Impact of post-emergent imazethapyr on morpho-physiological and biochemical responses in lentil (Lens culinaris Medik.)

Abstract

Yield reduction in lentil crop due to weed infestation is a key hindrance to its growth due to poor weed-crop competition. Imazethapyr (IM), a selective herbicide, target acetolactate synthase (ALS) which catalyzes the first reaction in biosynthesis of branched chain amino acids, required for plant growth and development. The objective of the present study was to investigate the impact of IM treatment on weeds, ALS enzyme activity, antioxidant capacity, osmolyte accumulation, growth and yield related parameters in lentil genotypes. Two IM tolerant (LL1397 and LL1612) and two susceptible (FLIP2004-7L and PL07) lentil genotypes were cultivated under weed free, weedy check and IM treatments. Weed control efficiency reached its peak at 21 days after spray (DAS). Imazethapyr treatment decreased chlorophyll and carotenoid content up to 28 DAS with higher reduction in susceptible genotypes. FLIP2004-7L and PL07 had reduced plant height and lower number of pods under IM treatment which resulted in decreased seed yield. Higher ALS activity in LL1397 and LL1612 at 21 DAS, higher antioxidant capacity and glycine betaine content both at 21 and 28 DAS and lower decrease in relative leaf water content might be mediating herbicide tolerance in these genotypes that led to higher seed yield. The identified IM tolerance mechanism can be used to impart herbicide resistance in lentil.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12298-022-01244-x.

Introduction

Lentil (Lens culinaris Medik.), a fast growing rabi legume crop, provides high-quality protein, carbohydrates, fibres, vitamins and micronutrients (Khazaei et al. 2019). Lentil is estimated to be harvested in total area of 1.35 million hectares in India, with production of 1.18 million tonnes and yield of 871.5 kg/ha (FAO 2020). Lentil productivity in India accounts for about 18% of global productivity (6.54 million tonnes).

Short statured lentil having slower early development experiences huge incursion of weeds during the crop season that cause damage to crop resulting in yield reduction (Jaswal and Menon 2020). Depending upon environmental conditions, weed density and diversity, the yield losses in lentil vary from 20 to 80% (Balech et al. 2022). Management of weeds at an appropriate time is important to increase the productivity of lentil. The most prevalent weeds in the field such as grassy and broadleaf compete with lentil plants for light, nutrients and other resources. Poor competitivity of lentil toward weeds limits the crop yield (Jeet et al. 2020). Crop rotations, proper sowing, cover crops and variety selection are used to reduce the weed biomass and increase lentil yield. However, these measures are insufficient for effective weed management (Pala et al. 2018). Mechanical weeding, such as hand weeding is used to manage weeds in the field, but it is time-consuming, difficult to use in large production areas and expansive due to high cost of labour (Pala et al. 2019).

Herbicide treatment still appears to be the most effective, fast and economical method for weed control (Singh and Singh 2017). Pre-emergence herbicides such as pendimethalin are recommended for weed management in lentil fields but they show their effect only for initial period. Because lentil is long duration crop and weeds that emerge late and compete with the crop so the use of post-emergence herbicides is recommended to control prominent weeds such as broadleaf, grassy weeds and perennial grasses (Singh et al. 2014). Imazethapyr (IM) has been recommended for pulse crops to control wide spectrum of weeds (Duary et al. 2016). IM, a post-emergence herbicide from the imidazolinones family, has very low human toxicity and is environmental friendly. It selectively controls a variety of weed species at low application rates (Presotto et al. 2012). IM treatment effectively minimize the weed competition by reducing weed density and increasing yield by 62% in IM treated blackgram (Aggarwal et al. 2014).

Imazethapyr affects the plant growth primarily by inhibiting the plant enzyme acetolactate synthase (ALS) which catalyse the first reaction in the biosynthetic pathway of branched chain amino acids (BCAAs): valine (Val), leucine (Leu) and isoleucine (Ile) (Qian et al. 2015). Val and Leu are synthesized in two parallel pathways involving four enzymes that use different substrates (Binder 2010). These four enzymes are: acetolactate synthase (ALS), also called as acetohydroxyacid synthase (AHAS, EC 4.1.3.18) ketol acid reductoisomerase (KARI, EC 1.1.1.86), dihydroxyacid dehydratase (DHAD, EC 4.2.1.9) and branched-chain aminotransferase (BCAT, EC 2.6.1.42). Threonine deaminase (TD, EC 4.2.1.16) catalyzes the deamination and dehydratation of threonine resulting in the formation of ammonia and 2-oxobutyrate (starting substrate for Ile synthesis), the latter along with pyruvate are the substrates for ALS. Acetolactate synthase (ALS) catalyse the first step in the parallel pathways in biosynthesis of Val and Leu and towards Ile. ALS catalyzes the conversion of two molecules of pyruvate into 2-acetolactate (Val and Leu) and from one molecule of pyruvate and of 2-oxobutarate into 2-aceto-2-hydroxybutyrate (Ile), using thiamine pyrophosphate (TPP), flavin adenine dinucleotide (FAD) and Mg²⁺ as cofactors. In last step, BCAAs are formed by transferring amino group to ketoacid in a transamination reaction, 3-methyl 2-oxobutanoate, the last intermediate that is transaminated to Val, lead to the synthesis of Leu (Supplementary Fig. 1).

Imazethapyr inhibits ALS, disrupts protein synthesis, inhibits cell division, and accumulates in meristematic regions of the plant after foliar treatment. These effects ultimately result in decreased plant growth (Rad and Aivazi 2020). The plant is damaged by the herbicide within 7 to 20 days of spraying due to IM's persistence in the soil, which affects both the sprayed weeds and newly emerging weeds (Sultani et al. 2019).

Nitrogen metabolism is affected by ALS inhibition, de novo protein synthesis is reduced as a result of transitory lack of BCAAs, which increased the amino acid pool (Zabalza et al. 2006). IM treatment reduced photosynthesis resulted in decreased stomatal conductance, reduced transpiration via the xylem, which suppressed N uptake and, as a result, decreased nitrogen content (Orcaray et al. 2010).

Chloroplasts are the main sites of photosynthesis. Imazethapyr treatment inhibited plant growth, damaged leaf cell structures especially chloroplast and reduced photosynthetic efficiency in Arabidopsis thaliana (Liu et al. 2019). Carotenoid is a non enzyme antioxidant that quenches singlet oxygen and protects chlorophyll from lipid peroxidation induced membrane damage. Chloroplast is the site of accumulation of reactive oxygen species (ROS) under stress conditions which are signalling molecule at low concentration and cause of oxidative stress at higher concentration (Lokdarshi et al. 2022). The tolerance mechanism involves scavenging of these ROS. One of the tolerance mechanisms involves accumulation of osmolytes such as glycine betaine, proline, soluble sugars and polyamines which help in maintaining cell turgor pressure, reducing reactive oxygen species and preventing cellular damage under oxidative stress (Pattnaik et al. 2021). Glycine betaine is metabolically more stable than any other osmoprotectant, such as proline or sugars (Jain et al. 2021). It is produced in chloroplast as a non enzyme antioxidant, helps in maintaining photosynthetic ability and proper folding of proteins by acting as a chaperone under stress conditions (Hasanuzzaman et al. 2019; Kurepin et al. 2015). Antioxidant capacity in leaves measured by it’s ability to scavenge 2, 2-diphenyl-1-picryl hydrazyl (DPPH) free radical also reduce ROS under stress conditions (Mei et al. 2020).

Herbicide-tolerant crops make weed control easier, more effective, and less expensive with minimal crop damage. Identification of herbicide tolerance mechanism in lentil will be of potential use for developing herbicide tolerant lentil varieties. The present study has been executed to evaluate the effect of imazethapyr treatment on physiological and biochemical responses that impart herbicide tolerance in lentil genotypes.

Material and methods

A field experiment was conducted during rabi season of 2019–2020 and 2020–2021 at the Research Farm of Pulse Section, Department of Plant Breeding and Genetics, Punjab Agricultural University Ludhiana (30° 54′ N latitude and 75° 48′ E longitude, altitude 247 m above the mean sea level), India. The soil of the experimental site was loamy sand in texture with low organic carbon (0.31%) and available nitrogen (111.6 kg ha⁻¹) and medium in available phosphorus (14.1 kg ha⁻¹) and potassium (164.4 kg ha⁻¹). The fertilizers mixed in soil during experimental period were N = 12.5 kg ha⁻¹ and P = 40 kg P₂O₅ ha⁻¹ applied through urea and single superphosphate, respectively. Total rainfall received during the crop-growing season in 2019–2020 and 2020–2021 was 170.1 and 44.2 mm, respectively. The weekly mean minimum and maximum air temperature ranged from 4.9 to 18.4 °C and 10.3 to 35.5 °C in 2019–2020 and from 3.5 to 15.7 °C and 14.0 to 32.6 °C in 2020–2021, respectively. Four lentil genotypes [Two herbicide-tolerant (LL1612 and LL1397) and two herbicide-susceptible (FLIP 2004_7L and PL07)] were sown under three weed control treatments; weed free (T1) by using hand weeding, weedy check (T2) in which weeds are intact and sprayed (T3) with Imazethapyr 10 SL @ 75 g a.i. ha⁻¹ (750 mL/hectare). The estimated cost of spraying imazethapyr was found to be Rs. 1500–1600 ha⁻¹. The experiment was laid in factorial RBD design with three replications. LL1397 and LL1612 are advanced breeding lines from PAU, Ludhiana. FLIP2004-7L is a rust resistant advanced breeding line from ICARDA and PL07 is a released variety from GBPUAT, Pantnagar. The samples from T1, T2 and T3 were analysed at different days after spray (DAS).

Estimation of acetolactate synthase activity

Fresh young upper leaf samples (200 mg) were extracted in ice cold 0.1 M potassium phosphate buffer (pH-7.5) containing 0.5 mM MgCl₂, 10 μM FAD, 1 mM sodium pyruvate, 0.5 mM TPP and 10% v/v glycerol and then centrifuged at 25,000 rpm for 25 min. ALS activity was measured by using modified method of Ray (1984). The reaction mixture consisting of 20 mM potassium phosphate buffer (pH-7.0) containing 1 mM sodium pyruvate, 0.5 mM MgCl₂, 0.5 mM TPP and 10 μM FAD. The reaction was initiated by adding an enzyme extract (0.2 mL) followed by incubation of reaction mixture at 30 °C for 60 min under dark condition. ALS catalysis was terminated with the addition of 6 N H₂SO₄. The reaction mixture was then incubated at 60 °C for 15 min to convert acetolactate to acetoin. The acetoin formed was quantified by dark incubation with 0.5 mL of creatinine and 0.5 mL of α-naphthol in 2.5 N NaOH for 30 min at 60 °C. The absorbance was recorded at 525 nm. The protein content was estimated by the method of Lowry et al. (1951).

Estimation of photosynthetic pigments (Hiscox and Israelstam 1979)

Leaf samples (50 mg) were dipped in DMSO for the extraction of photosynthetic pigments, incubated at 60 °C in water bath for 2 h and then cooled to room temperature. The absorbance was read at 480, 645 and 665 nm and photosynthetic pigments were calculated by the following equation:

$$\text{Total}\text{chlorophyll}\left(\text{mg}/\text{mL}\right)=20.2{\text{A}}_{645}+8.02{\text{A}}_{665}$$

$$\text{Carotenoid}\text{content}\left(\text{mg}/\text{mL}\right)={\text{A}}_{480}+0.114{\text{A}}_{665}-0.638{\text{A}}_{645}$$

Estimation of nitrogen and protein content

Dried leaf samples were ground to fine powder. Powdered samples (200 mg) were digested using 3 g of potassium sulphate and 0.5 g of copper sulphate with 10 mL of conc. H₂SO₄ on a digestor (Pelican KEL-PLUS KES 20L VA DLS TS) followed by distillation with 40% NaOH and 4% boric acid on distillation assembly (KELPLUS CLASSIC-DX VATS (E)). The titration was carried out with 0.1 N HCl by using titrator (Metrohm 877 Titrino plus). Nitrogen (%) content was calculated and converted to protein (%) content.

Estimation of weed control efficiency and weed index

Weeds collected from the base near the surface of soil in both weedy check and weed free fields, were oven dried at 60 °C for 72 h till their constant dry weight was achieved. Weed-control efficiency (WCE) was measured (Mani et al. 1973) as:

$$\text{WCE}\left(\%\right)=\frac{\text{Dry weight of weeds in weedy check}-\text{Dry weight of weeds in treatment}}{\text{Dry weight of weeds in weedy check}}\times 100$$

Weed index which provides yield loss (%) due to weed infestation, is used to determine the efficiency of herbicide treatment on weeds by comparing the yield of both weed free and IM sprayed field. Weed index (WI) was measured by using the following formula (Gill and Kumar 1969):

$$\text{WI}\left(\%\right)=\frac{\text{Yield from weed free area}-\text{Yield from IM treated area}}{\text{Yield from weed free area}}\times 100$$

Estimation of relative leaf water content (RLWC)

Fresh leaf samples were weighed and immersed in distilled water in test tubes. The leaves were taken out after 6 h, wiped with the tissue paper and turgid weight was recorded. Samples were then oven dried for 48 h at 80 °C (Ali and Wasfi 2016). Relative leaf water content (RLWC) was calculated using the following formula:

$$\text{RLWC}\left(\%\right)=\frac{\text{Fresh Weight}-\text{Dry Weight}}{\text{Turgid Weight}-\text{Dry Weight}}\times 100$$

Estimation of DPPH (2, 2-diphenyl-1-picryl hydrazyl) free radical scavenging activity

Leaf tissue (400 mg) was refluxed with 80% methanol (5 mL) for 1 h, filtered and supernatant was collected. The pellet was again refluxed with 80% methanol, both the supernatants were pooled and final volume was made and used for measuring antioxidant activity in terms of DPPH free radical scavenging activity by the method of Blois (1958). The sample extract was mixed with 0.1 mM DPPH reagent and incubated in dark for 30 min. The reaction mixture that had methanol in place of sample was used as control. The discoloration of DPPH was measured against reagent blank at 517 nm.

Estimation of glycine betaine content

Leaf tissue (100 mg) was homogenized with distilled water. The homogenate was filtered through Whatman filterpaper and filtrate was used for analysis. Glycine betaine was estimated by the method of Grieve and Grattan (1983). The test tubes containing samples and 2N H₂SO₄ were placed in an ice bath for 60 min followed by addition of KI_3. The tubes were again kept in ice bath for 90 min. After that, 1, 2-dichloroethane and chilled distilled H₂O were added. The absorbance of lower organic layer was measured at 365 nm by using distilled water as a blank. The standard curve was prepared by using betaine hydrochloride (50–100 μg) as a standard.

Statistical analysis

Data was provided as mean ± SD of three independent biological replications (n = 3) in each treatment. Graphical representations were prepared by using GraphPad Prism software (version 8.4.3). Statistix 10.0 software was used to analyse data by using two-way ANOVA with Tukey HSD all pairwise comparisons test (p < 0.05) so to analyze significant differences between biochemical parameters and genotypes among different treatments at different days after spray. Correlation matrix was prepared by using GraphPad Prism software to determine Pearson's correlation between morpho-physiological parameters and ALS activity. F- values at 5% level of significance were obtained by applying two-way ANOVA with multiple comparison using GraphPad Prism software (version 8.4.3).

Results

Weed flora

The prominent weed species observed in experimental lentil field were Oenothera drumundii, Gnaphalium purpureum, Coronopus didymus (jangali halan), Cyperus rotundus (motha), Spergula arvensis, Sisymbrium irio (jangli sarson), Medicago denticulate, Fumaria parviflora (pit papara) along with Rumex dentatus (jungli palak), Malwa neglecta (button weed) and Chenopodium album (bathu). Among them, the population of Oenothera drumundii was observed as the highest and effectively controlled by the application of IM which was followed by yellowing of leaves i.e., chlorosis and ultimately necrosis.

Effect of herbicide treatment on ALS activity

The key target for IM is the ALS enzyme, and the effect of IM on ALS activity was investigated in young upper leaves for two consecutive years of rabi season (2019–2020 and 2020–2021) at 5, 7, 14, 21, and 28 DAS. ALS activity was decreased with the progress of growth in all the treatments. In 2019–2020, ALS activity was reduced from 5 to 7 DAS by 26.59%, 24.5%, 43% and 49.4%, from 7 to 14 DAS by 13.75%, 5.6%, 6.7% and 15%, from 14 to 21 DAS by 31.6%, 47.93%, 50.77% and 60% and from 21 to 28 DAS by 80.5%, 75%, 87% and 77% in LL1397, LL1612, FLIP2004-7L and PL07, respectively, as depicted in Fig. 1A.

Fig. 1.

Effect on acetolactate synthase activity in upper leaves of lentil genotypes under different treatments T1,T2 & T3, representing control, weedy check and sprayed with imazethapyr, respectively, at 5, 7, 14, 21 and 28 days after spray (DAS) during 2019–2020 (A) & 2020–2021 (B). Bars represent the mean ± SD; n = 3. Different letters above bars represent significant differences among different genotypes under different treatments at p < 0.05 by using Tukey’s HSD test (Statistix 10.0). A represents significant differences between different genotypes under control and different treatments individually, B represents significant differences of a particular genotype under three different treatments and AB represents overall interaction between genotypes and treatments at different days after spray at F (5%) value by multiple comparison analysis using two-way ANOVA (GraphPad Prism software version 8.4.3)

During 2020–2021, ALS activity was reduced from 5 to 7 DAS by 32.44%, 44.91%, 55.78% and 67.94% from 7 to 14 DAS by 26.5%, 10.86%, 48.32% and 97.4% in LL1397, LL1612, FLIP2004-7L and PL07, respectively. However, from 14 to 21 DAS, activity was decreased by 35.6%, 55.32%, 61.2% in LL1397, LL1612 and FLIP2004-7L, respectively, but activity was slightly increased in PL07 by 7% showing some recovery. ALS activity was decreased in all the genotypes from 21 to 28 DAS (Fig. 1B). It was observed that reduction in ALS activity of susceptible genotypes was significantly higher compared to tolerant genotypes after 7 DAS during two consecutive years.

Effect of IM spray on plant height, seed yield, pods per plant and plant biomass

Plant height (cm), pods per plant and seed yield per plant (kg/ha) were observed at the time of harvesting (Fig. 2A–C). Plant height of LL1397, LL1612, FLIP2004-7L and PL07 in T1 treatment was measured 30, 29.33, 25.33 and 27.67 cm, respectively. After herbicide spray, plant height was reduced significantly to 27.67, 26.33, 23.0 and 20.33 cm, respectively, in these genotypes (Fig. 2A). Application of IM, significantly decreased the number of pods per plant to 52, 55, 14 and 16 in LL1397, LL1612, FLIP2004-7L and PL07, respectively. In comparison to weed-free treatment, this represents a reduction in these genotypes of 37.3%, 36.78%, 53.74%, and 59.73%, respectively. Pods per plant was also reduced to 57, 55, 35 and 42 in LL1397, LL1612, FLIP2004-7L and PL07, respectively, under weedy check treatment due to presence of greater weed-crop competition (Fig. 2B). Weeds had a significant impact on plant height and seed yield in FLIP2004-7L due to increased weed-crop competition. Seed yield was observed to be 1420, 1387, 956 and 1126 kg/ha in LL1397, LL1612, FLIP2004-7L and PL07, respectively, under T1 treatment. Herbicide treatment (T3) reduced seed yield to 870, 790, 380 and 420 kg/ha in LL1397, LL1612, FLIP2004-7L and PL07, respectively (Fig. 2C).

Fig. 2.

Effect on various growth parameters such as plant height (A), number of pods per plant (B), seed yield (C) of both tolerant and susceptible lentil genotypes under different treatments T1, T2 & T3 indicates control, weedy check & sprayed with imazethapyr, respectively, at the time of harvesting and dry weight of weeds (D) at 14, 21and 28 DAS (days after spray under control (T1) and herbicide treatment (T3). Bars represent the mean ± SD; n = 3. Different letters above bars represent significant differences among different genotypes under different treatments at p < 0.05 by using Tukey’s HSD test (Statistix 10.0). A represents significant differences between different genotypes under control and different treatments individually, B represents significant differences of a particular genotype under three different treatments and AB represents overall interaction between genotypes and treatments at different days after spray at F (5%) value by multiple comparison analysis using two-way ANOVA (GraphPad Prism software version 8.4.3)

Effect of herbicide treatment on photosynthetic pigments

Total chlorophyll content was measured highest in all the genotypes at 14 DAS and then decreased thereafter. The decrease in chlorophyll content in LL1397, LL1612, FLIP2004-7L and PL07 at 28 DAS was 1.86, 1.68, 2.09, and 2.07-fold in T1 treatment; 2.50, 2.38, 2.48 and 2.46-fold in T2 treatment and 1.79, 1.82, 2.31, and 2.86-fold under T3 treatment, respectively (Fig. 3A). The chlorophyll content was significantly affected in susceptible genotypes after 7 DAS under herbicide treatment but non-significantly in tolerant genotypes. Similarly, carotenoid content was observed to be highest at 14 DAS and then decreased in LL1397, LL1612, FLIP2004-7L and PL07 by 1.24, 1.1, 1.13, and 1.12-fold in control, 1.32, 1.37, 1.30 and 1.24-fold due to interference of weed and by 1.32, 1.31, 1.59 and 1.72-fold after herbicide spray from 14 to 28 DAS, respectively (Fig. 3B).

Fig. 3.

Effect on photosynthetic pigments such as chlorophyll content (A) and carotenoid content (B) in leaves of lentil genotypes under different treatments T1, T2 & T3 representing control, weedy check and sprayed with imazethapyr, respectively, at 7, 14, 21 and 28 days after spray (DAS). Bars represent the mean ± SD; n = 3. Different letters above bars represent significant differences among different genotypes under different treatments at p < 0.05 by using Tukey’s HSD test (Statistix 10.0). A represents significant differences between different genotypes under control and different treatments individually, B represents significant differences of a particular genotype under three different treatments and AB represents overall interaction between genotypes and treatments at different days after spray at F (5%) value by multiple comparison analysis using two-way ANOVA (GraphPad Prism software version 8.4.3)

Effect of herbicide treatment on protein and nitrogen content

The nitrogen and protein content in leaves was observed as reduced by 22.33%, 13.67%, 32.67% and 31% after herbicide application in LL1397, LL1612, FLIP2004-7L and PL07, respectively, at 28 DAS compared to control. During weedy check treatment, the content was reduced by 24.19%, 25.74%, 38.53% and 14.32% in LL1397, LL1612, FLIP2004-7L and PL07, respectively, at 28 DAS compared to weed free treatment. Herbicide stress significantly reduced the nitrogen and protein contents in FLIP2004-7L and PL07 at 21 and 28 DAS as compared to tolerant genotypes (Fig. 4A, B).

Fig. 4.

Effect on nitrogen content (%) (A) and protein content (%) (B) in leaves of lentil genotypes under different treatments T1, T2 and T3 representing control, weedy check and sprayed with imazethapyr, respectively, at 7, 14, 21 and 28 days after spray (DAS). Bars represent the mean ± SD; n = 3. Different letters above bars represent significant differences among different genotypes under different treatments at p < 0.05 by using Tukey’s HSD test (Statistix 10.0). A represents significant differences between different genotypes under control and different treatments individually, B represents significant differences of a particular genotype under three different treatments and AB represents overall interaction between genotypes and treatments at different days after spray at F (5%) value by multiple comparison analysis using two-way ANOVA (GraphPad Prism software version 8.4.3)

Effect of IM spray on weed control efficiency and weed index

Weed control efficiency was measured on the basis of dry weight of weeds in both weedy check and sprayed treatments (Fig. 2D). Weed efficiency was 4.8%, 49.1% and 26.7% at 14, 21 and 28 DAS, respectively, in T3 treatment compared with weed free treatment. Weed index was calculated compared with control for all genotypes to check the impact of weeds on lentil yield which was found to be 38.7% and 43% LL1397 and LL1612 (tolerant genotypes) and 60% and 62.7% (susceptible genotypes) in FLIP2004-7L and PL07, respectively.

Effect of IM spray on relative leaf water content

The results showed that there is a decrease in RLWC in both T3 and T2 plants compared to control plants. Decrease in RLWC after herbicide treatment was found to be 8.5%, 9.5%, 12.6% and 6.6% in LL1397 and 7.3%, 8.4%, 10% and 8.62% in LL1612 at 7, 14, 21 and 28 DAS, respectively. However, RLWC was reduced after IM spray to a greater extent by 14%, 18.5%, 21.4% and 11.9% in FLIP2004-7L and 10.9%, 16.5%, 17.4%, 11% in PL07 at 7, 14, 21 and 28 DAS, respectively. RLWC was significantly reduced at 14 and 21 DAS in all genotypes showing higher reduction in susceptible genotypes compared to their respective controls (Fig. 5A).

Fig. 5.

Effect on relative leaf water content (RLWC, A), 2,2-diphenyl-1-picryl hydrazyl (DPPH, B) and glycine betaine content (C) in leaves of lentil genotypes under different treatments T1, T2 and T3 indicates control, weedy check and sprayed with imazethapyr, respectively, at 7, 14, 21 and 28 days after spray (DAS). Bars represent the mean ± SD; n = 3. Different letters above bars represent significant differences among different genotypes under different treatments at p < 0.05 by using Tukey’s HSD test (Statistix 10.0). A represents significant differences between different genotypes under control and different treatments individually, B represents significant differences of a particular genotype under three different treatments and AB represents overall interaction between genotypes and treatments at different days after spray at F (5%) value by multiple comparison analysis using two-way ANOVA (GraphPad Prism software version 8.4.3)

Effect of IM spray on DPPH radical scavenging activity and glycine betaine content

DPPH free radical scavenging activity was measured to check the total antioxidant activities in both tolerant and susceptible genotypes in all treatments. It was observed tolerant genotypes (LL1397 and LL1612) showed significantly higher DPPH free radical scavenging activity compared to control (i.e. 14 DAS), in contrast to susceptible genotypes (FLIP2004-7L and PL07). After IM treatment, DPPH radical scavenging activity in LL1397 and LL1612 was comparable to their respective controls (T1) at 21 and 28 DAS but scavenging activity in susceptible genotypes was significantly lower than control indicating that significantly reduced total antioxidant capacity at 21 and 28 DAS in susceptible genotypes might be due to ROS overproduction in susceptible genotypes after IM treatment (Fig. 5B).

Glycine betaine content increased by IM spray in LL1397 and LL1612 (tolerant) genotypes by 8.9% and 20.6% at 7 DAS, 65.3% and 73.5% at 14 DAS, 60.1% and 110% at 21 DAS and 52.6% and 46.15%, respectively, at 28 DAS (Fig. 5C). However such increase was not observed in susceptible (FLIP2004-7L and PL07) genotypes. Glycine betaine content in FLIP2004-7L and PL07 was found to be significantly lower at 21 and 28 DAS.

Correlation analysis

Correlation analysis carried out to study the relationship between the biochemical morpho-physiological parameters in different treatments at different days after spray during both the years (Fig. 6). A significant positive correlation of ALS activity was observed with nitrogen (r = 0.68, 0.55), protein content (r = 0.68, 0.55), and chlorophyll (r = 0.52, 0.45) under different treatments. ALS activity showed negative but non-significant correlation with plant height and seed yield. Correlation of ALS activity with carotenoid content (r = − 0.49, − 0.59) and RLWC (r = − 0.34, − 0.47) was found to be negative with p value of 0.01 at both the years. A positive and significant (p < 0.01) correlation of plant height was obtained with seed yield (r = 0.85) and pods per plant (r = 0.85) depicted that IM spray affect the growth of plant with the reduction in plant height that leads to yield reduction by reducing the number of pods in susceptible genotypes compared to tolerant genotypes. DPPH radical scavenging activity show positive correlation (r = 0.62, 0.61) with ALS activity.

Fig. 6.

Correlation matrix representing Pearson’s correlation coefficient (r) between different morpho-physiological parameters and ALS activity in lentil genotypes under different treatments at 7, 14, 21 and 28 days after spray (DAS) by using GraphPad Prism software (version 8.4.3). The mean values (n = 3) were used for correlation analysis. The correlation values are significant at p < 0.01. ALS Acetolactate synthase, RLWC Relative leaf water content, DPPH 2,2-diphenyl-1-picryl hydrazyl

Discussion

The present study showed that application of IM effectively reduced all prominent weed species in lentil field. IM application has been reported to effectively control all type of weed species in Egyptian clover (Govindasamy et al. 2021). In the present study, in both years, the activity of ALS enzyme was reduced by herbicide treatment in all genotypes from 5-7 DAS (Fig. 1A, B). During 2019–2020, recovery was observed in all genotypes from 7 to 14 DAS, afterwards activity was reduced upto 28 DAS in all genotypes with higher reduction in susceptible genotypes. During 2020–2021, recovery was observed only in tolerant genotypes, but activity was greatly reduced in susceptible genotypes from 7 to 14 DAS.

The main site of BCCA biosynthesis by ALS is meristematic tissues (Zhou et al. 2007). The decrease in ALS activity at 28 DAS under all treatments suggest that ALS (branch point enzyme) activity might have attained a basal level in plants at this stage, which coincides with the transition from vegetative to reproductive development. Cell division in the stem and leaves also stopped at this stage in order to divert assimilates to developing reproductive sinks (Anbessa et al. 2007). Higher expression of ALS in young and rapidly dividing plant organs provide branched chain amino acids to growing areas of the plant and enzyme expression was lowest in plant organs in which growth has ceased (Keeler et al. 1993). Cao et al. (2022) reported that alteration in ALS gene sequences provide resistance to ALS inhibition. IM mainly affected the young and growing meristematic tissues resulting in extension of branches with late onset of flowering and poor pod setting in chickpea (Gaur et al. 2013).

Plant height and seed yield were significantly reduced after IM treatment in susceptible genotype (FLIP2004-7L and PL07) compared to tolerant genotypes (LL1397 and LL1612), implying that IM may reduce the weed-crop competition in tolerant genotypes, resulting in yield enrichment. It was observed that IM treatment led to the reduction in plant height and grain yield in faba bean (Khater et al. 2021) and in lentil (Sharma et al. 2018). Highest number of pods per plant was recorded in weed free treatment, however pods per plant were reduced to greater extent in susceptible genotypes after IM spray compared to tolerant genotypes suggesting that herbicide spray was effective in controlling weed flora in tolerant genotypes during initial stages led to reduced competition between weeds and crop resulting in better yield attributes and seed yield of lentil tolerant genotypes (Fig. 2B). Sukumar et al. (2018) reported better yield related parameters and effective weed control in blackgram after treatment with IM. Higher seed yield observed in weedy check treatment compared to IM treated genotypes might be due to low weed density observed because of low rainfall and low temperature during the crop season, as high temperature boosts the spread of weeds (Dhuppar et al. 2012).

The decrease in dry weight in shoots of susceptible genotypes might be due to decreased number of branches with delayed flowering, resulting in reduction in pod number and size in susceptible genotype. Similar trend was also observed by Mehdizadeh and Khiavi (2020).

IM treatment affected the leaf morphology, decreased chlorophyll content, damaged photosystem resulting in impaired photosynthetic efficiency in plants (Liu et al. 2019). LL1397 and LL1612 with higher chlorophyll and carotenoid content have better photosynthetic performance compared to susceptible genotypes after IM spray. Moreover higher accumulation of glycine betaine in these tolerant genotypes might be responsible for maintaining photosynthetic system under herbicide stress as glycine betaine not only provide osmoregulation but also protects photosynthetic apparatus by maintaining the structure and functional integrity of CO₂ fixing enzymes and decreasing accumulation of ROS in chloroplast under stressed conditions (Kurepin et al. 2015).

One of the initial physiological effects by IM application is change in the free amino acid pool by disrupting protein synthesis (Monreal et al. 2020). The positive correlation observed between ALS activity and protein content in the present study suggest that inhibition of ALS causes shortage of BCAA resulting in accumulation of free amino acid thereby affecting protein turnover. Decrease in leaf protein content was observed after imazamox treatment in sunflower plants (Balabanova et al. 2020).

Weed biomass reduced to a greater extent at 14 DAS and the efficacy of IM was further reduced due to the emergence of new weeds indicating that the critical period for weed management by IM in lentil might be 14 to 21 DAS. A negative correlation observed between weed control efficiency and weed index demonstrated that the improvement of crop performance due to suppression of intrusion by weeds. IM treatment was effective in increasing weed control efficiency and reducing weed biomass in blackgram (Duary et al. 2016). The decreased weed index by IM reduce weed crop competition and increase crop yield (Chicham et al. 2020; Sarin et al. 2021).

The decrease in RLWC in lentil genotypes after IM treatment indicate significant water loss in the leaves, with susceptible genotypes being more affected (Fig. 5A). IM treatment decreased RLWC content and alter the morphology of leaf surfaces by changing the structure of epidermal and mesophyll cells (Liu et al. 2019). RLWC is positively correlated with chlorophyll and carotenoid content (Fig. 6). Zhao et al. (2020) observed suppression of photosynthetic capacity along with reduction in RLWC in Arabidopsis thaliana after the IM application.

Higher DPPH free radical scavenging activity (Fig. 5A) in tolerant genotypes (LL1397 and LL1612) compared to susceptible genotypes (FLIP2004-7L) might be reducing herbicide induced stress as represented by the positive correlation of DPPH with ALS activity. Radwan et al. (2019) reported that higher DPPH scavenging activity in peanut leaves protect them from herbicide stress by scavenging accumulated ROS.

Imazethapyr altered protein turnover by inhibiting ALS enzyme led to accumulation of free amino acids that might be due to osmotic stress (Huang and Jander 2017). Lower osmotic potential due to osmotic stress resulted in reduced water accessibility that led to decreased biomass production, plant growth and reduced photosynthetic ability (Munns and Tester 2008). The accumulation of glycine betaine observed in tolerant lentil genotypes (LL1397 and LL1612) as compared to susceptible lentil genotypes (FLIP2004-7L and PL07) might be protecting them from the deleterious effects of herbicide stress by maintaining turgor pressure, photosynthetic ability and reducing oxidative stress (Jain et al. 2021; Kurepin et al. 2015).

Herbicide stress affected all genotypes but tolerant genotypes had the ability to recover due to morpho-physiological and biochemical traits. These genotypes showed flowering and pods development at their respective time. Susceptible genotypes showed delay in flowering and reproductive phase and further rise in temperature during crop development affected pod number and seed yield to a greater extent. Application of herbicide lead to transient growth arrest by delaying the onset of flowering followed by delayed pod initiation that ultimately postponed the maturity of crop (Sharma et al. 2018).

Conclusion

Imazethapyr (IM) an ALS-inhibiting herbicides has adversely affected plant height, ALS activity, photosynthetic pigments, nitrogen and protein content, weed control efficiency, weed index, RLWC, osmolyte accumulation and antioxidant capacity in susceptible genotypes, FLIP2004-7L and PL07 compared to tolerant genotypes, LL1397 and LL1612. All these morpho-physiological and biochemical responses might have contributed to the higher yield loss in susceptible genotypes compared to tolerant genotypes suggested antagonistic relationship between susceptibility and seed yield of genotypes. A direct relationship was observed between ALS activity and morpho-physiological parameters depending on the level of tolerance or sensitivity to IM treatment.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

SKG and RSG deigned the experiments; S performed experiments, analysed data and wrote first draft of manuscript; SKG supervised the analysis and finalized the manuscript; RSG contributed research materials and helped in manuscript editing; HKV helped in spraying in the field and in manuscript editing: RDB helped in data analysis and manuscript editing.

Declarations

Conflict of interest

The authors declare that they have no conflict of interests.