nitrogen has been found to delay leaf senescence and improves yield (Das and Jana, 2015). Foliar application of nutrients at flowering and podding stage allows adequate nutrient supplementation essential for enhancing the number of floral buds and preventing floral shedding by maintaining optimum bio-physiological conditions in plants (Maheswari and Karthik, 2017).
 

 

Foliar application of common agro-chemicals to lentil crop proved to have vast potential in bringing the underutilized rice-fallow lands elsewhere for successful cultivation of rabi crops by physiologically mitigating the repercussions of terminal moisture stresses on crops. Besides, the foliar spray of 2% KCl at both 50% flowering + 50% podding stages of lentil crop was found to be effective in sustaining the seed yield under rice-fallow condition.

The authors are grateful and would like to thank the Director, ICAR Research Complex for NEH Region, Umiam, Meghalaya, India for extending all support in conducting the present work.