The Combined Effects of Gibberellic Acid and Rhizobium on Growth, Yield and Nutritional Status in Chickpea (Cicer arietinum L.)

Abstract

Plant growth regulators and Rhizobium are actively involved in the regulation of flowering, pod formation, nodulation, and ultimately the growth and yield of legumes. However, very limited information is available on the combined effect of gibberellic acid (GA₃) and Rhizobium on growth attributes and yield of legume crops. This experiment was designed to fill this gap by studying the performance of chickpea under exogenous application of GA₃ (10⁻⁴ and 10⁻⁵ M) alone and in combination with Rhizobium. Exogenous application of GA₃ (10⁻⁵ M) combined with rhizobium inoculation gave the highest values for number of nodules per plant (16) and their dry biomass (0.22 g). Moreover, GA₃ application and seed inoculation with Rhizobium, when applied singly, significantly enhanced chickpea growth. However, the most promising results were obtained by the inoculation of Rhizobium accompanied with GA₃ (10⁻⁵ M). Plant height, grain and stover yield, and chlorophyll contents were enhanced up to 35%, 39%, 21%, and 51%, respectively. Likewise, the bioaccumulation of macronutrients (N, P and K) was maximum in plants receiving both Rhizobium inoculation and GA₃ application. It is concluded that the combined application of Rhizobium and GA₃ has synergistic effects on the growth, yield, and nutrient contents of chickpea.

1. Introduction

Agricultural revolution and crop intensification are among the recent challenges to supply food and fiber for ever increasing human population [1,2]. However, poor soil fertility and low fertilizer use efficiency hinders sustainable crop production [3,4]. It has now become a great dilemma for the scientific community to increase per hectare yield and utilization of existing arable lands to sustain food production. For this, the use of chemical fertilizers has gained popularity among farmers to improve soil fertility and increase crop yields [5]. At present, about 40–60% crop (cereal) production relies on synthetic fertilizers and it is estimated that by 2050 the crop production will have to rely on fertilizers up to 110% [6]. The use of inappropriate agro-technology by the farmers on the other hand has further excavated this problem in the form of environmental pollution, deterioration of soil quality and loss of biodiversity [7,8]. This is especially true for Pakistan, where the unwise use of agrochemicals, imbalanced fertilization, utilization of untreated wastewater and farm wastes are in common practice for agricultural production [9]. There is a significant difference between the quantity of applied fertilizers and that utilized by the plants. Only a small fraction of the applied fertilizer is utilized by the plants and the rest is lost in the environment and hence causes negative effects on the environment. Therefore, the application of alternative approaches that are environmentally sound, cost effective, and farmer friendly are required to overcome these constraints both agronomically and economically [10].

Chickpea is the most vital grain legume of Leguminosae family cultivated around the world and an important source of fiber, oil, fat, ash and protein [11]. Biological nitrogen fixation (BNF) is a physiological process that facilitates agricultural crop production by adequate N supply [12]. Being a leguminous plant, chickpea establishes a symbiotic relationship with rhizobia present in its root nodules and thus support BNF [2]. These plants are associated with an array of bacteria having the ability to reduce atmospheric (inert N₂) into a form available for plants through BNF [13]. A lack of suitable rhizobial strains causing poor nodulation, flower drop and pod shedding [14] is among the main causes of lower production of chickpea [15,16]. In soils, deprived of suitable rhizobial strains, the artificial inoculation of chickpea seed is an effective approach for enhancing root nodulation and ultimately yield [2,17]. It is well established that rhizobia associate with or adhere nearby the roots of legumes and promote growth through phosphate solubilization, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity, siderophore production, indole-3-acetic acid (IAA) production, catalase, oxidase, etc. [18,19,20,21].

Root nodulation is a complex and sensitive phenomenon requiring synchronized bacterial infection, plant organogenesis and root growth [22,23,24]. Signaling pathways of nodulation factors are stimulated through Ca²⁺ nuclear oscillations and spiking to initiate root nodulation in cortical cells [25,26]. Almost all signals and organogenesis measures are extensively affected by the hormones balance in plants [27]. These hormones affect root nodulation and nitrogen fixation in legumes both positively and negatively. The stimulating effects of phytohormones, i.e., auxins and cytokinins in root nodulation are well documented. Moreover, auxins, cytokinins, and gibberellins (gibberellic acids, GAs), through crosstalk with cytokinins signaling paths, regulate the root nodulation in legumes [28].

Legume–Rhizobium symbiosis is mostly reliant on environmental contexts, soil fertility, and plant growth stages. Owing to these factors, plants may be incapable to biosynthesize adequate amount of these phytohormones endogenously. Exogenous application of these phytohormones results in enhanced crop yield by regulating pod formation and flower drop [16,29]. The plants respond to exogenous application of phytohormones and among plant hormones, GA₃ is an important growth hormone and exogenous application of GA₃ in legumes regulates nodule formation [28].

The nodulation process is sensitive to environmental fluctuations and further subject to soil conditions that might affect nodule formation. In this regard, the exogenous application of gibberellic acid (GA₃) could be an important strategy to support nodulation process and sustain crop production. Moreover, the combined use of gibberellic acid (GA₃) and Rhizobium sp. has already been shown to improve growth, physiology and yield of crops [30]. Various reports are available where alone application of Rhizobium or plant growth regulators (PGRs) have been investigated to enhance growth and yield of various leguminous and non-leguminous crops [2,31,32,33,34]. However, their combined effects on crops have rarely been studied. Taking this in view, we hypothesized that combined application of gibberellic acid (GA₃) and rhizobia may enhance growth, nodulation and yield of chickpea, while their effects may vary based on the concentration levels. Therefore, the aim of the present study was to evaluate the combined effect of Rhizobium sp. and exogenously applied GA₃ on growth, root nodulation, and ultimately the yield of chickpea and to evaluate the possible correlations among applied treatments and studied parameters.

2. Materials and Methods 2.1. Collection of Nodulated Chickpea Roots and Isolation of Rhizobium Species Several healthy nodulated plants of chickpea were collected from diverse fields of Ayub Agricultural Research Institute (AARI), Faisalabad, Pakistan. These plants were uprooted carefully and selected, based on pink colored healthy nodules, transported to the Soil Bacteriology Section, Agri. Biotechnology Research Institute, Faisalabad, Pakistan in polythene bags.

Rhizobium sp. was isolated in yeast extract mannitol agar (YEMA) medium [35]. Pink, firm, healthy, and unbroken nodules were rinsed under sterilized water to eliminate adhering soil particles. Nodules surface sterilization process was carried out by dipping in 70% ethyl alcohol, followed by repeated washing in sterilized water, and then a 3% sodium hypochlorite solution was used for further surface sterilization [36]. The nodules were surface washed three times with sterile water and crushed in a crucible. Suspension of crushed nodules was plated on the YEMA medium having 1% Congo-red dye followed by 24 h incubation at 28 ± 2 °C. Purified growth was shifted to the broth medium of yeast mannitol (YM) and kept in a shaker (revolving at 75 rpm) at 28 ± 2 °C and preserved in the refrigerator at 4 °C.

2.2. Identification and Phenotypic Characteristics of Isolates

Ten pure isolated cultures were made to perform Gram reaction and then subjected to biochemical tests and morphological traits. The potential of rhizobial cultures to grow on various salt concentrations was examined by streaking on YEM broth with 0.5, 1, 1.5, 2, and 2.5% (w/v) NaCl [37].

The ability to grow on different pH levels of these bacterial isolates was determined by adjusting pH values between 4 and 9 in triplicates [38]. Glucose peptone agar and lactose assays were used to test the ability of the microbes for glucose and lactose utilization as a sole carbon source. Furthermore, biochemical tests such as gelatin hydrolysis, fluorescence assay, triple sugar ion agar, lipase [39], lysine decarboxylase, citrate, urease, starch hydrolysis, and bromothymol blue tests [40] were also performed.

The selected isolates of Rhizobium sp. were identified and confirmed through Biolog^® identification system (Microlog System Release 4.2; Biolog Inc., Hayward, CA, USA) (Table 1). The Biolog^® identification system is equally effective as 16S rRNA gene sequencing for identification of Rhizobium sp. [41].

2.3. Detection of IAA and GA in Broth Culture

Auxin production of Rhizobium was determined calorimetrically in terms of IAA equivalents. Five days old starter cultures of Rhizobium were used in Yeast Mannitol broth (150 mL) and placed on a shaker revolving at speed of 75 rpm at 28 ± 2 °C for 72 h followed by centrifugation (10,000 rpm for 20 min). After centrifugation, 3 mL of supernatants were used with 2 mL of Salkowski’s reagent, i.e., 2 mL 0.5 M FeCl₃ and 98 mL 35% HClO₄. This mixture was incubated at room temperature (30 min). Spectrophotometric absorbance was recorded at 535 nm. The standardized curve [42] was used to determine auxin concentration. The isolates giving the highest values of auxin were selected for further study.

The gibberellin contents were measured calorimetrically by using a standard procedure [43] with slight modifications. In this method, 2 mL zinc acetate reagent were added in 15 mL of supernatant. After 2 min, 2 mL potassium ferrocyanide was added in the mixture followed by centrifugation for 15 min (2000 rpm). After centrifugation, 5 mL HCl (30%) was added in the supernatant (5 mL) and incubated for 75 min at 20 °C. A standard curve was prepared using GA₃ (Hi-media) in concentrations ranging from 100 to 1000 µg m⁻¹. Spectrophotometric absorbance was recorded at 254 nm. In the case of blank, 5% HCl was used.

2.4. Seed Bacterization of Cicer Arietinum L. The inoculum of selected Rhizobial isolate R4 (based on high GA production and other growth promoting traits) was prepared in 200 mL Erlenmeyer flask containing yeast extract mannitol (YEM) medium (Yeast, 0.5 g; mannitol, 10.0 g; K₂HPO₄, 0.5 g; MgSO₄·7H₂O, 0.2 g; NaCl, 0.1 g; distilled water, 1000 mL; pH, 6.8), incubated at 28 °C in a shaking incubator at 160 rpm for 2 days. An optical density of 0.5 (OD_0.5), measured with a spectrophotometer at λ 600 nm, was achieved by dilution to maintain a uniform cell density (10⁸ CFU mL^–1) prior to seed inoculation. Surface sterilized healthy seeds were inoculated with Rhizobium broth (10 mixed with 10% jaggery solution and sterilized peat. Seeds were mixed thoroughly until a fine layer of inoculum appeared on them and spread on the polythene sheet to dry in the laboratory overnight. 2.5. Pot Experiment Effect of Rhizobium inoculant alone or combined with two different concentrations of GA₃ i.e., 10⁻⁴ M and 10⁻⁵ M on growth and yield parameters of chickpea was studied in a wire house pot experiment at the research area of the Soil Bacteriology Section, AARI, Faisalabad, Pakistan. The soil used for the experiment was air-dried, thoroughly mixed and sieved (2 mm). The soil was sandy clay loam having 7.89 pH, 1.36 dS m⁻¹ EC, 0.77% organic matter, 0.034% total nitrogen, 7.66 mg kg⁻¹ available phosphorus 112 mg kg⁻¹ extractable potassium. Each pot contained 16 kg soil. The research experiment contained the following six treatments: (1) Control, (2) Rhizobium inoculation, (3) GA₃ (10⁻⁴ M), (4) GA₃ (10⁻⁵ M), (5), Rhizobium + GA₃ (10⁻⁴ M), (6), and Rhizobium + GA₃ (10⁻⁵ M). Surface sterilized chickpea seeds were inoculated with Rhizobium sp. R4 using slurry as described previously. In control, seed was coated with sterilized LB broth slurry. Four chickpea seeds were sown in each pot and later thinned to two plants after 7 days of sowing (DAS). The trial was conducted in CRD design with four replications. Recommended dosages of chemical fertilizers (N, P & K) were applied. 2.6. Agronomic Traits Agronomic parameters like plant height, pods plant⁻¹, nodules plant⁻¹, nodule’s weight, and yield (grain and stover) were observed before and after harvesting chickpea plants. For nodulation data, one plant from each replicate was pulled out, with roots, washed in distilled water and mature nodules per plant were weighed and the average was computed. At harvesting (100 DAS after seedling emergence) pods from each plant were taken, counted, and threshed to get grain yield, and the average was worked out. Fifty seeds weight was calculated from the seed lot of each treatment and then mean value was computed. 2.7. Physiological Traits

Physiological attributes were recorded at mid-day (between 10:00 and 14:00) after 60 DAS. For the determination of chlorophyll contents, fully green leaves were selected for the measurement of photosynthetic pigments. To determine chlorophyll contents (a and b), plant leaves were crushed in acetone. The mixture was centrifuged for 10 min at 1000 rpm and absorbance at 645 nm for chlorophyll “a” and 663 nm for chlorophyll “b” was recorded on spectrophotometer [44].

To determine the volume of pink bacteroid tissue of nodules, root nodules were taken at the flowering stage and a minor section of nodules (5 μL) made by using sharp. The amount of pink bacteroid tissue comprising of the leghaemoglobin present in the nodule cortex was noted following the method described by Sadasivan and Manickam [45].

2.8. Physicochemical Analysis of Plant and Soil

Pre and post sowing analysis of soil regarding N, P, and K was performed following the procedure of Mehlich and Mehlich [46]. After harvesting, the representative samples of seeds and stovers were oven dried for 24 h at 65 °C and ground. Nitrogen contents were determined by micro Kjeldahl’s method, phosphorus contents were measured by yellow method and K content by Flame photometric method described by Jackson [47].

Protein and carbohydrate contents in the dry seeds were estimated as described by Lowry et al. [48].

2.9. Statistical Analysis

The collected data regarding growth, yield, and biochemical parameters were subjected to analysis of variance [49]. The significance of differences among treatment means was tested through LSD test (IBM SPSS Statistics 19, USA). Directional relationship among observed parameters and treatments were estimated through Pearson correlation and principal component analysis (R Software^® 4.0.2).

3. Results 3.1. Biochemical Characterization

The microscopic studies showed that colonies were rod-shaped gram-negative. All isolates were non-spore forming and motile rods. The colonies of all isolates were circular, 2–4 mm, milky white, transparent (Table 1). Some Rhizobium strains can grow under high salt concentration. In the present investigation, it was found that all the strains showed growth at 0.5% (w/v) NaCl and continued grown up to 2.5% (w/v) NaCl concentrations. All isolates could grow in the yeast extract mannitol agar (YEMA) medium with pH range 4–9. In the present study, optimum pH for Rhizobium isolates growth was found to be 6–7. The minimal growth of isolates was exhibited at pH 4 and pH 9. All isolates were streaked on Bromothymol blue added YEMA selective media for further confirmation. All four strains showed growth in two days and turned YEMA media from blue to yellow, confirming their nature of being fast growers and acid producers. A starch hydrolysis assay was examined to determine the production of reducing sugar from starch in bacteria. The clear zone around colonies was observed after the addition of iodine. Lysine decarboxylase test was performed using Bromocresol purple Falkow media. Results showed a change in the color of medium inoculated with Rhizobium sp. Lipase Test was found to be positive for Rhizobial isolates R2-R7 and R10. Glucose, sucrose, and starch proved equally good for growth of strains under study (Table 1). Rhizobial cells growth on GPA medium depicted the capability of Rhizobium to use Glucose as Carbon source (Table 1). However, no growth was observed in case of lactose. Moreover, the solidification of gelatin medium at 4 °C for 30 as well as 60 min depicted that Rhizobial cells didn’t produce gelatinase. Furthermore, Rhizobium cells failed to fluoresce on King’s medium under the UV (Table 1). Production of indole acetic acid (IAA) was common to all Rhizobium isolates, but the highest value was recorded in R₃ (6.86 µg mL⁻¹). Moreover, in the case of gibberellic acid, maximum concentration was produced by R₄ (11.36 mg L⁻¹). Biolog identification showed that all isolates exhibited similarity to Mesorhizobium ciceri. However, the maximum similarity index (80%) with Mesorhizobium ciceri was recorded in the case of R₂ isolate compared to the others.

3.2. Growth and Yield Parameters

Growth and yield parameters of chickpea were increased significantly with foliar GA₃ application and Rhizobium inoculation either alone or combined (Figure 1 and Figure 2).

Rhizobium inoculation significantly enhanced the plant height, number of nodules plant⁻¹, number of pods plant⁻¹ grain yield, and fresh and dry weight of nodules over the control (Figure 1 and Figure 2). Foliar spray of GA₃ on Rhizobium inoculated plants had an additive effect on plant height, nodules plant⁻¹, and fresh and dry weight of nodules by 35, 55%, 16%, and 12%, respectively (Figure 1). Rhizobium supplemented with GA₃ (10⁻⁵ M) gave the maximum increase in stover biomass, i.e., 21%, compared to control. Statistically significant increase in grain yield was recorded with the alone applications of Rhizobium and GA₃ (10⁻⁵, 10⁻⁴ M). Rhizobium seed treatment enhanced grain yield up to 10% over control (Figure 1). The combined application of Rhizobium and GA₃ (10⁻⁴ M) increased the yield by 39% than control. The least increase in grain yield i.e., 9%, was recorded with the alone application of GA₃ (10⁻⁴ M). Rhizobium inoculation supplemented with foliar application of GA₃ increased the pods plant⁻¹ and dry pod weight. Maximum increase (41% and 57%, respectively) was recorded for GA₃ at 10⁻⁴ M on Rhizobium inoculated treatments (Figure 2).

3.3. Physiological and Quality Parameters

Rhizobium inoculation along with foliar GA₃ application positively increased the chlorophyll a and b contents (Figure 2). Rhizobium and GA₃ (10⁻⁵ M) combined gave the maximum increase of 51%. Alone application of Rhizobium resulted in an increase of 15% in chlorophyll contents over control.

It was observed that the highest leghaemoglobin contents (0.40 mg/g) were recorded with the combined application of GA₃ (10⁻⁵ M) with Rhizobium inoculation in comparison to the control treatment (Figure 2).

Data regarding seed carbohydrate and protein contents were positively increased by the inoculation of Rhizobium but this response was further enhanced with GA₃ application. The highest increase of seed carbohydrate (16%) and protein contents (12%) were recorded for GA₃ (10⁻⁴ M) with Rhizobium inoculation (Figure 2).

3.4. Chemical Analysis of Soil and Plant

Application of Rhizobium or GA₃ improved nitrogen (N), phosphorous (P), and potassium (K) contents individually (Figure 3). A higher increase in stover N, P, and K contents by 18%, 47%, and 15% was attained with the mutual application of Rhizobium and GA₃ (10⁻⁵ M). Similarly, Rhizobium along with GA₃ (10⁻⁵ M) enhanced seed N, P, and K contents by 4%, 21%, and 34%, respectively (Figure 3). Foliar GA₃ application and Rhizobium both increase nutrient contents when applied individually. Maximum gain in N, P, and K contents (0.037%, 8.03 mg kg⁻¹, 119 mg kg⁻¹) was recorded application of GA₃ (10⁻⁵ M) and Rhizobium (Figure 3).

3.5. Pearson Correlation and Principal Component Analysis

All agronomic traits, i.e., plant height, nodules weight (fresh and dry), pod dry weight, pods number, and yield (grain and stover) exhibited strong positive correlation with physiological parameters (chlorophyll contents), biochemical attributes (leghemoglobin contents, seed carbohydrate, protein contents), and mineral contents of the plant (stover K, seed N, stover P, seed P, stover N, and seed K) (Figure 4).

The score and biplots for some important traits of chickpea are shown in Figure 5. Of the extracted components, PC₁ and PC₂ explained maximally and accounted for 91.3% of total observed variation in the dataset. The first component PC₁ accounted for (80.9%) while the second component accounted for (10.4%) of the total variance (Figure 5A,B). Thus, both of these components accounted for most of the positive effects of combined application of GA₃ and Rhizobium on growth and yield of chickpea plants. Principle component analysis further confirmed that growth hormone alone or in combination with Rhizobium significantly improve studied attributes of chickpea plants. All the six applied treatments were successfully separated by the principal components (Figure 5A). The control, Rhizobium inoculation and GA₃ (10⁻⁴ M) were clustered in PC₁ whereas, GA₃ (10⁻⁵ M), Rhizobium + GA₃ (10⁻⁴ M) and Rhizobium + GA₃ (10⁻⁵ M) were clustered in PC₂. This distribution of treatments showed combined application of GA₃ and Rhizobium had positive effects on growth and yield determining attributes of chickpea (Figure 5A). Moreover, the vectors representing the tested attributes of chickpea plants were displaced away from the point of origin and reached to the edges, further indicating that there exists a strongly positive correlation among these variables as indicated by the sharp angles of vectors (located close to each other) (Figure 5B). PC₁ was defined as a collective function of grain yield (GY), stover yield (SY), chlorophyll contents (Chl), number of pods (N pod), seed carbohydrate contents (SC), plant height (PH), leghaemoglobin contents (leg HB), dry pod Weight (DPW), protein contents (protein), weight of nodules (Wt Nod), dry weight of nodules (DWN), stover K (STK), seed N (SDN), stover P (STP), seed P (SDP), stover N (STN), and seed K (SDK).

4. Discussion

Biological nitrogen fixation (BNF) as an important source of nitrogen (N) is key process to modern sustainable agriculture. Enduring sustainability in agriculture can be achieved by using effective management of resources. It is irrational to consider agricultural sustainability on a broader scale without biological nitrogen fixation. Plant growth promoting rhizobacteria (PGPR) are important to plant growth through producing phytohormones, fixing N₂ symbiotically, inorganic phosphate solubilizing, organic phosphate mineralization, and nutrients supplementation [50,51,52,53,54]. We found higher growth and yield attributes of chickpea under the application of Rhizobium and gibberellic acid GA₃ in a pot trial (Figure 1 and Figure 2).

The beneficial effects of Rhizobium alone and supplemented with other growth stimulators in the present study are in accordance with the previous reports [55,56,57]. Application of plant growth regulators (PGRs) especially GA₃ exert stimulatory and beneficial effects on plant growth and development which is well studied in green gram, mungbean, chickpea, tomato, cowpea, and china aster [58,59]

The results of this study confirm the alteration in plant height in response to foliar application of GA₃ (Figure 1). In chickpea, shoot length significantly increases by inoculation with Rhizobium [51,52,58]. Likewise, the application of GA₃ has a positive effect on shoot and root length in chickpea [60]. It might be due to the role of gibberellins in cell multiplication, elongation, and metabolic pathways of protein synthesis. It has been observed that plants have potential to store excess of exogenously applied hormones as reversible conjugates and are released during the growth period as active hormone where and when plants need them. Earlier, it has been found that PGRs play important role in source and sink dynamics of field crops through improving distribution and transportation of accumulates [29,61].

In the present study, yield parameters were also increased significantly with foliar application of plant growth regulator inoculated with rhizobia. The foliar application of PGRs improves inflorescence, pod development, seed index, harvest index, biomass, and yield in chickpea [62]. Neelima et al. [63] observed that yield components of chickpea i.e., flower retention, number of pods, pod setting, and seed index increase significantly by foliar application of GA₃ and Cycocel^®. In the present study, results depicted that nodulation is improved in Rhizobium treated chickpea alone and complemented with exogenous application of GA₃. This may be attributed to increased N-fixation by Rhizobium due to the increasing contents of nitrogenase [2]. This premise is supported by the increased accumulation of N in the plant biomass (Figure 3).

The longevity and efficacy of root nodules are favorably influenced by the exogenous application of PGRs [64]. In the present study, an increasing trend in nodule dry weight was observed after inoculation with Rhizobium alone or in combination with PGR. Also, Rhizobium inoculation supplemented with GA₃ had more positive effect and produced the maximum pods and straw yield than only Rhizobium inoculated plants. GA₃ is known to increase number of pods, pods yield per plant (fresh and dry) and seed index in chickpea, green gram and cow pea [14,59,65], whereas chickpea sown in soil treated with Rhizobium produced pods with more seeds [66]. In present study, foliar application of GA₃ on Rhizobium inoculated chickpea also resulted in increased chlorophyll contents [14]. These results are further substantiated by the findings of Tripathi et al. [67].

In the present study, protein contents increased with the alone or combined application of Rhizobium and GA₃ (Figure 2). Earlier, it has been found that exogenous GA₃ spray promote protein contents of tomato [68]. Protein content of seed is improved through application of Rhizobium in the present study (Figure 2). These results are in line with Rabbani et al. [55] who reported increased biochemical constituents in pea seeds. Jain et al. [69] reported the response of growth regulators (GA_3, IAA, and Kinetin) on leghaemoglobin synthesis and noted that about all PGRs expressed overall stimulatory effect on the production of leghaemoglobin. Nitrogenase activity of root nodules is raised by the increasing volume of pink bacteroid tissue. The application of GA₃ resulted in enhanced nodule bacteroid regions and leghaemoglobin contents over untreated control.

In this study, carbohydrate contents of chickpea seeds were enhanced with the combined application of GA₃ and Rhizobium. Under drought, the amylase activity of cotyledons is affected severely which is significantly improved with the alone application GA₃ [70]. The application of GA₃ and rhizobium is a win-win strategy to enhance agricultural production.

In the current study, an increasing trend in the nutrient contents of grain, stover, and soil was observed. These findings are supported by Gupta et al. [71] who observed increased contents of N, P and P and higher fertilizer use efficiency under the application of PGRs. This attribute may be due to enhanced root growth and higher nodulation observed with the combined application GA₃ and Rhizobium in the present study. Moreover, the higher nutrient concentrations in chickpea seeds in the present study may also be due to the increased supply of nutrients due to applied rhizobia and GA₃ [72,73].

Rhizobia are well recognized plant growth promoting bacteria that enhance plant growth through plenty of mechanisms, such as nutrient solubilization, production of secondary metabolites, enhanced nitrogen fixation, and production of plant growth hormones [12,15]. The application of gibberellic acid on the other hand enhances plant growth by mediating of root nodulation process in legumes through cross talk with cytokinins signaling pathways [28]. This synergy was observed in the present study, evidenced by the enhanced root nodulation under the application of gibberellic acid and rhizobium in combination. Moreover, the applied rhizobia have shown the capacity to synthesize gibberellic acids and IAA along with other growth promoting activities (Table 1), which might have contributed to enhanced growth, yield, and nutritional quality parameters of chickpea.

5. Conclusions The present study clearly showed that the combined application of Rhizobium and gibberellic acid (GA₃) played a significant role in improving the growth, yield, physiological, and biochemical attributes of chickpea. It is worthy to mention that by comparing the independent application of both, the combined application showed more promising results. The combined use of Rhizobium and plant growth regulator would be an effective approach to enhance legume productivity. However, multi-sites field trials are required to warrant the successful performance of the technology under natural field conditions.

Figure 1. Effect of plant growth hormone and Rhizobium sp. On growth (A,B), nodulation (C,D) and yield (E,F) attributes of chickpea. T1: untreated control, T2: Rhizobium sp. Inoculation, T3: GA3 (10-4 M), T4: GA3 (10-5 M), T5: Rhizobium + GA3 (10-4 M), T6: Rhizobium +GA3 (10-5 M). Quantities sharing similar letters are not statistically different from each other at p > 0.05.

Figure 2. Effect of plant growth hormone and Rhizobium sp. On some yield (A-D) and quality (E-G) parameters of chickpea. T1: untreated control, T2: Rhizobium sp. Inoculation, T3: GA3 (10-4 M), T4: GA3 (10-5 M), T5: Rhizobium + GA3 (10-4 M), T6: Rhizobium +GA3 (10-5 M). Quantities sharing similar letters are not statistically different from each other at p > 0.05.

Figure 3. Effect of plant growth hormone and Rhizobium sp. On plant (A-C) and soil (D-F) nutrient contents of chickpea. T1: untreated control, T2: Rhizobium sp. Inoculation, T3: GA3 (10-4 M), T4: GA3 (10-5 M), T5: Rhizobium + GA3 (10-4 M), T6: Rhizobium + GA3 (10-5 M). Quantities sharing similar letters are not statistically different from each other at p > 0.05.

Figure 4. Represents correlation among measured parameters, where abbreviations of correlation matrix are as Grain Yield (GY), Stover Yield (SY), Chlorophyll contents (Chl), Number of pods (N pod), Seed carbohydrate contents (SC), Plant height (PH), Leghaemoglobin contents (Leg HB), Dry pod Weight (DPW), Protein contents (Protein), Weight of nodules (Wt Nod), dry weight of nodules (DWN), Stover K (STK), Seed N (SDN), Stover P (STP), Seed P (SDP), Stover N (STN), and Seed K (SDK). Dark red = highly positive, Light red = less positive.

Figure 5. Score plots (A) and loading plots (B) of PCA on different attributes of chickpea by application of plant growth hormone alone and in combination with Rhizobium sp. Score plots (A) represents separation of treatments as (1) Control, (2) Rhizobium inoculation, (3) GA3 (10-4 M), (4) GA3 (10-5 M), (5) Rhizobium inoculation + GA3 (10-4 M), (6) Rhizobium inoculation + GA3 (10-5 M). The abbreviations of loading plots (B) are as Grain Yield (GY), Stover Yield (SY), Chlorophyll contents (Chl), Number of pods (N pod), Seed carbohydrate contents (SC), Plant height (PH), Leghaemoglobin contents (Leg HB), Dry pod Weight (DPW), Protein contents (Protein), Weight of nodules (Wt Nod), dry weight of nodules (DWN), Stover K (STK), Seed N (SDN), Stover P (STP), Seed P (SDP), Stover N (STN), and Seed K (SDK).

Figure 5. Score plots (A) and loading plots (B) of PCA on different attributes of chickpea by application of plant growth hormone alone and in combination with Rhizobium sp. Score plots (A) represents separation of treatments as (1) Control, (2) Rhizobium inoculation, (3) GA3 (10-4 M), (4) GA3 (10-5 M), (5) Rhizobium inoculation + GA3 (10-4 M), (6) Rhizobium inoculation + GA3 (10-5 M). The abbreviations of loading plots (B) are as Grain Yield (GY), Stover Yield (SY), Chlorophyll contents (Chl), Number of pods (N pod), Seed carbohydrate contents (SC), Plant height (PH), Leghaemoglobin contents (Leg HB), Dry pod Weight (DPW), Protein contents (Protein), Weight of nodules (Wt Nod), dry weight of nodules (DWN), Stover K (STK), Seed N (SDN), Stover P (STP), Seed P (SDP), Stover N (STN), and Seed K (SDK).

Characteristics	Rhizobial Isolates
	R1	R2	R3	R4	R5	R6	R7	R8	R9	R10
Phenotypic and physiological characterization
Colony color	White	Milky white	Milky white	Milky white	Milky white	Milky white	Milky white	Milky white	Milky white	Milky white
Shape	Circular	Circular	Circular	Circular	Circular	Circular	Circular	Circular	Circular	Circular
Size (mm)	2.5	3.1	2.1	1.8	3.0	2–3	2.6	2–4	2–4	2.5
Opacity	Transparent	Transparent	Transparent	Translucent	Transparent	Transparent	Transparent	Transparent	Transparent	Transparent
Gram reaction	Negative	Negative	Negative	Negative	Negative	Negative	Negative	Negative	Negative	Negative
Bacterium shape	Rod	Rod	Rod	Rod	Rod	Rod	Rod	Rod	Rod	Rod
Motility test	Motile	Motile	Motile	Motile	Motile	Motile	Motile	Motile	Motile	Motile
Bacterial growth conditions a,b
NaCl
0.5%	++	+	+	++	++	++	++	++	+	+
1.0%	++	+	+	+	++	++	++	++	++	++
1.5%	+	+	+	+	+	++	++	++	++	+
2.0%	+	++	+	+	++	+	++	++	++	+
2.5%	++	++	++	++	+	+	++	+	+	++
pH
4	−	+	+	−	+	+	+	+	+	+
5	+	+	+	+	+	+	+	+	−	+
6	+	+	+	+	+	+	+	+	+	+
7	+	+	+	+	+	+	+	+	+	+
8	+	+	+	+	+	+	+	−	+	−
9	−	−	+	+	+	−	+	+	+	+
Carbon utilization potential
GPA assay	+	−	+	+	+	+	+	+	+	+
Lactose Assay	−	+	−	−	−	−	−	−	−	−
Biochemical Characterization
Gelatin Hydrolysis	−	−	−	−	−	−	−	−	−	−
Fluorescence assay	−	−	−	−	−	−	−	−	−	−
Triple sugar Ion	+	+	+	+	+	+	+	+	+	+
Lipase	−	+	+	+	+	+	+	−	−	+
Lysine	+	+	+	+	+	+	−	+	−	+
Citrate	+	+	+	+	+	+	+	−	+	+
Urease	+	+	+	+	+	+	+	+	+	−
Starch Hydrolysis	−	−	−	+	+	+	−	−	+	+
Bromothymol blue	+	+	+	+	+	+	+	+	+	+
Auxin (IAA equivalents µg mL−1)	4.34	5.67	6.86	4.23	3.01	2.34	4.45	5.68	4.21	2.47
Gibbrellic acid (mg L−1)	5.37	8.34	4.87	11.36	7.42	7.87	6.11	8.75	8.99	7.01
Biolog identification and similarity index %
Mesorhizobium ciceri	76	80	74	76	74	73	72	78	76	74

a −, absent; +, present b +, low efficiency; ++, high efficiency.

Author Contributions

Conceptualization, M.N. and M.R.; methodology, M.R.; software, A.M. and M.M.; validation M.N. and S.A.; formal analysis, S.K. and M.R.; investigation, M.N. and H.M.A.; resources, H.M.A. and M.H.S.; data curation, M.R. and A.M.; writing-original draft preparation M.R., M.N.; review, and editing, M.N., M.H.S., M.H.S., and M.Z.M.S.; supervision S.A. and M.N.; funding acquisition, H.M.A. All authors have read and agreed to the published version of the manuscript.

Funding

Researchers Supporting Project number (RSP-2020/123), King Saud University, Riyadh, Saudi Arabia.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

The authors extent their appreciation to the Researchers Supporting Project number (RSP-2020/123), King Saud University, Riyadh, Saudi Arabia.

Conflicts of Interest

The authors declare no conflict of interest.

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