Foliar bio-stimulant application at flower initiation optimizes chickpea productivity and grain quality in rainfed environments

Abstract

Bio-stimulants are emerging as effective agents to improve crop performance under abiotic stress, particularly in rain-fed systems. This two-year field study evaluated the effects of foliar-applied bio-stimulant (Actibion) (1250 mL ha^− 1) composed of various amino acids including aspartic acid (1.21%), glutamic acid (3.14%), proline (1.15%) at different growth stages—flower initiation (FI), pod initiation (PI), and both (FPI) on chickpea (Cicer arietinum L.) growth, yield, and grain quality under rain-fed conditions in Pakistan. Treatments were compared with water spray and an unsprayed control in a randomized complete block design. Foliar application at the flower initiation stage significantly enhanced key growth parameters, increasing plant height by ~ 22%, pod length by ~ 14%, leaf area index by ~ 23%, and chlorophyll content by ~ 19%, relative to control. Seed yield improved by over 50% across both seasons. Grain nutrient contents (N, P, K) and protein concentration also increased significantly with bio-stimulant application at FI. This treatment yielded the highest net economic returns, demonstrating both agronomic and financial benefits. These findings suggest that foliar application of bio-stimulant at flower initiation is a promising, sustainable approach to enhance chickpea productivity and nutritional quality under rain-fed conditions.

Graphical abstract

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-026-08129-1.

Introduction

Chickpea (Cicer arietinum L.) is a temperate, herbaceous annual legume widely cultivated across the globe. It plays a significant role in human nutrition, offering a high-protein alternative to animal-based sources. Chickpea seeds contain approximately 20–25% protein, 60% carbohydrates, and essential minerals including Fe, Zn, and Mg [1, 2]. In Pakistan, chickpea is grown on approximately 794,000 hectares, yielding around 230,000 tonnes, with 92% of the cultivation occurring in the rainfed “Thal” region (GOP, 2023–24). However, actual yields remain well below potential due to soil nutrient depletion, erratic rainfall, and limited agronomic intervention [3].

Despite its nutritional importance and adaptability, a substantial gap exists (up to ~ 50%) between the potential and actual yield of chickpea [2]. This yield gap arises primarily from drought stress, poor nutrient management, and limited biological nitrogen fixation [4, 5]. Among these, nutrient deficiency and water stress are the most critical factors limiting productivity [6]. As a predominantly rainfed crop, chickpea is highly vulnerable to environmental fluctuations. Soils in rainfed areas often lack fertility and are prone to degradation, further worsening crop performance [7].

Among adaptive management strategies, foliar application of plant growth-promoting substances and amino-acid-based biostimulants has emerged as a practical means of improving legume performance under stress [8, 9]. Foliar feeding offers several advantages, including faster nutrient uptake and improved efficiency, particularly under arid conditions where root absorption is often compromised [10]. Among these strategies, the use of plant bio-stimulants has gained considerable attention. Bio-stimulants are substances or microorganisms that, when applied to plants or soil, enhance plant growth, development, and resilience to abiotic stresses without supplying direct nutrients [11]. These include amino acids, enzymes, vitamins, hormones, and microelements [12, 13]. Unlike conventional fertilizers, bio-stimulants activate natural physiological processes, such as improved nutrient absorption, enhanced root development, and increased stress tolerance in olive and two varieties of bean [14, 15].

With growing environmental concerns, bio-based inputs are increasingly used as sustainable alternatives or complements to mineral fertilizers [11, 12]. Recent studies suggest that bio-stimulants not only promote plant health and productivity but also ensure safer and more environmentally sustainable crop production [16, 17]. These products, often marketed as protein hydrolysates, can be applied via foliar sprays, seed treatments, or soil drenches [18]. Foliar application, in particular, is cost-effective and efficient, especially during critical crop growth stages when nutrient demand is high and soil nutrient availability is limited in Loquat [19–21].

Bio-stimulants enriched with amino acids – such as aspartic acid, glutamic acid, and proline – contain a broad spectrum of essential compounds that support plant metabolic activities and stress resilience [22–24]. Recent findings demonstrate that amino-acid-based foliar sprays improve photosynthetic rate, nitrogen metabolism, and grain nutrient composition in legumes including cowpea, soybean, and chickpea [5, 25, 26].

Although earlier studies have reported positive effects of amino acid–based or seaweed-derived biostimulants in legumes [5, 25, 26], these investigations were generally confined to irrigated systems or pot trials and did not explore the influence of application timing under field-based rainfed conditions. Very few studies have tested amino-acid-based formulations under true rainfed field conditions, where water stress, nutrient limitation, and environmental variability strongly influence crop responsiveness. More importantly, no published work has compared the effectiveness of a commercial amino-acid biostimulant (Actibion) applied at distinct phenological stages to identify the most responsive growth window in chickpea. By integrating two years of field data, physiological measurements, nutrient profiling, and economic analysis, this study provides the first field-based evidence identifying flower initiation as the optimum timing for amino-acid biostimulant application in rainfed chickpea. This directly advances current knowledge by linking application timing with physiological response and farmer-relevant profitability under semi-arid rainfed conditions.

Materials and methods

Experimental site and meteorological description

The field experiments were conducted in the Thal region of District Mianwali, Punjab, Pakistan (32.36° N, 71.69° E; elevation 200 m above sea level), during two consecutive cropping seasons: 2018–2019 and 2019–2020. This region falls under agro-ecological zone III and is characterized by sandy soils and an arid to semi-arid climate. The average minimum and maximum temperatures during the study period were 11 °C and 22 °C, respectively. Detailed meteorological data, including rainfall and temperature trends during the crop growth periods, are presented in Fig. 1. The soil at the experimental site was predominantly sandy, and its physico-chemical properties for both seasons are detailed in Table 1. Although soil moisture and reference evapotranspiration (ETo) were not directly recorded, the coarse soil texture, low organic matter, and rainfall patterns indicate that the crop likely experienced intermittent moisture stress under natural rainfed conditions. These data collectively describe the semi-arid, low-organic-matter conditions typical of the Thal region’s rainfed chickpea-growing environment.

Fig. 1.

Weekly average maximum or minimum temperature and rainfall of experimental site throughout the chickpea growing seasons (a) 2018-19 (Year I) and (b) 2019-20 (Year II)

Table 1.

The physico-chemical properties of the experimental field during the growing seasons of 2018-19 (Year I) and 2019-20 (Year II)

Characteristics	Values
	Year I	Year II
Sand (g kg− 1)	836	839
Silt (g kg− 1)	90	91
Clay (g kg− 1)	74	70
Soil texture class	Sandy Soil	Sandy Soil
Soil pH	8.47	8.51
EC (dS m− 1)	3.04	3.19
Saturation percentage	22.1	20.8
Organic matter (%)	0.27	0.23
Organic C content (%)	0.157	0.134
Total soil N content (%)	0.014	0.012
Available P (mg kg− 1)	4.16	3.86
Extractable K (mg kg− 1)	219	207

Experimental design and treatments

The experiment was conducted using a Randomized Complete Block Design (RCBD) with four replications to evaluate the effect of biostimulant (Actibion) application at different chickpea growth stages. Seven treatments were tested: (1) control (no spray), (2) one foliar application of Actibion @ 1250 mL ha^− 1 at flower initiation (FI), (3) one water spray at FI, (4) one foliar application of Actibion @ 1250 mL ha^− 1 at pod initiation (PI), (5) one water spray at PI, (6) two foliar applications of Actibion @ 1250 mL ha^− 1 – first at FI and second at PI (FPI), and (7) two water sprays applied at both FI and PI (FPI). The bio-stimulant used was “Actibion,” which is a commercial product and containing a blend of amino acids including alanine (0.52%), arginine (1.73%), aspartic acid (1.21%), cystine (0.78%), glycine (0.78%), histidine (0.22%), leucine (0.78%), glutamic acid (3.14%), lysine (0.72%), phenylalanine (0.54%), methionine (0.08%), proline (1.15%), threonine (1.14%), serine (1.51%), tyrosine (0.26%), isoleucine (0.59%), and valine (0.85%). The applied dose of 1250 mL ha^− 1 was selected according to the manufacturer’s recommended foliar rate for field crops and has been previously validated in agronomic trials as an effective and farmer-relevant concentration. This ensured that the treatment reflected a practical and scalable dosage rather than an experimentally optimized one. The chickpea cultivar ‘Bittal-2016’ was used as the test crop. Certified seeds of this cultivar were obtained from the Pulses Research Institute, Ayub Agricultural Research Institute (AARI), Faisalabad, Punjab, Pakistan. ‘Bittal-2016’ is a released and widely cultivated variety recommended for rainfed environments by the Pakistan Agricultural Research Council (PARC).

The plant material used in this study was not collected from the wild; therefore, no collection permits or access permissions were required. Formal identification of the cultivar was ensured through certified seed documentation and varietal descriptors provided by AARI at the time of seed procurement. As the study involved a registered commercial cultivar grown under standard agronomic field conditions, the preparation and deposition of herbarium voucher specimens were not applicable.

Experimental protocol

Prior to sowing, the field was prepared by cultivating the soil 2–3 times with a tractor-mounted cultivator followed by planking. Sowing was performed using a single-row hand drill at a seed rate of 60 kg ha^− 1 with 30 cm row spacing. Each experimental plot measured 5 m × 3 m (15 m²) with consistent row spacing throughout. Seeds were treated with fungicide before sowing. After emergence, the seedlings were thinned twice to maintain a plant-to-plant spacing of 10 cm. Weeds were controlled manually as needed.

The experiment was conducted under rainfed conditions with no supplemental irrigation. Foliar application of the bio-stimulant (Actibion) was applied at a rate of 1250 mL ha^− 1 according to the respective treatment plans. Water sprays were applied as control treatments at corresponding growth stages. Foliar sprays were applied using a knapsack sprayer calibrated to deliver 300 L ha^− 1, corresponding to 4.5 L per plot. Sprays were applied uniformly until foliage was fully wetted but without runoff.

The first season’s experiment began on October 10, 2018, and was harvested on March 15, 2019, while the second season’s trial started on November 3, 2019, and ended on March 28, 2020.

Procedure for data collection

During both years of the study, the chickpea crop was harvested when approximately 90% of the pods had reached maturity, and grains were separated manually. The following procedure was employed to collect data related to the growth, yield, and quality attributes of chickpea.

Yield attributes

The average number of pods per plant was determined by counting the pods on five randomly selected plants. Pod length (cm) was measured individually for each pod from five selected plants, and the average pod length was calculated. The number of seeds per pod was recorded by manually threshing pods from five plants, and the total seeds were divided by the number of pods to calculate the average. To determine the number of seeds per plant, seeds from five plants were counted and the mean value was taken. For counting the number of nodules per plant, five plants were carefully uprooted after moistening the soil, and the roots were washed thoroughly under running water. The nodules on each plant were counted, and the average was calculated. The 100-seed weight (g) was determined by weighing 100 randomly selected seeds from each treatment using a digital balance.

Seed yield (kg ha^{− 1}) was determined by harvesting the entire plot manually with a sickle, threshing the plants, weighing the seed on a digital field balance, and converting the weight into kilograms per hectare. Biological yield (kg ha^{− 1}) was calculated by weighing the total biomass (plant bundles) from each treatment and converting it to kg ha^{− 1}. The harvest index (%) was then calculated using the formula by Donald and Hamblin [27].

Physiological parameters

Leaf area index (LAI) was calculated using the formula by Sesták et al. [28] as follows:

Leaf area duration (LAD) was determined by the method of Power et al. [29].

where LAI represents leaf area index at two time points, and t is the time interval between measurements.

Crop growth rate (CGR; g m⁻² d⁻¹) was calculated after 90 days after sowing (DAS) using following formula.

where W₁ and W₂ are the initial and final dry weights, and T₁ and T₂ represent the sampling dates.

Chlorophyll content (SPAD value) was measured using a chlorophyll content meter on five leaves from each treatment, and the average reading was recorded.

Determination of nutrient contents in Chickpea grain

Grain samples were oven-dried and ground into a fine powder using a Willey mill before nutrient analysis. Grain nitrogen content (%) was determined using the Kjeldahl method as described by Estefan et al. [30]. The percentage of nitrogen was calculated using the formula:

where V₁ is the volume of acid used for the sample (mL), V₂ is the volume of acid for the blank (mL), N is the normality of standard acid, and W is the sample weight (g).

Phosphorus content (%) was determined following Estefan et al. [30], using the formula.

where V₁ is the total digest volume (mL), V₂ is the digest volume used for measurement (mL),

and Wt is the dry sample weight (g).

Potassium content (ppm) was measured using flame photometry according to Estefan et al. [30] using the formula.

where C is the potassium concentration from the calibration curve, V is the final volume of digested sample (mL), and W is the sample weight (g).

Protein content (%) in grain was estimated using the Lowry method for protein determination [31]. Zinc content was measured using an atomic absorption spectrophotometer, while boron content was estimated through flame photometry at 420 nm [30].

Economics analysis

Net economic return (Rs. ha^− 1) was calculated by subtracting the total cost of cultivation from the gross income. The Benefit-Cost Ratio (BCR) was computed by dividing gross income by total cost of cultivation.

Statistical analysis

All collected data were subjected to statistical analysis using Statistix 8.1 software (Analytical Software, Tallahassee, FL, USA). Treatment means were compared using Tukey’s Honest Significant Difference (HSD) test at a 5% probability level. Correlation analyses were performed using replicated treatment values of both cropping seasons (Table S1). The randomized complete block design (RCBD) with four replications helped minimize within-plot variation and ensured statistical robustness.

Results

Growth and physiological attributes

The foliar application of bio-stimulant at different growth stages of chickpea showed a significant impact on growth and physiological traits compared to water sprays and the no-spray control during both growing seasons (2018–2019 and 2019–2020), as presented in Table 2. Application of bio-stimulant consistently enhanced growth attributes across both years. No significant differences were observed between the control and water sprays applied at flower initiation (FI) and flower + pod initiation (FPI) stages for most traits.

Table 2.

Impact of foliar applied bio-stimulant (Actibion) on growth and physiological parameters of Chickpea under rain-fed conditions during the growing seasons of 2018-19 (Year I) and 2019-20 (Year II)

Treatments	Plant height (cm)		Leaf area index (LAI)		Leaf area duration (days)		Crop growth rate (g m− 2 d− 1)		Chlorophyll content (SPAD)		Nodules plant− 1
	Year I	Year II	Year I	Year II	Year I	Year II	Year I	Year II	Year I	Year II	Year I	Year II
T1	45.25 de	43.50de	3.02 d	3.10 d	39.83 d	33.84 d	1.70 d	1.80 d	42.32 f	42.44 e	13.7 d	15.5 d
T2	61.50 a	61.25 a	4.96 a	5.03 a	57.35 a	57.62 a	3.02 a	3.12 a	55.97 a	55.89 a	19.5 a	21.5 a
T3	47.00 d	46.00 d	3.13 d	3.23 d	41.05 d	44.36 d	1.80 d	1.87 d	43.40 e	43.65 d	14.2 d	15.5 d
T4	55.25 c	53.25 c	3.70 c	3.83 c	48.90 c	50.14 c	2.42 c	2.55 c	50.32 c	50.77 c	16.0 c	17.5 c
T5	43.75 e	42.75 e	3.02 d	3.18 d	40.55 d	43.68 d	1.72 d	1.77 d	43.87 d	44.29 d	14.0 d	15.5 d
T6	58.23 b	58.00 b	4.24 b	4.48 b	52.53 b	53.81 b	2.67 b	2.76 b	52.91 b	52.88 b	17.7 b	19.5 b
T7	46.75 d	45.00 de	3.08 d	3.16 d	40.73 d	42.55 d	1.77 d	1.82 d	43.50 de	42.52 e	14.2 d	15.5 d
HSD (0.05)	2.77	3.23	0.17	0.22	1.77	2.67	0.22	0.18	0.37	0.76	1.70	1.58

Means sharing the same letter did not differ significantly at p ≤ 0.05 according to Tukey’s HSD

T₁ = No spray (control), T₂ = Bio-stimulant @ 1250 mL ha^− 1 at flower initiation, T₃ = Water spray at flower initiation, T₄ = Bio-stimulant @ 1250 mL ha^− 1 at pod initiation, T₅ = Water spray at pod initiation, T₆ = Bio-stimulant @ 1250 mL ha^− 1 at flower and pod initiation and T₇ = Water spray at flower and pod initiation

Plant height increased significantly with bio-stimulant application, with the tallest plants recorded when the bio-stimulant @ 1250 mL ha^− 1 was sprayed at the flower initiation stage (61.50 cm in 2018 and 61.25 cm in 2019) over control and all biostimulant sprayed at other stages. The shortest plants were observed in the no-spray control (45.25 cm in 2018 and 43.50 cm in 2019). Similarly, the leaf area index (LAI) improved significantly, with the highest LAI (4.96 in 2018 and 5.03 in 2019) observed under the same treatment, followed by application at both FI and PI stages and then application at only pod initiation stage. The lowest LAI was recorded in the control treatment.

Leaf area duration (LAD) was also enhanced by the foliar application of the bio-stimulant, with the maximum LAD (57.35 days in 2018 and 57.62 days in 2019) observed at the flower initiation stage. Crop growth rate (CGR) followed a similar trend, with the highest CGR values (3.02 g m^− 2 d^− 1 in 2018 and 3.12 g m^− 2 d^− 1 in 2019) recorded for the same treatment. Whereas biostimulant application at FPI stage and at PI produced LAI and CGR lower than flower initiation stage, while CGR values under water spray treatments were statistically similar to the control.

Chlorophyll content, measured in SPAD units, significantly improved under bio-stimulant application. The highest SPAD values (55.97 in 2018 and 55.89 in 2019) were observed with bio-stimulant applied at the flower initiation stage, whereas the lowest values (42.32 and 42.44 in 2018 and 2019, respectively) were found in the control treatment. Furthermore, the number of nodules per plant also increased significantly with the bio-stimulant application, peaking at 19.5 (2018) and 21.5 (2019) nodules per plant when the treatment was applied at the flower initiation stage. Water sprays at various stages had no significant effect and remained statistically similar to the control.

Yield attributes

The application of bio-stimulant @ 1250 mL ha^− 1 at different growth stages significantly influenced yield and yield-related attributes compared to the control and water spray treatments, as shown in Table 3. The highest number of pods per plant (36 in 2018 and 37 in 2019) was recorded under bio-stimulant application at the flower initiation stage, followed by the combined application at both flower and pod initiation stages which is followed by biostimulant application at pod initiation stage. The control treatment resulted in the lowest pod count.

Table 3.

Impact of foliar applied bio-stimulant (Actibion) on yield and yield components of Chickpea under rain-fed conditions during the growing seasons of 2018-19 (Year I) and 2019-20 (Year II)

Treatments	No. of pod plant− 1		Pod length (cm)		No. of seeds pod− 1		100-seed weight (g)		Seed yield (kg ha− 1)		Biological yield (kg ha− 1)		Harvest index (%)
	Year I	Year II	Year I	Year II	Year I	Year II	Year I	Year II	Year I	Year II	Year I	Year II	Year I	Year II
T1	29.5 d	30.5 d	2.15 d	29.5 d	1.07 c	1.12 c	13.5 d	14.0 d	1414.3 d	1437.5 d	4444.0 d	4576.7 d	30.69 d	31.04 d
T2	36.0 a	37.0 a	2.80 a	36.0 a	1.87 a	1.92 a	17.0 a	17.5 a	2126.5 a	2163.0 a	5815.0 a	5842.5 a	37.00 a	36.73 a
T3	30.2 d	31.2 d	2.12 d	30.2 d	1.17 c	1.17 c	13.7 d	14.2 d	1484.5 d	1493.3 d	4413.2 d	4778.3 d	31.75 d	31.15 d
T4	32.0 c	33.0 c	2.40 c	32.0 c	1.45 b	1.50 b	15.0 c	15.5 c	1613.0 c	1631.0 c	4913.5 c	5010.3 c	33.97 c	33.00 c
T5	30.0 d	31.0 d	2.10 d	30.0 d	1.10 c	1.17 c	13.2 d	13.7 d	1461.3 d	1470.0 d	4326.7 d	4718.5 d	32.07 d	31.05 d
T6	34.0 b	35.0 b	2.60 b	34.0 b	1.60 b	1.65 b	16.0 b	16.5 b	1836.5 b	1861.5 b	5324.8 b	5426.8 b	35.48 b	34.86 b
T7	29.7 d	30.5 d	2.15 d	29.7 d	1.15 c	1.15 c	14.0 d	14.5 d	1430.0 d	1458.3 d	4384.3 d	4759.5 d	32.14 d	30.63 d
HSD (0.05)	1.44	1.44	0.19	1.44	0.23	0.24	0.84	0.84	94.6	112.7	373.7	212.8	1.47	1.79

Means sharing the same letter did not differ significantly at p ≤ 0.05 according to Tukey’s HSD

T₁ = No spray (control), T₂ = Bio-stimulant @ 1250 mL ha^− 1 at flower initiation, T₃ = Water spray at flower initiation, T₄ = Bio-stimulant @ 1250 mL ha^− 1 at pod initiation, T₅ = Water spray at pod initiation, T₆ = Bio-stimulant @ 1250 mL ha^− 1 at flower and pod initiation and T₇ = Water spray at flower and pod initiation

Pod length was also enhanced, with the longest pods (2.80 cm in 2018 and 2.82 cm in 2019) observed when bio-stimulant was applied at flower initiation. The shortest pods were recorded in the control treatment (2.15 cm in 2018 and 2.10 cm in 2019), which were statistically similar to the water spray treatments. Similarly, the maximum number of seeds per pod (1.67 in 2018 and 1.90 in 2019) was achieved with bio-stimulant application at the flower initiation stage.

The 100-seed weight also improved significantly under bio-stimulant treatments, with the highest values recorded at flower initiation (17.0 g in 2018 and 17.50 g in 2019), followed by the FPI stage. Control and water spray treatments showed statistically similar and lower 100-seed weights. Grain yield increased significantly under all bio-stimulant treatments compared to control and water sprays. The maximum seed yield was recorded with bio-stimulant applied at flower initiation, resulting in increases of 712.2 kg ha^− 1 (50.38%) in 2018 and 725.5 kg ha^− 1 (50.46%) in 2019 over control. This was followed by application at the FPI stage (29.87% and 29.49% yield increase over control in 2018 and 2019, respectively).

The highest biological yield (5815.0 kg ha^− 1 in 2018 and 5842.5 kg ha^− 1 in 2019) and harvest index (37.0% in 2018 and 36.73% in 2019) were also observed with bio-stimulant application at the flower initiation stage, followed by application at FPI. The lowest biological yield and harvest index were recorded in the control and water spray treatments.

Nutrient and quality attributes

Nutrient analysis of chickpea grains (Table 4; Fig. 2) demonstrated significant improvements in nitrogen (N), phosphorus (P), and potassium (K) content with bio-stimulant application across both seasons. The highest nutrient concentrations were recorded with foliar application of bio-stimulant @ 1250 mL ha^− 1 at the flower initiation stage, yielding N contents of 3.72% (2018) and 3.95% (2019), P contents of 0.17% and 0.18%, and K contents of 1.75% and 1.85%, respectively. These values were followed by the application at both FI and PI stages (N contents of 3.35% 3.57%, P contents of 0.150%, 0.157% and K contents of 1.52% and 1.62% during 1st and 2nd year respectively). The lowest N, P, and K contents were found in the control and water spray treatments.

Table 4.

Economic analysis of Chickpea production in growing seasons of 2018-19 (Year I) and 2019-20 (Year II) under foliar application of bio-stimulant @1250mLha^− 1 at different growth stages

Treatments	Gross income (Rs.ha− 1)		Total variable Cost (Rs.ha− 1)		Net benefits (Rs.ha− 1)		Benefit over control		Benefit-cost ratio
	Year I	Year II	Year I	Year II	Year I	Year II	Year I	Year II	Year I	Year II
T 1	76,823	84,180	17,170	18,315	59,653	65,865	0	0	4.47	4.6
T 2	182,877	198,996	20,357	21,477	162,520	177,519	102,867	111,654	8.98	9.27
T 3	80,750	87,924	17,270	18,415	63,480	69,509	3,827	3,644	4.68	4.77
T 4	137,105	148,239	20,357	21,477	116,748	126,762	57,095	60,897	6.74	6.9
T 5	84,974	88,807	17,270	18,415	67,704	70,392	8,051	4,527	4.92	4.82
T 6	156,102	170,108	23,453	24,564	132,649	145,544	72,996	79,679	6.66	6.93
T 7	87,422	93,104	17,270	18,415	70,152	74,689	10,499	8,824	5.06	5.06

T₁ = No spray (control), T₂ = Bio-stimulant @ 1250 mL ha^− 1 at flower initiation, T₃ = Water spray at flower initiation, T₄ = Bio-stimulant @ 1250 mL ha^− 1 at pod initiation, T₅ = Water spray at pod initiation, T₆ = Bio-stimulant @ 1250 mL ha^− 1 at flower and pod initiation and T₇ = Water spray at flower and pod initiation

Fig. 2.

Impact of foliar applied bio-stimulant on N (a & b), P (c & d) and K (e & f) contents in chickpea grain during the growing seasons of 2018-19 (Year I) and 2019-20 (Year II), respectively. T₁ = No spray (control), T₂ = Bio-stimulant @ 1250 mL ha^− 1 at flower initiation, T₃ = Water spray at flower initiation, T₄ = Bio-stimulant @ 1250 mL ha^− 1 at pod initiation, T₅ = Water spray at pod initiation, T₆ = Bio-stimulant @ 1250 mL ha^− 1 at flower and pod initiation and T₇ = Water spray at flower and pod initiation. Means sharing the same letter did not differ significantly at p ≤ 0.05 according to Tukey’s HSD

Similarly, foliar application of bio-stimulant significantly increased Zn and B contents in chickpea grains (Table 5). The highest concentrations were obtained from treatments applied at flower initiation, with notable improvements also seen with applications at PI and FPI. In contrast, the lowest Zn and B values were recorded in the control and water spray treatments at all stages.

Table 5.

Impact of foliar applied bio-stimulant (Actibion) on Zn, B and crude protein contents of Chickpea under rain-fed conditions during the growing seasons of 2018-19 (Year I) and 2019-20 (Year II)

Treatments	Zn Contents (mg kg− 1)		B Contents (mg kg− 1)		Crude Protein (%)
	Year I	Year II	Year I	Year II	Year I	Year II
T1	17.00 d	17.25 d	17.25 d	16.75 d	15.46 d	16.24 d
T2	26.25 a	27.25 a	27.75 a	27.21 a	23.27 a	24.68 a
T3	18.00 d	18.25 d	18.50 d	18.94 d	16.09 d	16.71 d
T4	21.25 c	21.75 c	22.75 c	23.22 c	18.74 c	20.77 c
T5	16.75 d	17.50 d	17.75 d	17.25 d	15.62 d	16.31 d
T6	23.75 b	24.50 b	25.25 b	25.73 b	20.93 b	22.34 b
T7	17.50 d	18.00 d	18.00 d	17.75 d	15.77 d	16.56 d
HSD (0.05)	1.42	1.31	1.29	1.93	1.33	0.84

Means sharing the same letter did not differ significantly at p ≤ 0.05 according to Tukey’s HSD

T₁ = No spray (control), T₂ = Bio-stimulant @ 1250 mL ha^− 1 at flower initiation, T₃ = Water spray at flower initiation, T₄ = Bio-stimulant @ 1250 mL ha^− 1 at pod initiation, T₅ = Water spray at pod initiation, T₆ = Bio-stimulant @ 1250 mL ha^− 1 at flower and pod initiation and T₇ = Water spray at flower and pod initiation

Protein content in chickpea grains (Table 5) was also significantly affected by bio-stimulant applications. The highest protein levels (23.27% in 2018 and 24.68% in 2019) were obtained with application at flower initiation, followed by application at FPI. In comparison low protein was noted with biostimulant application at all other stages application FPI, PI. The lowest protein contents (15.46% and 16.24%) were observed in the no-spray control, which was statistically similar to water spray treatments.

Correlation analysis

The correlation matrices for Year I and Year II reveal a clear pattern: most growth, yield, and nutritional traits moved together, and the relationships were generally very strong (Fig. 3). In both seasons, coefficients commonly sat above 0.80, and several trait pairs crossed 0.90–0.99, showing that improvements in one parameter were usually accompanied by gains in others. Vegetative growth indicators (plant height, leaf area index, leaf area duration, crop growth rate, and chlorophyll content) showed strong positive links with yield components such as pods per plant, pod length, seeds per pod, and 100-seed weight, ultimately aligning with higher seed and biological yield. Nutrient contents (N, P, K, Zn, B) and protein also correlated closely with both growth and yield attributes, reflecting better nutrient uptake and deposition in the seed. The similarity in correlation structure between the two years suggests that these relationships were stable, reinforcing that Actibion application tends to enhance growth, productivity, and nutritional quality in a coordinated manner.

Fig. 3.

Pearson’s correlation matrix among growth, yield, and nutritional traits of chickpea following biostimulant (Actibion) application at different growth stages over two years

Economic analysis

The economic evaluation (Table 5) revealed that all bio-stimulant treatments generated higher gross income, net returns, and benefit-cost ratio (BCR) compared to control and water spray treatments. The most economically viable treatment was the foliar spray of bio-stimulant @ 1250 mL ha^− 1 at the flower initiation stage, which produced the highest gross income (Rs. 182,877 ha^− 1 in 2018 and Rs. 198,996 ha^− 1 in 2019), net benefits (Rs. 162,520 ha^− 1 and Rs. 177,519 ha^− 1), and BCR (8.98 and 9.27). The next most profitable treatment was the application at FPI stage, followed by application at PI stage. The control and water spray treatments resulted in the lowest economic returns.

Discussion

The application of bio-stimulants significantly enhanced the growth, yield, and quality of chickpeas across both growing seasons. Foliar application of Actibion at 1250 mL ha^{− 1} significantly improved plant height, leaf area index, and crop growth rate compared with the control, consistent with recent reports in chickpea [3] and soybean [25]. Such enhancement reflects the synergistic role of amino acids and plant growth regulators that stimulate cell elongation, photosynthetic activity, and biomass accumulation [12, 32].

Among all treatments, the flower-initiation stage showed the strongest physiological response, indicating this phase as the most responsive to foliar nutrition [3, 5]. The foliar application of bio-stimulant at the flower initiation (FI) and flowering + pod initiation (FPI) stages enhanced the leaf area index (LAI), leaf area duration (LAD), and crop growth rate (CGR). These improvements can be linked to the nutrients and amino acids present in the bio-stimulant, which stimulate plant metabolism, cell division, and photosynthesis. Chickpea shown highest improvement in all growth, yield and quality parameters under the application of biostimulant at flower initiation stage as compared to other stages and control. The improvement may be attributed to the best time of application of biostimulant. At this time chickpea plant was at its early reproductive phase and had maximum time for physiological response to biostimulant application as compared to when applied at pod initiation and both flower and pod initiation stage which are later stages. Carillo et al. [33] confirmed that amino acids like aspartate, alanine, and glutamate present in bio-stimulants enhance chlorophyll content and photosynthetic activity. Similarly, Sharma et al. [3] observed increased leaf area due to improved cell division and elongation induced by Panchagavya. Sujatha and Vijayalakshmi [34] reported a 46.62% rise in total chlorophyll content in black gram with foliar stimulant application. These results suggest that the exogenous supply of amino acids and plant growth regulators enhances photosynthetic capacity and vegetative vigor.

An increase in the number of root nodules per plant was also observed following foliar application of the bio-stimulant at 1250 mL ha^{− 1}. This may be due to the improved availability of nutrients and amino acids that stimulate root development and nodulation [35]. Carvalho et al. [36] noted that plant regulators enhance root growth and seedling establishment, especially under stress conditions. Kadam et al. [37] found similar results in peas, where Dasagavya application led to increased nodules due to enhanced cell division. Sharanya et al. [38] also attributed increased nodule numbers to better root growth and nutrient absorption resulting from bio-stimulant application.

Our study further revealed that foliar application of bio-stimulant increased the number of pods per plant and pod length. This could be attributed to enhanced carbohydrate and protein synthesis and translocation at critical reproductive stages. Matthews et al. [39] suggested that bio-stimulant sprays improve nutrient uptake efficiency, delay senescence, and reduce flower drop, ultimately increasing pod number and size. Similar findings were reported by Sahu et al. [5], who found maximum pod numbers with foliar application of humic acid-based bio-stimulants. El Sheikha et al. [40] also observed improved pod traits in snap beans and common beans, respectively, following foliar application of moringa leaf extract and L-glutamic acid.

The observed increase in 100-seed weight and seed yield can be attributed to enhanced enzymatic activity and efficient translocation of photo-assimilates to the reproductive organs, facilitated by bio-stimulant application [41, 42]. Silva et al. [43] found that Glutamin Extra improved seed weight due to its role in metabolism and amino acid synthesis. Abdeen and Hefni [44] reported increased faba bean yield with foliar-applied amino acid solutions. Similarly, Shafiq et al. [45] emphasized the role of amino acid-based bio-stimulants in maximizing seed yield in chickpeas. Enhanced photosynthetic activity, LAI, and chlorophyll content contributed to the overall biological yield. This aligns with findings by Asif et al. [46] in sorghum and Sadoyan et al. [47] in chickpeas using bio-stimulants. A higher harvest index under bio-stimulant treatments was also observed, consistent with results by [48] in rice and Shafiq et al. [45] in black gram.

Significant improvement in seed nutrient content (N, P, K) was also recorded. This may be due to the presence of amino acids, ascorbic acid, and riboflavin in the bio-stimulant, which facilitate nutrient uptake and assimilation. Carillo et al. [33], Di Mola et al. [49], and Tsouvaltzis et al. [50] all reported increased uptake of macronutrients in crops treated with amino acid-based stimulants. Sharma et al. [3] documented increased phosphorus in chickpea grains following Panchagavya spray. Octavia et al. [51] and Al-Karaki and Othman [26] confirmed higher NPK content in Allium cepa L. and lettuce leaves, respectively, with foliar-applied bio-stimulants.

Our findings also showed improved micronutrient (Zn and B) content in chickpea grains following bio-stimulant application. This may be due to modified root architecture and enhanced nutrient solubility, mobility, and uptake [52, 53]. According to du Jardin [11] and Ribeiro et al. [54], bio-stimulants improve nutrient acquisition and plant resilience. El Sheikha et al. [40] and Octavia et al. [51] similarly reported increased Zn and B content in crops treated with yeast extract and amino acid-based bio-stimulants.

The increase in grain protein content may be attributed to elevated nitrogen accumulation, which supports amino acid and protein synthesis. As nitrogen is a primary component of protein, its increased availability promotes higher protein biosynthesis [55]. Jagtap et al. [56] noted that organic treatments increased nitrogen content and protein yield in grains. Boghdady et al. [57] also reported enhanced protein profiles in chickpea seeds treated with seaweed extract.

The positive responses observed in this study can be explained by several well-established physiological mechanisms associated with amino-acid-based biostimulants. Actibion contains high proportions of glutamic acid, aspartic acid, and proline, which function as precursors for chlorophyll synthesis and key intermediates in nitrogen assimilation pathways. Glutamate and aspartate drive amino-acid and protein biosynthesis and stimulate Rubisco activity, thereby enhancing photosynthetic capacity and biomass accumulation [33]. Proline acts as an osmoprotectant, stabilizing membranes and enzymes under transient water stress that commonly occurs in rainfed systems, which may explain the higher chlorophyll retention and CGR observed in our study. Amino acids also act as signaling molecules that promote root system expansion, increasing the plant’s ability to acquire N, P, K, Zn, and B from nutrient-poor sandy soils [11, 12]. Enhanced nodulation under Actibion application supports this mechanism, as amino acids such as arginine and serine increase N-fixation efficiency. The combined improvement in nutrient uptake and carbon assimilation likely contributed to greater assimilate allocation to reproductive organs, resulting in higher pod number, seed weight, and protein concentration.

Overall, the results confirm that targeted foliar application of amino-acid-based biostimulants at flower initiation enhances physiological performance, nutrient assimilation, and economic returns in rainfed chickpea, offering a sustainable option for smallholder farmers in semi-arid regions. However, it is important to acknowledge that the response to biostimulant application can vary with soil fertility, climatic conditions, and management practices. The Thal region of Pakistan, where this study was conducted, typifies semi-arid rainfed conditions common to many chickpea-growing areas in South Asia, the Middle East, and parts of Africa. While absolute yield gains may differ across environments, the consistent physiological response to foliar amino-acid application at the flower initiation stage suggests that the underlying mechanism is broadly applicable. Nevertheless, multi-location and multi-year trials would be valuable for refining regional recommendations.

Conclusion

The present study demonstrated that foliar application of bio-stimulant at 1250 mL ha^− 1, particularly when applied at the flower initiation stage, significantly improved the growth, physiological traits, yield attributes, seed quality, and nutrient (macro and micronutrient) content of chickpea grown under rain-fed conditions. Among all treatments, application at the flower initiation stage resulted in the highest increases in plant height, chlorophyll content, pod and seed development, and 100-seed weight. This treatment also recorded the greatest economic returns, indicating its superiority in both agronomic performance and profitability. These findings suggest that a single foliar spray of bio-stimulant at 1250 mL ha^− 1 during the flower initiation stage offers a cost-effective, sustainable, and practical approach for enhancing chickpea productivity and nutritional quality under rain-fed environments. Because the physiological mechanisms underlying these responses—enhanced N assimilation, osmoprotection, and increased nutrient uptake—are not region-specific, the findings have relevance for chickpea production across semi-arid rainfed environments globally. The identification of flower initiation as the optimal spray stage provides a transferable, timing-based strategy for biostimulant use, independent of location.

Abbreviations

FI

Flower initiation

PI

Pod initiation

FPI

Flower + pod initiation

LAI

Leaf area index

LAD

Leaf area duration

CGR

Crop growth rate

BCR

Benefit–cost ratio

PARC

Pakistan Agricultural Research Council

AARI

Ayub Agricultural Research Institute. The standardized unit–symbol glossary used throughout the manuscript has been provided in Table S2.

Authors’ contributions

All authors contributed significantly to the conceptualization, experimental design, data collection, analysis, interpretation, manuscript writing, and critical revision. All authors read and approved the final manuscript.

Funding

This research was supported by the Ongoing Research Funding (ORF) program, King Saud University, Riyadh, Saudi Arabia (Project No. ORF-2026-218).

Data availability

All data generated or analyzed during this study are included within this published article.

Declarations

Ethics approval and consent to participate

Not applicable. This study did not involve human participants or animals and was conducted using a cultivated crop species under standard agronomic field conditions.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.