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Article

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

by
Munazza Rafique
1,
Muhammad Naveed
2,*,
Adnan Mustafa
3,
Saleem Akhtar
1,
Muneeb Munawar
4,
Sadia Kaukab
5,
Hayssam M. Ali
6,
Manzer H. Siddiqui
6 and
Mohamed Z. M. Salem
7
1
Soil Bacteriology Section, Agricultural Biotechnology Research Institute, AARI, Faisalabad 38000, Pakistan
2
Institute of Soil and Environmental Sciences, University of Agriculture Faisalabad, Faisalabad 38040, Pakistan
3
National Engineering Laboratory for Improving Quality of Arable Land, Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing 100081, China
4
Vegetable Research Institute, AARI, Faisalabad 38000, Pakistan
5
Pulses Research Institute, AARI, Faisalabad 38000, Pakistan
6
Botany and Microbiology Department, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia
7
Forestry and Wood Technology Department, Faculty of Agriculture (El-Shatby), Alexandria University, Alexandria 21545, Egypt
*
Author to whom correspondence should be addressed.
Agronomy 2021, 11(1), 105; https://doi.org/10.3390/agronomy11010105
Submission received: 20 November 2020 / Revised: 18 December 2020 / Accepted: 1 January 2021 / Published: 8 January 2021
(This article belongs to the Special Issue Effects of Biostimulants and Bioeffectors on Plant Growth)
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Versions Notes

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 (OD0.5), 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 (GA3, 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.

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.

References

  1. Wik, M.; Pingali, P.; Broca, S. Background Paper for the World Development Report: Global Agricultural Performance: Past Trends and Future Prospects; World Bank: Washington, DC, USA, 2008. [Google Scholar]
  2. Ahmad, M.; Naseer, I.; Hussain, A.; Zahid, M.M.; Mustafa, A.; Hilger, T.H.; Zahir, Z.A.; Minggang, X. Appraising endophyte–plant symbiosis for improved growth, nodulation, nitrogen fixation and abiotic stress tolerance: An experimental investigation with chickpea (Cicer arietinum L.). Agronomy 2019, 9, 621. [Google Scholar] [CrossRef] [Green Version]
  3. Aziz, M.Z.; Yaseen, M.; Abbas, T.; Naveed, M.; Mustafa, A.; Hamid, Y.; Saeed, Q.; Xu, M. Foliar application of micronutrients enhances crop stand, yield and the biofortification essential for human health of different wheat cultivars. J. Integr. Agric. 2019, 18, 1369–1378. [Google Scholar] [CrossRef]
  4. Ashraf, M.N.; Aziz, T.; Maqsood, M.A.; Bilal, H.M.; Raza, S.; Zia, M.; Mustafa, A.; Xu, M.; Wang, Y. Evaluating organic materials coating on urea as potential nitrification inhibitors for enhanced nitrogen recovery and growth of maize (Zea mays). Intl. J. Agric. Biol. 2019, 22, 1102–1108. [Google Scholar]
  5. Pingali, P.L. Green Revolution: Impacts, limits, and the path ahead. Proc. Natl. Acad. Sci. USA 2012, 109, 12302–12308. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  6. Aziz, M.Z.; Yaseen, M.; Naveed, M.; Wang, X.; Fatima, K.; Saeed, Q.; Mustafa, A. Polymer-Paraburkholderia phytofirmans PsJN coated diammonium phosphate enhanced microbial survival, phosphorous use efficiency, and production of wheat. Agronomy 2020, 10, 1344. [Google Scholar] [CrossRef]
  7. Verma, P.; Yadav, A.N.; Kazy, S.K.; Saxena, A.K.; Suman, A. Elucidating the diversity and plant growth promoting attributes of wheat (Triticum aestivum) associated acid tolerant bacteria from southern hills zone of India. Natl. J. Life Sci. 2013, 10, 219–226. [Google Scholar]
  8. Mustafa, A.; Naveed, M.; Abbas, T.; Saeed, Q.; Hussain, A.; Ashraf, M.N.; Minggang, X. Growth response of wheat and associated weeds to plant antagonistic rhizobacteria and fungi. Ital. J. Agron. 2019, 14, 191–198. [Google Scholar] [CrossRef]
  9. Bashir, M.A.; Naveed, M.; Ahmad, Z.; Gao, B.; Mustafa, A.; Núñez-Delgado, A. Combined application of biochar and sulfur regulated growth, physiological, antioxidant responses and Cr removal capacity of maize (Zea mays L.) in tannery polluted soils. J. Environ. Manag. 2020, 259, 110051. [Google Scholar] [CrossRef]
  10. Shakya, M.S.; Patel, M.M.; Singh, V.B. Knowledge level of chickpea growers about chickpea production technology. Indian Res. J. Ext. Educ. 2008, 8, 65–68. [Google Scholar]
  11. Jukanti, A.; Gaur, P.M.; Gowda, C.L.; Chibbar, R. Nutritional Quality and Health Benefits of Chickpea (Cicer arietinum L.). Br. J. Nutr. 2012, 108, S11–S26. [Google Scholar] [CrossRef] [Green Version]
  12. Ali, M.A.; Naveed, M.; Mustafa, A.; Abbas, A. The good, the bad, and the ugly of rhizosphere microbiome. In Probiotics and Plant Health; Springer: Singapore, 2017; pp. 253–290. [Google Scholar]
  13. Umar, W.; Ayub, M.A.; ur Rehman, M.Z.; Ahmad, H.R.; Farooqi, Z.U.R.; Shahzad, A.; Rehman, U.; Mustafa, A.; Nadeem, M. Nitrogen and Phosphorus Use Efficiency in Agroecosystems. In Resources Use Efficiency in Agriculture; Springer: Singapore, 2020; pp. 213–257. [Google Scholar]
  14. Mazid, M.; Khan, T.A.; Mohammad, F. Medicinal Plants of rural India: A review of use by Indian folks. Indo Glob. J. Pharm. Sci. 2012, 2, 286–304. [Google Scholar]
  15. Kantar, F.; Hafeez, F.Y.; Shivakumar, B.G.; Sundaram, S.P.; Tejera, N.A.; Aslam, A.; Bano, A.; Raja, P. Chickpea: Rhizobium management and nitrogen fixation. In Chickpea Breeding and Management; CABI: London, UK, 2007; pp. 179–192. [Google Scholar]
  16. Mazid, M.; Zeba, H.K.; Quddusi, S. Significance of Sulphur nutrition against metal induced oxidative stress in plants. J. Stress Physiol. Biochem. 2011, 7, 165–184. [Google Scholar]
  17. Khattak, S.; Khan, D.F.; Shah, S.H.; Madani, M.S.; Khan, T. Role of Rhizobial inoculation in the production of chickpea crop. Soil Environ. 2006, 25, 143–145. [Google Scholar]
  18. Ditta, A.; Arshad, M.; Zahir, Z.A.; Jamil, A. Comparative efficacy of rock phosphate enriched organic fertilizer vs. mineral phosphatic fertilizer for nodulation, growth and yield of lentil. Int. J. Agric. Biol. 2015, 17, 589–595. [Google Scholar] [CrossRef]
  19. Ditta, A.; Khalid, A. Bio-organo-phos: A sustainable approach for managing phosphorus deficiency in agricultural soils. In Organic Fertilizers—From Basic Concepts to Applied Outcomes; Larramendy, M., Soloneski, S., Eds.; InTech: London, UK, 2016; pp. 109–136. [Google Scholar]
  20. Sarfraz, R.; Hussain, A.; Sabir, A.; Fekih, I.B.; Ditta, A.; Xing, S. Role of biochar and plant growth-promoting rhizobacteria to enhance soil carbon sequestration—A review. Environ. Monit. Assess. 2019, 191, 251. [Google Scholar] [CrossRef] [PubMed]
  21. Zahir, Z.A.; Shah, M.K.; Naveed, M.; Akhter, M.J. Substrate-dependent auxin production by Rhizobium phaseoli improves the growth and yield of Vigna radiata L. under salt stress conditions. J. Microbiol. Biotechnol. 2010, 20, 1288–1294. [Google Scholar] [CrossRef] [PubMed]
  22. Madsen, E.B.; Madsen, L.H.; Radutoiu, S.; Olbryt, M.; Rakwalska, M.; Szczyglowski, K.; Stougaard, J. A receptor kinase gene of the LysM type is involved in legume perception of rhizobial signals. Nature 2003, 425, 637–640. [Google Scholar] [CrossRef]
  23. Limpens, E.; Franken, C.; Smit, P.; Willemse, J.; Bisseling, T.; Geurts, R. LysM domain receptor kinases regulating rhizobial Nod factor-induced infection. Science 2003, 302, 630–633. [Google Scholar] [CrossRef]
  24. Radutoiu, S.; Madsen, L.H.; Madsen, E.B.; Felle, H.H.; Umehara, Y.; Gronlund, M.; Stougaard, J. Plant recognition of symbiotic bacteria requires two LysM receptor-like kinases. Nature 2003, 425, 585–592. [Google Scholar] [CrossRef]
  25. Gleason, C.; Chaudhuri, S.; Yang, T.; Munoz, A.; Poovaiah, B.W.; Oldroyd, G.E. Nodulation independent of rhizobia induced by a calcium-activated kinase lacking autoinhibition. Nature 2006, 441, 1149–1152. [Google Scholar] [CrossRef]
  26. Tirichine, L.; Imaizumi-Anraku, H.; Yoshida, S.; Murakami, Y.; Madsen, L.H.; Miwa, H.; Stougaard, J. Deregulation of a Ca2+/calmodulin-dependent kinase leads to spontaneous nodule development. Nature 2006, 441, 1153–1156. [Google Scholar] [CrossRef] [PubMed]
  27. Romdhane, S.B.; Tajini, F.; Trabelsi, M.; Aouani, M.E.; Mhamdi, R. Competition for nodule formation between introduced strains of Mesorhizobium cicer and the native populations of rhizobia nodulating chickpea (Cicer arietinum L.) in Tunisia. World J. Microb. Biotechnol. 2007, 23, 1195–1201. [Google Scholar] [CrossRef]
  28. Maekawa, T.; Maekawa-Yoshikawa, M.; Takeda, N.; Imaizumi-Anraku, H.; Murooka, Y.; Hayashi, M. Gibberellin controls the nodulation signaling pathway in Lotus japonicus. Plant J. 2009, 58, 183–194. [Google Scholar] [CrossRef] [PubMed]
  29. Mustafa, A.; Hussain, A.; Naveed, M.; Ditta, A.; Nazli, Z.; Sattar, A. Response of okra (Abelmoschus esculentus L.) to soil and foliar applied L-tryptophan. Soil Environ. 2016, 35, 76–84. [Google Scholar]
  30. Gana, A.S. The role of synthetic growth hormones in crop multiplication and improvement. Afr. J. Biotechnol. 2010, 10, 10330–10334. [Google Scholar] [CrossRef]
  31. Bourebaba, Y.; Durán, D.; Boulila, F.; Ahnia, H.; Boulila, A.; Temprano, F.; Palacios, F.M.; Imperial, J.; Ruiz-Argüeso, T.; Rey, L. Diversity of Bradyrhizobium strains nodulating Lupinus micranthus on both sides of the Western Mediterranean: Algeria and Spain. Syst. Appl. Microbiol. 2016, 39, 266–274. [Google Scholar] [CrossRef]
  32. Chen, J.; Hu, M.; Ma, H.; Wang, Y.; Wang, E.T.; Zhou, Z.; Gu, J. Genetic diversity and distribution of bradyrhizobia nodulating peanut in acid-neutral soils in Guangdong Province. Syst. Appl. Microbiol. 2016, 39, 418–427. [Google Scholar]
  33. Dall’Agnol, R.F.; Bournaud, C.; Faria, S.M.; Béna, G.; Moulin, L.; Hungria, M. Genetic diversity of symbiotic Paraburkholderia species isolated from nodules of Mimosa pudica (L.) and Phaseolus vulgaris (L.) grown in soils of the Brazilian Atlantic Forest (Mata Atlântica). FEMS Microbiol. 2017, 93, 1–15. [Google Scholar] [CrossRef] [Green Version]
  34. Costa, E.M.; Guimarães, A.A.; Carvalho, T.S.; Rodrigues, T.L.; Ribeiro, P.R.A.; Lebbe, L.; Willems, A.; Moreira, F.M.S. Bradyrhizobium forestalis sp. nov., an efficient nitrogen-fixing bacterium isolated from nodules of forest legume species in the Amazon. Arch. Microbiol. 2018, 200, 743–752. [Google Scholar] [CrossRef]
  35. Rajendran, G.; Singh, F.; Desai, A.J.; Archana, G. Enhanced growth and nodulation of pigeon pea by coinoculation of Bacillus strains along with Rhizobium sp. Bioresour. Technol. 2008, 99, 4544–4550. [Google Scholar] [CrossRef]
  36. Saeed, Z.; Naveed, M.; Imran, M.; Bashir, M.A.; Sattar, A.; Mustafa, A.; Hussain, A.; Xu, M. Combined use of Enterobacter sp. MN17 and zeolite reverts the adverse effects of cadmium on growth, physiology and antioxidant activity of Brassica napus. PLoS ONE 2019, 14, e0213016. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  37. Graham, P.; Sadowsky, M.J.; Keyser, H.H. Proposed minimal standards for the description of new genera of roots and stem nodulating bacteria. Syst. Bacteriol. 1991, 41, 582–587. [Google Scholar] [CrossRef] [Green Version]
  38. Naveed, M.; Mitter, B.; Yousaf, S.; Pastar, M.; Afzal, M.; Sessitsch, A. The endophyte Enterobacter sp. FD17: A maize growth enhancer selected based on rigorous testing of plant beneficial traits and colonization characteristics. Biol. Fertil Soils 2014, 50, 249–262. [Google Scholar] [CrossRef]
  39. Kumari, B.S.; Ram, M.R.; Mallaih, K.V. Studies on nodulation, biochemical analysis and protein profiles of Rhizobium isolates from Indigofera species. Malays. J. Microbiol. 2009, 133–139. [Google Scholar]
  40. Vincent, J.M. A Manual of Practical Study of Root Nodule Bacteria; Blackwell Scientific Publication: Oxford, UK, 1970. [Google Scholar]
  41. Flores-Vargas, R.D.; O’Hara, G.W. Isolation and characterization of rhizosphere bacteria with potential for biological of weeds in vineyards. J. Appl. Microbiol. 2006, 100, 946–954. [Google Scholar] [CrossRef] [PubMed]
  42. Sarwar, M.; Arshad, M.; Martens, W.T.; Frankenberger, J.R. Tryptophan-dependent biosynthesis of auxins in soil. Plant Soil 1992, 147, 207–215. [Google Scholar] [CrossRef]
  43. Holbrook, A.A.; Edge, W.L.W.; Bailey, F. Spectrophotometric Method for Determination of Gibberellic Acid in Gibberellins; ACS Publications: Washington, DC, USA, 1961; pp. 159–167. [Google Scholar]
  44. Arnon, I.D. Copper enzymes in isolated chloroplast. Polyphenol oxidase in Beta vulgaris. Plant Physiol. 1949, 24, 11–15. [Google Scholar] [CrossRef] [Green Version]
  45. Sadasivan, S.; Manickam, A. Biochemical Methods, 3rd ed.; New Age International: New Delhi, India, 2008. [Google Scholar]
  46. Mehlich, A. Mehlich 3 soil test extractant: A modification of Mehlich 2 extractant. Commun. Sci. Plant Anal. 1984, 15, 1409–1416. [Google Scholar] [CrossRef]
  47. Jackson, M.L. Soil Chemical Analysis; Prentice Hall of India: New Delhi, India, 1967; pp. 183–192. [Google Scholar]
  48. Lowry, O.H.; Rosebrough, N.J.; Farr, A.L.; Randall, R.J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1951, 193, 265–275. [Google Scholar] [CrossRef]
  49. Steel, R.G.D.; Torrie, J.H.; Dicky, D.A. Principles and Procedures of Statistics—A Biometrical Approach, 3rd ed.; McGraw-Hill Book International Co.: Singapore, 1997. [Google Scholar]
  50. Dey, R.; Pal, K.K.; Bhatt, D.M.; Chauhan, S.M. Growth promotion and yield enhancement of peanut (Arachis hypogaea L.) by application of plant growth promoting rhizobacteria. Microbiol. Res. 2004, 159, 371–394. [Google Scholar] [CrossRef]
  51. Ditta, A.; Imtiaz, M.; Mehmood, S.; Rizwan, M.S.; Mubeen, F.; Aziz, O.; Qian, Z.; Ijaz, R.; Tu, S. Rock phosphate enriched organic fertilizer with phosphate solubilizing microorganisms improves nodulation, growth and yield of legumes. Commun. Soil Sci. Plant Anal. 2018, 49, 2715–2725. [Google Scholar] [CrossRef]
  52. Ditta, A.; Muhammad, J.; Imtiaz, M.; Mehmood, S.; Qian, Z.; Tu, S. Application of rock phosphate enriched composts increases nodulation, growth and yield of chickpea. Int. J. Recycl. Org. Waste Agric. 2018, 7, 33–40. [Google Scholar] [CrossRef] [Green Version]
  53. Hussain, A.; Zahir, Z.A.; Ditta, A.; Tahir, M.U.; Ahmad, M.; Mumtaz, M.Z.; Hayat, K.; Hussain, S. Production and implication of bio-activated organic fertilizer enriched with zinc-solubilizing bacteria to boost up maize (Zea mays L.) production and biofortification under two cropping seasons. Agronomy 2020, 10, 39. [Google Scholar] [CrossRef] [Green Version]
  54. Ullah, N.; Ditta, A.; Khalid, A.; Mehmood, S.; Rizwan, M.S.; Mubeen, F.; Imtiaz, M. Integrated effect of algal biochar and plant growth promoting rhizobacteria on physiology and growth of maize under deficit irrigations. J. Soil Sci. Plant Nutr. 2020, 20, 346–356. [Google Scholar] [CrossRef]
  55. Rabbani, M.G.; Solaiman, A.R.M.; Hossain, K.M.; Hossain, T. Effects of Rhizobium inoculants, nitrogen, phosphorus and molybdenum on nodulation, yield and seed protein in pea. Korean J. Crop. Sci. 2005, 50, 112–119. [Google Scholar]
  56. Nishita, G.; Joshi, N.C. Growth and yield response of chickpea (Cicer arietinum) to seed inoculation with Rhizobium sp. Nat. Sci. 2010, 8, 232–236. [Google Scholar]
  57. Ogutcu, H.; Kasimoglu, C.; Elkocae, E. Effects of rhizobium strains isolated from wild chickpeas on the growth and symbiotic performance of chickpeas (Cicer arietinum L.) under salt stress. Turk. J. Agric. For. 2010, 34, 361–371. [Google Scholar]
  58. Kumar, A.; Biswas, T.K.; Singh, N.; Lal, E.P. Effect of gibberellic acid on growth, quality and yield of tomato (Lycopersicon esculentum Mill.). IOSR J. Agric. Vet. Sci. 2014, 7, 28–30. [Google Scholar] [CrossRef]
  59. Nabi, A.J.; Hasan, M.N.; Alam, M.M.; Islam, M.S.; Islam, M.R. Responses of gibberellic acid (GA3) on growth and yield of cowpea cv. BARI Falon-1 (Vigna unguiculate L.). J. Environ. Sci. Nat. Res. 2014, 7, 7–12. [Google Scholar]
  60. Thakare, U.; Patil, N.; Malpathak, N. Performance of chickpea under the influence of gibberellic acid and oxygenated peptone during germination. Adv. Biosci. Biotechnol. 2011, 2, 40–45. [Google Scholar] [CrossRef] [Green Version]
  61. Solaimalai, A.; Sivakumar, C.; Anbumani, S.; Suresh, T.; Arulmurugan, K. Role of plant growth regulators in rice production: A review. Agric. Rev. 2001, 22, 33–40. [Google Scholar]
  62. Rao, K.L.; Reddy, N.; Mahalakshmi, P.J.R. Effect of PGR and micronutrient on flower absorption, pod setting and yield of chickpea. Annals Plant Physiol. 2005, 19, 14–17. [Google Scholar]
  63. Neelima, A.; Ranjana, R.; Kaur, J. Alleviation of normal and late sown chickpea (Cicer arietinum L.) yield through foliar application of bio regulators. Environ. Ecol. 2006, 24S, 147–176. [Google Scholar]
  64. Fatima, Z.; Bano, A.; Aslam, M. Effect of plant growth regulators and Rhizobium inoculum on N2 fixation and yield of chickpea. In Proceedings of the 7th International Symposium of Nitrogen Fixation with Non-Legume, Faisalabad, Pakistan, 16–21 October 1996; Kluwer Academic Publisher: Dordrecht, The Netherlands; Boston, MA, USA, 1998; pp. 103–106. [Google Scholar]
  65. Akbari, N.; Barani, M.; Ahmadi, H. Effect of gibberellic acid (GA3) on agronomic traits of green gram (Vigna radiate L. Wilczek) irrigated with different levels of saline water. World Appl. Sci. J. 2008, 5, 199–203. [Google Scholar]
  66. Zaman, S.; Mazid, M.A.; Kabir, G. Effect of Rhizobium inoculation on nodulation, yield and yield traits of chickpea (Cicer arietinum L.) in four different soils of greater Rajshahi. J. Life Earth Sci. 2011, 6, 45–50. [Google Scholar] [CrossRef] [Green Version]
  67. Tripathi, D.K.; Singh, H.C.; Parihar, S.K.S. Effect of growth regulators on quality parameters in Chickpea (Cicer arietinum). Farm. Sci. J. 2003, 12, 156–157. [Google Scholar]
  68. Afrin, S. Effects of GA3 and IAA Alone and in Combination on BARI Tomato-14 (Lycopersicon esculentum Mill). Master’s Thesis, Jagannath University, Dhaka, Bangladesh, 2015; p. 93. [Google Scholar]
  69. Jain, R.K.; Jain, A.K.; Gera, V.K. Effect of growth regulators on leghaemoglobin synthase in chickpea nodules. Legume Res. 2008, 31, 303–305. [Google Scholar]
  70. Kaur, S.; Gupta, A.K.; Kaur, N. Seed priming increases crop yield possibly by modulating enzymes of sucrose metabolism in chickpea. J. Agron. Crop. Sci. 2005, 191, 81–97. [Google Scholar] [CrossRef]
  71. Gupta, B.; Shrivastava, G.K.; Annu, V. Response of plant growth regulators on nutrient uptake and protein yield of chickpea under vertisols of Chhattisgarh. Environ. Ecol. 2007, 25, 100–102. [Google Scholar]
  72. Idris, E.E.; Iglesias, D.J.; Talón, M.; Borriss, R. Tryptophan-dependent production of indole-3-acetic acid (IAA) affects level of plant growth promotion by Bacillus amyloliquefaciens FZB42. Mol. Plant-Microbe Interact. 2007, 20, 619–626. [Google Scholar] [CrossRef] [Green Version]
  73. Tuna, A.L.; Kaya, C.; Dikilitas, M.; Higgs, D. The combined effects of gibberellic acid and salinity on some antioxidant enzyme activities, plant growth parameters and nutritional status in maize plants. Environ. Exp. Bot. 2008, 62, 1–9. [Google Scholar] [CrossRef]
Agronomy 11 00105 g001 550
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 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.
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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 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.
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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 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.
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Agronomy 11 00105 g004 550
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 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.
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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).
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Table
Table 1. Physiochemical and growth promoting parameters of Rhizobium.
Table 1. Physiochemical and growth promoting parameters of Rhizobium.
CharacteristicsRhizobial Isolates
R1R2R3R4R5R6R7R8R9R10
Phenotypic and physiological characterization
Colony colorWhiteMilky whiteMilky whiteMilky whiteMilky whiteMilky whiteMilky whiteMilky whiteMilky whiteMilky white
ShapeCircularCircularCircularCircularCircularCircularCircularCircularCircularCircular
Size (mm)2.53.12.11.83.02–32.62–42–42.5
OpacityTransparentTransparentTransparentTranslucentTransparentTransparentTransparentTransparentTransparentTransparent
Gram reactionNegativeNegativeNegativeNegativeNegativeNegativeNegativeNegativeNegativeNegative
Bacterium shapeRodRodRodRodRodRodRodRodRodRod
Motility testMotileMotileMotileMotileMotileMotileMotileMotileMotileMotile
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)4.345.676.864.233.012.344.455.684.212.47
Gibbrellic acid (mg L−1)5.378.344.8711.367.427.876.118.758.997.01
Biolog identification and similarity index %
Mesorhizobium ciceri76807476747372787674
a −, absent; +, present b +, low efficiency; ++, high efficiency.
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Rafique, M.; Naveed, M.; Mustafa, A.; Akhtar, S.; Munawar, M.; Kaukab, S.; Ali, H.M.; Siddiqui, M.H.; Salem, M.Z.M. The Combined Effects of Gibberellic Acid and Rhizobium on Growth, Yield and Nutritional Status in Chickpea (Cicer arietinum L.). Agronomy 2021, 11, 105. https://doi.org/10.3390/agronomy11010105

AMA Style

Rafique M, Naveed M, Mustafa A, Akhtar S, Munawar M, Kaukab S, Ali HM, Siddiqui MH, Salem MZM. The Combined Effects of Gibberellic Acid and Rhizobium on Growth, Yield and Nutritional Status in Chickpea (Cicer arietinum L.). Agronomy. 2021; 11(1):105. https://doi.org/10.3390/agronomy11010105

Chicago/Turabian Style

Rafique, Munazza, Muhammad Naveed, Adnan Mustafa, Saleem Akhtar, Muneeb Munawar, Sadia Kaukab, Hayssam M. Ali, Manzer H. Siddiqui, and Mohamed Z. M. Salem. 2021. "The Combined Effects of Gibberellic Acid and Rhizobium on Growth, Yield and Nutritional Status in Chickpea (Cicer arietinum L.)" Agronomy 11, no. 1: 105. https://doi.org/10.3390/agronomy11010105

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