Growth and Yield of Okra Exposed to a Consortium of Rhizobacteria with Different Organic Carriers under Controlled and Natural Field Conditions

Abstract

Crop yields, soil fertility, and soil quality decline due to the overuse of chemical fertilizers and other agrochemicals. The damaging effects of these agrochemicals on the environment can be minimized by integration with eco-friendly approaches, i.e., biofertilizers. These eco-friendly biofertilizers containing plant growth-promoting rhizobacteria, (PGPR) not only solubilize mineral nutrients for crop uptake but also release phytohormones for their growth improvements. The objective of this research is to use these PGPR’s capacity to promote growth in order to increase okra production. For this purpose, different organic carriers were used, i.e., Press mud, Charcoal, Biochar, Peat, and Compost for PGPR’s inoculation. Before being used as a consortium with various carrier materials, the pre-isolated and characterized PGPR strains (AN-35, ZM-27, and ZM-63) were tested for compatibility against one another. The PGPR consortium and carriers were applied in the following treatments, i.e., T₀: (control), T₁: PGPR, T₂: Peat + PGPR, T₃: Pressmud+ PGPR, T₄: Compost + PGPR, T₅: Charcoal + PGPR, and T₆: Biochar + PGPR in the present pot and field studies. Under the pot experiment, the results depicted that all treatments showed a significant increase in okra growth, nutrient contents, and yield of okra along with increasing the microbial biomass in the soil but the treatment containing PGPR consortium with peat caused the maximum increase. Similarly, the results of the field experiment also showed a significant increase under all treatments but the maximum increase in nutrient contents, growth attributes, and yield of okra was found under the treatment containing PGPR consortium with peat (T₂). Therefore, this study recommends the use of peat and studied the PGPR consortium as a suitable carrier to develop carrier-based biofertilizers for sustainable okra production.

1. Introduction

Okra has been listed as a highly nutritious vegetable because it contains vitamins, minerals, and antioxidants. In economically underdeveloped regions of Asia and Africa, this crop is typically grown and can encourage food security in times of environmental changes. Fresh fruit of okra is a good source of dietary fiber as well as vitamins, minerals, and plant proteins [1]. Mucilage of okra is used as a medicine, and it contains about 20% edible oil and protein. Moreover, vitamins such as vitamin C, B₆, K, and other mineral contents pyridoxine, folates, calcium, iron, and phosphorus help to maintain physiological functions in the body [2]. The production of okra is severely lost by nutrient losses, diseases, environmental conditions, and abiotic stresses [3].

The green revolution in agricultural production has increased the use of agrochemicals (pesticides, herbicides, and fertilizers) to enhance crop productivity to meet food security is posing harmful effects on our environment [4]. Moreover, salinity, drought, and inadequate nutrients caused less production in agriculture [5]. These chemicals are also damaging the environment by adding up heavy metal residues, which ultimately enter the food chain by using contaminated food and water [6,7]. However, the use of these agrochemicals is mandatory for higher-quality crop production. Therefore, scientists are trying to find out alternate economical and eco-friendly approaches for improving crop productivity through organic agriculture, biofertilizers, and their integration with chemical fertilizers [8].

Biofertilizers also called microbial inoculants or bioformulations are the use of microorganisms or their products specifically for crop production [9]. Biofertilizers are defined as fertilizers containing microorganisms (MOs) or their products applied to seeds, leaves, roots, or soil to boost rhizosphere colonization to reduce nutrient deficiencies or a cluster of microorganisms residing in the soil, the rhizosphere/rhizoplane, and the phyllosphere that does exceptionally well under certain conditions [10]. The microorganisms used in the biofertilizer formulations not only enhance the rhizosphere colonization of certain crops but also provide additional benefits like minerals solubilization, nutrients uptake, pathogen protection, phytohormones production (Auxins, gibberellins), induction of systemic resistance, production of antibiotics and abiotic stresses amelioration (heat, salinity, and drought) in crops [11,12].

The provided benefits by these phyto-friendly creatures urged the scientist to formulate certain biofertilizers/bioinoculants for sustainable crop production. Mostly the bio-inoculants contain living cells in some organic material called carriers [13]. Carriers can be organic or inorganic but must meet the criteria defined as they should be nontoxic, have high water holding capacity, and be biodegradable and economical [14]. Numerous carrier materials are being used for the preparation of the biofertilizers including organic and inorganic carriers. Pressmud, an organic carrier is the by-product of the sugarcane industry [15,16]. Biochar is an organic material produced by the process of pyrolysis and provides higher ion exchange capacity, enhances soil water retention, improves soil physical properties, boosts microbial activity as a carbon source, enhances crop nutrition, and helps in the amelioration of abiotic stresses especially metals and salinity [17].

Composting is a process of decomposing organic matter into a stabilized form called compost. Compost enhances the physiochemical and biological properties of soils, boosting water holding capacity, and as a conditioner improves plant growth and yield under abiotic stress like drought [18]. It is not only beneficial for enhancing soil organic matter, but compost also increases the availability of macro- and micronutrients to crops [19]. Moreover, it supports microbial communities by providing carbon and a variety of nutrients for their exponential growth [20].

Peat provides a nutrient-rich environment, allowing different microorganisms to grow and establish themselves and build microcolonies on particle surfaces. It is a very porous substance with a large surface area, so it provides a good environment for inoculants to thrive. Peat also has a high retention capacity for water, is easy to use, and is generally accepted [21]. The most popular carrier used in the Rhizobium inoculation industry for the development of formulation is peat [22]. Charcoal provides a favorable microenvironment for microbes to survive and populate because it buffers pH and protects them against heat and dehydration [23]. Due to its high water-holding capacity and high porosity, charcoal has the ability to protect microbes from desiccation during seasonal droughts and to increase biomass production [24,25]. After a thorough study of the above-mentioned facts, the present study aims to optimize the PGPR consortium against different organic carriers (peat, pressmud, compost, charcoal, and biochar) to develop carrier-based biofertilizers for sustainable okra production.

2. Materials and Methods

2.1. Experimental Location

The pot and field experiments were conducted at the wirehouse and farm area of the Department of Soil Science, the Islamia University of Bahawalpur, respectively. The pre-sowing soil sample was collected from the respective field and analyzed for the basic physicochemical characterization as per Ryan et al. [26]. The physicochemical attributes of the soil are already described in our previous publication by Safdar et al. [27] in

Table 1. Pre-sowing physicochemical soil attributes.

Analysis	Unit	Soil Sample
Textural class		Sandy Clay Loam
ECe	dS m–1	0.63
pH		8.3
Saturation Percentage	%	34.0
Organic matter	%	0.48
Phosphorus	mg kg−1	5.53
Potassium	mg kg−1	153.0
Nitrogen	%	0.024

.

2.2. Collection of Bacteria and Organic Carriers

The rhizobacterial strains from the Bacillus genus used in the present study including Bacillus subtilis (ZM-63), Paenibacillus polymyxa (ZM-27), and Bacillus megaterium (AN-35) having accession numbers KX788861, KX788859, and MN005929, respectively, have been previously evaluated, characterized, and screened for plant growth promotion and biofortification were taken from the culture collection of Soil Microbiology and Biotechnology Laboratory (SMBL), Department of Soil Science, the Islamia University of Bahawalpur. The collection of the organic carriers, i.e., peat, pressmud, compost, charcoal, and biochar, and their chemical characterization is described in our recent publication [27].

2.3. Testing Compatibility and Consortium Preparation

The consortium of the strains was prepared firstly by testing the compatibility of the prescribed strains for this purpose and was streaked on general-purpose medium (GPM) agar plates in a crisscross fashion to observe the clearing zone. The combinations not showing growth inhibition (clearing zones) were considered compatible and their equal volumes of broth were mixed and homogenized on a vortex for 30 s [16].

2.4. Formulation Development and Seed Inoculation

The various organic carriers were autoclaved for 20 min at 121 °C and 15 psi of pressure to sterilize them. Afterward, the fresh cultures of the microbial strains were mixed to make a consortium. The consortium, organic carriers, and clay were mixed in 3:6:1, respectively, on a sterile surface and incubated for 48 h at 28 ± 2 °C. A total of 5 formulations were developed including Peat + PGPR, Press mud + PGPR, Compost + PGPR, Charcoal + PGPR, and Biochar + PGPR. The okra seed was surface disinfected with 5% sodium hypochlorite (NaOCl) solution for 3 min after the incubation period, followed by a 1 min ethanol wash and 5-times of distilled-autoclaved water rinsing. The seed was then subjected to a sugar solution (20%) and mixed with the formulated product at 1 kg product per 8 kg of seed [28].

2.5. Pot Trial

By conducting a pot experiment at the Departmental wirehouse, the study’s objective was achieved. The soil used in the present study belongs to the Aridisols order as per the United States Department of Agriculture’s (USDA) taxonomy. The air-dried and sieved 12 kg soil was filled into each pot. The treatments of the pot experiment were made as per the developed formulations as follows; T₀: (control), T₁: PGPR, T₂: Peat + PGPR, T₃: Press mud + PGPR, T₄: Compost + PGPR, T₅: Charcoal + PGPR, and T₆: Biochar + PGPR. Through a completely randomized design (CRD), the treatments were distributed in triplicate. The recommended dose (80:60:40 kg/acre) of NPK was applied through urea, diammonium phosphate (DAP), and muriate of potash (MOP) before sowing while Nitrogen is divided into two splits before sowing and after first picking. Agronomic practices were adopted throughout the crop period. The growth and physiological parameters were measured at the maturity of the okra and nutritional status and yield parameters were measured after harvest.

2.6. Field Trial

The authenticity of the pot experiment results was tested in field trials of okra. The experiment was carried out at the farm area Department of Soil Science. The same treatment set as described in the pot trial was used but the treatments allocation to experimental units was done using a randomized complete block design and three replications. The recommended fertilizers application and agronomic practices were also adopted as per pot studies and the data collection was also as per the prescribed time in the pot experiment.

2.7. Determination of Growth Parameters

Okra plants at harvesting were subjected to the following growth parameters, i.e., plant height through meter rod, shoot and root fresh weight by analytical balance, and shoot and root dry weight by analytical balance. Leaf relative water contents (RWC) were calculated with the formula from Lazcano Ferrat and Lovatt [29].

RWC (%) = (Fresh weight − Dry weight/Fully Turgid Weight − Dry weight) × 100

(1)

2.8. Samples Preparation and Determination of Plant Nutrients

The plant samples after harvesting were air dried followed by oven drying in the hot air at 65 ± 2 °C for 24 h. Afterward, the 0.5 g plant sample was digested by Wolf’s [30] method using sulfuric acid and hydrogen peroxide until the colorless endpoint. The digested sample was then filtered and diluted with distilled water up to 50 mL the volume. The sample after dilution was analyzed for nitrogen through distillation at the Kjeldahl apparatus, and for potassium through a Flame photometer [31]. Phosphorus was determined by the colorimetric method on the spectrophotometer. The determination of the micronutrients such as iron (Fe) and zinc (Zn) from roots, shoots, and okra fruits was done by following the method of Ryan et al. [26]. The digested samples were examined for this purpose using an atomic absorption spectrophotometer (PerkinElmer, Analyst 100, Waltham, MA, USA), and the concentration values were determined by constructing a standard curve using known concentration standards.

2.9. Microbial Biomass Carbon and Nitrogen Determination

The microbial biomass nitrogen and carbon contents were determined from the rhizosphere soil samples taken at harvest through chloroform fumigation and extraction method described for carbon by Okalebo [32] and for nitrogen by Anderson and Ingram [33], respectively.

2.10. Determination of Microbial Population

The microbial population was determined from the rhizosphere of okra at harvest. Each treatment’s rhizosphere samples were collected and brought to the lab at 4 °C. The colony forming units (CFU) g⁻¹ dry soil were determined by serial dilution and pour plate methods as described by Alexander [34].

2.11. Statistical Analysis

The observed parameters data were analyzed through a one-way variance analysis (ANOVA) using Statistix^® 8.1. software (Analytical Software, Tallahassee, FL, USA) for determination of treatments significance with control. While the least significant difference (LSD) test was used to calculate the treatment means for differences at p ≤ 0.05 [35].

3. Results

3.1. Pot Trial

3.1.1. Effect of Different Carrier Materials on Physiological Parameters of Okra in the Pot Trial

The data regarding the effect of organic carriers on the physiology of okra are presented in (

Table 2. Effect of different carrier materials on physiological parameters of okra in pot trial.

Treatment	Shoot Fresh Weight (g)	Root Fresh Weight (g)	Shoot Dry Weight (g)	Root Dry Weight (g)	Shoot Length (cm)	Root Length (cm)	Bacterial Population CFU × 104 g−1 Soil	Microbial Biomass C (mg/kg)
Control	144.4 ± 0.188 g*	20.4 ± 0.210 f	71.5 ± 0.071 f	10.3 ± 0.044 f	56.7 ± 0.482 f	5.20 ± 0.0957 d	53.3 ± 0.2693 f	577.7 ± 0.754 g
PGPR	149.8 ± 0.336 f	21.4 ± 0.176 e	82.3 ± 0.140	11.8 ± 0.064 de	58.2 ± 0.092 e	5.32 ± 0.1126 d	58.7 ± 0.2693 e	599.2 ± 1.345 f
Peat + PGPR	180.7 ± 0.336 a	25.1 ± 0.026 a	92.1 ± 0.046 a	13.0 ± 0.003 a	71.0 ± 0.247 a	6.76 ± 0.1423 a	64.3 ± 0.2693 a	722.8 ± 1.345 a
Pressmud + PGPR	173.2 ± 0.123 c	23.3 ± 0.123 c	87.1 ± 0.046 c	12.2 ± 0.046 c	62.3 ± 0.56 c	5.84 ± 0.1185 bc	62.0 ± 0.4665 b	692.8 ± 0.494 c
Compost + PGPR	177.2 ± 0.071 b	24.2 ± 0.123 b	89.1 ± 0.046 b	12.6 ± 0.046 b	63.9 ± 0.246 b	6.01 ± 0.1368 b	63.0 ± 0.466 b	708.6 ± 0.285 b
Charcoal + PGPR	168.5 ± 0.22 e	22.2 ± 0.071 d	84.7 ± 0.499 d	11.6 ± 0.047 d	59.4 ± 0.474 de	5.54 ± 0.0118 cd	60.3 ± 0.269 d	674.1 ± 0.882 e
Biochar + PGPR	169.8 ± 0.097 d	22.6 ± 0.071 d	85.3 ± 0.047 d	11.9 ± 0.049 d	60.4 ± 0.118 d	5.78 ± 0.018 bc	61.3 ± 0.269 cd	679.8 ± 0.764 d
LSD (p ≤ 0.05)	0.6931	0.4834	0.7537	0.1737	1.3146	0.3561	1.2674	3.4393

* Values are mean ± standard error of means where n = 3. Mean values with the same lettering are statistically at par (p ≤ 0.05).

). The root and shoot fresh weight of okra was significantly improved by peat + PGPR formulation by 22 and 25%, respectively, as compared with the control. Similarly, the dry weight of the root and shoot of okra was also improved by the same treatment causing 26 and 28% improvements, respectively, as compared to the control plants. Whereas root length and shoot length of okra plants was increased by 25 and 30%, respectively, by the application of the combination of peat with PGPR as compared to the control. Moreover, the bacterial population and microbial biomass carbon (MBC) were also increased by 20 and 25% by the microbial consortium inoculated with peat as carrier material as compared to the respective control.

3.1.2. Effect of Different Carrier Materials on Nutrients Uptake of Okra in the Pot Trial

The data regarding the differential effect of organic carriers on the chemical parameters of okra are presented in (

Table 3. Effect of different carrier materials on chemical parameters of okra in pot trial.

Treatment	Microbial Biomass N (mg/kg)	Nitrogen in Roots (%)	Nitrogen in Shoots (%)	Potassium in Shoots (%)	Potassium in Roots (%)	Phosphorus in Shoot (%)	Phosphorus in Root (%)	Iron in Plant (mg/kg)	Zinc in Plant (mg/kg)
Control	2.28 ± 0.044 d*	1.26 ± 0.021 e	0.85 ± 0.017 d	1.14 ± 0.006 d	1.11 ± 0.006 d	0.52 ± 0.005 c	0.460 ± 0.005 e	5.41 ± 0.174 e	5.70 ± 0.109 e
PGPR	2.38 ± 0.005 c	1.3 ± 0.005 e	0.87 ± 0.016 cd	1.20 ± 0.023 cd	1.17 ± 0.023 cd	0.526 ± 0.027 bc	0.470 ± 0.005 de	5.6 ± 0.009 e	5.95 ± 0.0134 d
Peat + PGPR	2.81 ± 0.002 a	1.4 ± 0.010 a	1.00 ± 0.0001 a	1.38 ± 0.0035 a	1.34 ± 0.004 a	0.596 ± 0.039 a	0.540 ± 0.008 a	6.36 ± 0.027 a	7.02 ± 0.0046 a
Press mud + PGPR	2.56 ± 0.019 b	1.39 ± 0.005 bc	0.94 ± 0.021 b	1.243 ± 0.029 bc	1.21 ± 0.029 bc	0.553 ± 0.007 bc	0.496 ± 0.010 bc	5.98 ± 0.008 bc	6.40 ± 0.0466 b
Compost + PGPR	2.60 ± 0.019 b	1.42 ± 0.009 b	0.94 ± 0.021 b	1.28 ± 0.016 b	1.25 ± 0.016 b	0.570 ± 0.0046 b	0.509 ± 0.004 b	6.01 ± 0.0008 b	6.50 ± 0.0466 b
Charcoal + PGPR	2.41 ± 0.0119 c	1.34 ± 0.0027 d	0.90 ± 0.003 bc	1.23 ± 0.022 bc	1.20 ± 0.022 bc	0.540 ± 0.009 bc	0.484 ± 0.006 cd	5.73 ± 0.0123 c	6.03 ± 0.03 cd
Biochar + PGPR	2.48 ± 0.0018 c	1.36 ± 0.017 cd	0.93 ± 0.005 b	1.21 ± 0.005 c	1.18 ± 0.005 c	0.543 ± 0.01 bc	0.480 ± 0.005 c–e	5.81 ± 0.015 d	6.19 ± 0.0046 c
LSD (p ≤ 0.05)	0.0751	0.0422	0.0531	0.067	0.0663	0.0458	0.0234	0.2518	0.1879

* Values are mean ± standard error of means where n = 3. Mean values with the same lettering are statistically at par (p ≤ 0.05).

). The data depicted a significant increase in the nutritional contents of okra by all the inoculated treatments, however, the maximum effects were observed when the consortium was inoculated with peat. The microbial biomass nitrogen was improved by 23% as compared to the control. The potassium contents of roots and shoots of okra were improved by 21%. The nitrogen contents in the roots and shoots also increased, by 15% and 18%, respectively. While the phosphorus content in okra plant roots and shoots increased by 17 and 14%, respectively. The micronutrient contents of okra shoot also caused significant improvement by the same treatment as iron contents were increased by 17% while Zn contents by 23% as compared to the control.

3.1.3. Nutritional Status of Okra Fruit in the Pot Trial

The application of the PGPR consortium with different carrier materials significantly improved the nutritional status of okra fruit (Table 3). The maximum nitrogen, phosphorus, and potassium contents in okra fruit were observed as 17, 16 and 17%, more than the control (T₀), respectively. Moreover, the same treatment also caused significant improvement in the biofortification of micronutrients such as Fe and Zn (Figure 1). The magnitude of biofortification of Zn and Fe were recorded as 23 and 17.5% higher over control, respectively.

3.1.4. Effect of Different Carrier Materials on Yield Parameters of Okra in the Pot Trial

Table 4. Effect of different carrier materials on yield parameters of okra in pot trial. Values are mean ± standard error of three replications.

Treatment	Nitrogen in Fruit (%)	Phosphorus in Fruit (%)	Potassium in Fruit (%)	Fruit Diameter (cm)	Okra Yield (g/pot)
Control	1.72 ± 0.054 e*	0.382 ± 0.0035 e	1.24 ± 0.0057 d	0.99 ± 0.0490 d	8.83 ± 0.0971 d
PGPR	1.78 ± 0.0028 de	0.39 ± 0.0046 de	1.31 ± 0.0233 cd	1.43 ± 0.0374 bc	9.8 ± 0.1234 bc
Peat + PGPR	2.02 a ± 0.0087 a	0.446 ± 0.0053 a	1.46 ± 0.0061 a	1.54 ± 0.0117 a	10.6 ± 0.3703 a
Pressmud + PGPR	1.89 ± 0.0033 bc	0.412 ± 0.0072 bc	1.35 ± 0.0290 bc	1.49 ± 0.0257 bc	9.63 ± 0.2569 bc
Compost + PGPR	1.91 ± 0.001 b	0.423 ± 0.0033 b	1.38 ± 0.0159 b	1.51 ± 0.0356 ab	10.1 ± 0.2394 ab
Charcoal + PGPR	1.82 ± 0.0041 cd	0.403 ± 0.0048 cd	1.34 ± 0.0217 bc	1.45 ± 0.0239 bc	9.17 ± 0.0712 cd
Biochar + PGPR	1.86 ± 0.017 bc	0.399 ± 0.0039 de	1.32 ± 0.0046 bc	1.38 ± 0.0097 c	9.33 ± 0.0538 cd
LSD (p ≤ 0.05)	0.0813	0.0183	0.0667	0.1151	0.7241

* Values are mean ± standard error of means where n = 3. Mean values with the same lettering are statistically at par (p ≤ 0.05).

presents the information on the impact of various carrier materials on the yield parameters of okra. The maximum increase in the fruit diameter and fruit yield, which were 55 and 20%, respectively, as compared to the control were recorded under the PGPR application with peat in the pot trial.

3.2. Field Trial

3.2.1. Effect of Different Carrier Materials on the Growth and Microbial Parameters of Okra in Field Trial

The data on the effect of different carrier materials on the physiological parameters of okra depicted a significant increase in the growth of okra by application of the PGPR consortium with different organic carriers, however, the maximum increase was observed under consortium application with peat application (

Table 5. Effect of different carrier materials on physiological parameters of okra in the field trial. Values are mean ± standard error of three replications.

Treatment	Shoot Fresh Weight (g)	Root Fresh Weight (g)	Shoot Dry Weight (g)	Root Dry Weight (g)	Shoot Length (cm)	Root Length (cm)	Bacterial Population CFU × 104 g−1 Soil	Microbial Biomass C (mg/kg)
Control	288.7 ± 0.2693 g*	49.5 ± 0.1942 e	96.2 ± 0.0888 g	24.7 ± 0.0971 f	56.5 ± 0.2467 g	6.60 ± 0.5165 c	64.0 ± 0.4665 f	577.5 ± 0.7629 f
PGPR	299.3 ± 0.5387 f	53.3 ± 0.1425 d	99.8 ± 0.1804 f	26.7 ± 0.0712 e	61.4 ± 0.1810 f	7.30 ± 0.1007 bc	68.0 ± 0.4665 e	598.7 ± 1.5719 e
Peat + PGPR	361.4 ± 0.6729 a	62.9 ± 0.4788 a	120.5 ± 0.2258 a	31.4 ± 0.2394 a	70.4 ± 0.2467 a	8.63 ± 0.1540 a	78.0 ± 0.4665 a	739.3 ± 3.3257 a
Press mud + PGPR	346.4 ± 0.2468 c	59.1 ± 0.3885 c	115.5 ± 0.0811 c	29.5 ± 0.1942 c	66.8 ± 0.1185 c	7.56 ± 0.0246 b	73.3 ± 0.2693 bc	692.6 ± 0.4994 c
Compost + PGPR	354.3 ± 0.1425 b	60.9 ± 0.1089 b	118.1 ± 0.0473 b	30.5 ± 0.0544 b	68.7 ± 0.0684 b	7.59 ± 0.0080 b	75.0 ± 0.4665 b	708.5 ± 0.2883 b
Charcoal + PGPR	337.1 ± 0.4409 e	57.3 ± 0.9206 c	112.4 ± 0.1464 e	28.7 ± 0.4603 d	64.3 ± 0.1185 e	7.23 ± 0.0543 bc	71.0 ± 0.4665 d	673.9 ± 0.8921 d
Biochar + PGPR	339.7 ± 0.1942 d	58.1 ± 0.6201 c	113.2 ± 0.0653 d	29.1 ± 0.310 cd	65.9 ± 0.1810 d	7.43 ± 0.0182 b	72.3 ± 0.2693 cd	679.6 ± 0.7726 d
LSD (p ≤ 0.05)	1.5074	1.6752	0.5036	0.9200	0.6654	0.7831	1.5756	5.5814

* Values are mean ± standard error of means where n = 3. Mean values with the same lettering are statistically at par (p ≤ 0.05).

). The maximum increase in root fresh and dry weight, shoot fresh and dry weights, and root and shoot length were recorded as 26.5, 27, 25.3, 25, 24, and 30% higher than the uninoculated control treatment, respectively. The microbial population in terms of colony-forming units per gram (CFU g⁻¹ dry soil) and microbial biomass carbon (MBC) were also significantly enhanced by 22 and 28% with the same treatment, respectively as compared to the control.

3.2.2. Effect of Different Carrier Materials on Nutrients Uptake of Okra in Pot Trial

As clear from the data presented in

Table 6. Effect of different carrier materials on chemical parameters of okra in the field trial. Values are mean ± standard error of three replications.

Treatment	Microbial Biomass N (mg/kg)	Nitrogen in Roots (%)	Nitrogen in Shoots (%)	Potassium in Shoots (%)	Potassium in Roots (%)	Phosphorus in Shoot (%)	Phosphorus in Root (%)	Iron in Shoot (mg/kg)	Zinc in Shoot (mg/kg)
Control	3.60 ± 0.0472 f*	1.38 ± 0.0043 e	1.11 ± 0.0264 d	1.14 ± 0.0057 d	1.16 ± 0.0057 d	0.58 ± 0.0047 e	0.44 ± 0.00 f	6.87 ± 0.045 g	0.36 ± 0.0072 e
PGPR	3.73 ± 0.0321 e	1.47 ± 0.0047 d	1.16 ± 0.0264 c	1.20 ± 0.0236024 cd	1.22 ± 0.024 cd	0.59 ± 0.007 de	0.44 ± 0.0072 f	7.34 ± 0.014 f	0.37 ± 0.0053 de
Peat + PGPR	4.36 ± 0.0094 a	1.59 ± 0.0053 a	1.31 ± 0.0007 a	1.36 ± 0.0062 a	1.38 ± 0.0062 a	0.67 ± 0.0094 a	0.50 ± 0.0014 a	7.95 ± 0.010 a	0.42 ± 0.0009 bc
Press mud + PGPR	4.16 ± 0.0072 c	1.53 ± 0.0061 c	1.25 ± 0.0143 b	1.25 ± 0.0294 bc	1.27 ± 0.029 bc	0.64 ± 0.005 bc	0.48 ± 0.005 bc	7.74 ± 0.022 c	0.39 ± 0.002 bc
Compost + PGPR	4.26 ± 0.0047 b	1.55 ± 0.0041 b	1.24 ± 0.0038 b	1.28 ± 0.0161 b	1.30 ± 0.0161 b	0.64 ± 0.0024 b	0.48 ± 0.0027 b	7.83 ± 0.033 b	0.40 ± 0.0045 b
Charcoal + PGPR	3.93 ± 0.0047 d	1.48 ± 0.0058 d	1.20 ± 0.0018 bc	1.23 ± 0.022 bc	1.25 ± 0.022 bc	0.62 ± 0.005 cd	0.460.003 cd	7.48 ± 0.021 e	0.38 ± 0.0049 cd
Biochar + PGPR	3.98 ± 0.0094 d	1.52 ± 0.0034 c	1.20 ± 0.0022 bc	1.22 ± 0.005 bc	1.24 ± 0.005 bc	0.62 ± 0.007 bc	0.46 ± 0.005 de	7.59 ± 0.026 d	0.38 ± 0.005 b–d
LSD (p ≤ 0.05)	0.0834	0.0183	0.0566	0.0667	0.0667	0.0229	0.0163	0.0990	0.0176

* Values are mean ± standard error of means where n = 3. Mean values with the same lettering are statistically at par (p ≤ 0.05).

, a significantly higher nutrient uptake was recorded with the treatment receiving PGPR consortium and peat as carrier material compared to the un-inoculated control treatment. The maximum increase in microbial biomass nitrogen (21%) was caused by peat + PGPR formulation (T₂), followed by PGPR application with pressmud. The maximum increase in root and shoot potassium contents in both parts was 19%, whereas the nitrogen contents were 15 and 18% higher in root and shoot of okra, respectively, as compared to the control. The phosphorus contents in root and shoot were found significantly higher (15 and 16%, respectively) under peat-inoculated PGPR formulations compared to the control. Similarly, the micronutrient contents were also improved by the same treatment and there were 16 and 17% increases in Fe and Zn contents, respectively in comparison to the control treatment.

3.2.3. Nutritional Status of Okra Fruit in Field Trial

The application of PGPR consortium with different carrier materials significantly improved the nutritional status of okra fruit (

Table 7. Effect of different carrier materials on yield parameters of okra in the field trial. Values are mean ± standard error of three replications.

Treatment	Nitrogen in Fruit (%)	Phosphorus in Fruit (%)	Potassium in Fruit (%)	Fruit Diameter (cm)	Okra Yield (t/ha)
Control	1.71 ± 0.0250 e*	0.47 ± 0.0250 e	1.25 ± 0.0018 d	1.13 ± 0.0269 d	1.21 ± 0.247 d
PGPR	1.77 ± 0.0028 d	0.49 ± 0.0028 d	1.26 ± 0.0030 d	1.45 ± 0.0239 c	1.32 ± 0.289 cd
Peat + PGPR	1.98 ± 0.0138 a	0.54 ± 0.0138 a	1.44 ± 0.0059 a	1.61 ± 0.0093 a	1.73 ± 0.393 a
Press mud + PGPR	1.86 ± 0.0241 bc	0.51 ± 0.0241 bc	1.38 ± 0.0204 b	1.55 ± 0.0097 bc	1.46 ± 0.422 bc
Compost + PGPR	1.89 ± 0.0098 b	0.52 ± 0.0098 b	1.39 ± 0.0089 b	1.58 ± 0.0093 ab	1.66 ± 0.297 ab
Charcoal + PGPR	1.81 ± 0.0041 cd	0.50 ± 0.0041 cd	1.30 ± 0.0056 c	1.51 ± 0.0142 bc	1.52 ± 0.504 a–c
Biochar + PGPR	1.83 ± 0.0086 cd	0.49 ± 0.0086 cd	1.31 ± 0.0019 c	1.52 ± 0.0257 b	1.5 ± 0.406 bc
LSD (p ≤ 0.05)	0.0561	0.0192	0.0338	0.0691	0.2171

* Values are mean ± standard error of means where n = 3. Means followed by the same letters in a column are not statistically different at p ≤ 0.05 according to the least significant difference (LSD) test.

). The maximum nitrogen, phosphorus, and potassium contents in okra fruit were observed as 15, 16, and 16%, respectively as compared to the un-inoculated control (T₀). Moreover, the same treatment has also caused significant improvement in the biofortification of micronutrients such as Fe and Zn (Figure 2). The magnitude of biofortification of Zn and Fe were recorded as 18.2 and 17.6% higher than the control, respectively.

3.2.4. Effect of Different Carrier Materials on Yield Parameters of Okra in Field Trial

Table 7 presents the information on the impact of various carrier materials on the yield parameters of okra. The maximum increase of 42% in the fruit diameter and fruit yield was recorded in both the parameters under PGPR application with peat in the field trial as compared to the control.

4. Discussion

Microbial-based biofertilizer application for crop production has paved the way towards the fulfillment of the United Nations’ sustainable development goals (UN-SDGs) for food security. The present study also aimed to use the potential micronutrient (Fe & Zn) solubilizing bacteria for improving the yield and quality of okra [36]. The results depicted a significant increase in the growth, nutritional status, yield, and quality of okra. The enhancement was attributed to the synergistic impact of carrier material in boosting the microbial population in the rhizosphere by providing carbon for microbial growth [37]. Similar findings have also been reported by Hassan and Bano [38] who depicted that carrier-based bio formulations provide favorable microenvironments for bacteria to thrive even under harsh environmental conditions [39].

Different inoculation methods are being adopted to enhance the efficacy of the PGPR, i.e., liquid formulations, organic carrier-based formulations, peat formulation, and bio-encapsulation are prominent. A liquid formulation such as treatment T₁ (PGPR consortium) has caused the minimum increase in the growth and yield of okra similar findings were observed by Pacheco-Aguirre et al. [40]. It was also depicted by Shahzad et al. [41] that liquid inoculants have lesser growth promotion potential as compared to carrier-based formulations. Similar benefits of the carrier materials and lesser growth promotion by liquid inoculants were also proved by Khandare et al. [42].

The present study also showed that peat is a useful carrier for delivering inoculum, which not only increased microbial count, microbial biomass carbon, and microbial biomass nitrogen but also emerged as the best carrier in this study (Table 2 and Table 4). The increase in the microbial count was due to the microbial proliferation in the presence of organic carriers, which ultimately increase the MBC and MBN contents of soil a pool for plant’s nutrition upon their decomposition [43]. Albareda et al. [44] also compared the microbial activities in two organic carriers (peat and compost) and concluded that peat provides more nutrition and survival to microbes. The problem with the biofertilizers product is their shelf life because they are living entities and their survival depends upon the availability of the nutrition and energy source, but the organic carriers provide both nutrition as well as energy to the bacteria and enhance their shelf life from a few days to several months at 30 °C [45].

On the other hand, the nutritional status of the okra plant and okra fruit biofortification was also improved by consortium application under peat formulation. The bacteria enhance nutrient uptake and biofortification by decamping the organic residues [15,46] and releasing nutrient-solubilizing substances (organic acids) and developing root architecture aided by the production of IAA and other growth-promoting substances [47]. The carrier because of its organic origin contains higher nitrogen contents and upon its decomposition, it provides plants with available nitrogen forms which can be easily taken up by plants and enhance the quality of the fruit [48].

Furthermore, the higher yields of okra under carrier-based formulations are evidence of optimum utilization and uptake of the nutrients, which is responsible for proper vegetative growth, physiological attributes, and fruit formation in vegetables [49]. The results of the present study were in line with the findings of Choudhary et al. [50]. The increment in the okra yield might be due to the growth-promoting abilities of the PGPR strains which were enhanced by their application in the form of organic carrier-based formulations [27,51].

5. Conclusions

Based on the results recorded, the combined application of bacterial strains AN-35, ZM-27, and ZM-63 along with organic carriers (biochar, peat, and compost) significantly enhanced the growth, physiology, and mineral contents in fruit and bacterial population in the rhizosphere of okra. However, the application of peat as a carrier material with bacterial strains proved more promising compared to other treatments. The peat biofertilizer formulation significantly enhanced N, P, K, Zn, and Fe contents in okra, which further enhanced the growth and yield parameters of okra. In the future, the use of these novel strains could be used for the development of biofertilizers for okra and may be tested for other crops. The peat-based formulation at multi-location farmer’s field trials is necessary to authenticate the reported results and for the commercialization of the product.