Improving Growth and Nutrient Quality of Mustard Varieties Through Rhizobacterial Inoculation

doi:10.21203/rs.3.rs-5320810/v1

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Improving Growth and Nutrient Quality of Mustard Varieties Through Rhizobacterial Inoculation

https://doi.org/10.21203/rs.3.rs-5320810/v1

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The excessive feeding of chemical fertilizers into the soil has affected its fertility. The continued use of these fertilizers can impose serious environmental hazards on mankind. Biofertilizers are an alternative strategy and could improve the growth of different crops. The study was conducted in a completely randomized design to understand the effect of soil-borne rhizospheric bacteria (Bradyrhizobium japonicum & Burkholderia cepacia) on morphology, physiology, yield, antioxidants activity and protein content of four Mustard (Brassica juncea (L.) Czern.) varieties. Both the rhizospheric bacteria were cultured in LB (Luria-Bertani) broth to prepare the bacterial suspension. A single pot was inoculated with 25 ml of the bacterial suspension. The combined and individual treatments of both bacteria resulted in an increase in the plant heights, shoot and root lengths, shoot and root fresh weights, shoot and root dry weights and leaf yield, compared to control. The number of pods, weight of 1000 seeds, pod length and number of seeds per pod was also enhanced with these bacterial treatments. The B. japonicum treatment uplifted the total chlorophyll, carotenoids, and relative water content in mustard varieties. The treatment of B. cepacia raised the phenolic content, total soluble protein, and the CAT, and POD activity in Mustard plants. The results revealed that the use of B. japonicum and B. cepacia could be a better choice for plant breeders as a biofertilizer. However, the effects could vary depending upon the type of crops, as well as the varieties of the same crop.

Biological sciences/Plant sciences/Plant development

Biological sciences/Plant sciences/Plant physiology

Biological sciences/Plant sciences/Plant symbiosis

Antioxidants

Brassica

Carotenoid

Inoculant

Mustard

Brassica juncea (L.) Czern. (Mustard) is a biennial crop belonging to Brassicaceae. It is mostly cultivated in winter and ranges up to 3709 species of 338 genera [1]. B. juncea is hugely cultivated in Europe, Asia and America due to its higher oil content (nearly 44%) and resistance to various diseases [2, 3]. In arid regions, B. juncea is a more versatile oilseed crop than Brassica napus. Compared to B. napus, it grows seedlings more vigorously, covers ground more quickly, is more resistant to heat and drought, and is less susceptible to the blackleg fungus Leptosphaeri amaculans. Because of the thinner seed coat, B. juncea silique seeds may have a larger percentage of oil and protein and are non-shattering. Many countries in the northern hemisphere acknowledge the potential advantages of cultivating B. juncea [4].

Mustard extract and oil (Canola) contains anti-inflammatory, antioxidant, antidepressant, anti-mutative, anti-cancerous, anti-diabetic and antibacterial properties [5, 6]. The vegetative parts of mustard are used to cook a traditional food, named ‘Saag’, in all over subcontinent countries [7]. Mustard is serving human beings via fulfilling their different necessities, i.e., food, fodder, biofuel and especially by producing essential natural chemicals. From last 5000 years, its seeds are used as spices [8]. A significant volatile and spicy component of mustard, allyl isothiocyanate, is primarily responsible for the distinct and strong flavor of B. juncea. The glucosinolate found in B. juncea, sinigrin, is broken down by the enzyme myrosinase to release allyl isothiocyanate, which is what gives the food its strong flavor. Flavonoids, polyphenols, sulfur compounds, vitamin A and K are abundant in B. juncea [5, 9, 10]. Mustard plants were also found to be involved in phytoremediation of soils, contaminated with heavy metals [11].

Pakistan is an agricultural country. Its economy is highly based on agricultural productivity, however the gap between production and consumption of crops gets wider with every passing year [12]. Owing to the greater gap between the demand and production of edible oil, Pakistan imports 83% of its edible oil which cost us more than 192 billion PKR. The remaining 17% of edible oil is obtained from domestic production, via rapeseed and mustard [13]. Pakistan produces 0.573-million-tonnes of edible oil yearly, but the overall demand is raised to 3.20-million-tonnes. Pakistan obtains 32% of its domestic edible oil production from Brassica cultivars [14]. Due to ever-growing population, the food and edible oil demands are too increased rapidly [15]. Meeting the daily food demands, it is obligatory for farmers to use various synthetic fertilizers directly to the soil. The application of these chemical nutrients aid to reach more yielding crops in less time [16]. The enormous feeding of artificial fertilizers has levied critical effects to the soil microenvironment, atmosphere, and as well as to the underground water table. Generally, 40 to 50% of the total artificial fertilizer applied to land is been taken up by plants. The remaining chemicals sit into the soil, in an unavailable form for plants. The inaccessible form of nutrients affects the soil bio-environment and underground water sources [17]. Our Earth atmosphere is filled with 78% of nitrogen (N₂) and there are many microorganisms naturally converting this free nitrogen into ammonia and nitrates, which are the favorable forms of nutrients for plants to uptake directly form soil [18].

Various soil-borne bacteria improve plant growth while performing their obligatory biological activities. These bacteria are also enormously involved in improving soil fertility [19]. By using such bacteria as biofertilizers could be an alternative method to cope with much demanding issue of ever-increasing synthetic fertilizers [20]. Numerous rhizospheric bacteria are naturally involved in the activities like nitrogen-fixing and phosphate solubilizing. B. cepacia has the ability to dissolve phosphate (P) from insoluble soil, which plants can then use for a variety of metabolic processes. B. japonicum develop a symbiotic relationship that allows plants to absorb nitrogen from the atmosphere [21]. The beneficial properties of soil microbes are well known. However, the potential and capability to impart positive effect on quality and quantity of crop vary among varieties of even a single plant species. Therefore, the research was aimed to evaluate the potential and beneficial effects of soil beneficial bacteria on mustard varieties. Moreover, it was aimed to figure out the ecofriendly way to improve the quantity and quality of that economically important crop using more compatible biofertilizers.

Experimental design and layout

The experiments were conducted to evaluate the effects of rhizobacteria on growth and physiology of mustard varieties. Seeds of B. juncea L. Czern. varieties (RBF17008, Tail Mustard, AARI Canola, & Super Raya) were procured from Ayub Agricultural Research Institute, Faisalabad, Pakistan. The seeds were carefully washed with distilled water and then sterilized with 2% NaOCl solution [22]. Pots (30 cm x 30 cm) filled with loamy soil, were used for experiment. Total 12 seeds were sown in each pot and the pots were watered every morning, based on weather conditions. After germination only six of vigorously grown seedlings were kept. Depending on the species and strain, different processes allow bacteria to influence plant growth; PGPR can have a direct or indirect impact on plant growth. Plant growth can be encouraged in two different ways. Through their ability to supply nutrients (potassium, phosphorus, nitrogen, and other essential minerals) or by controlling plant hormone levels, rhizobacteria either directly or indirectly support plant growth. They do this by lessening the effects of various pathogenic microorganisms on plant development and growth, acting as environmental protectors, biocontrol agents, and root colonizers [23].

Inoculum Preparation

The rhizospheric bacteria (Bradyrhizobium japonicum & Burkholderia cepacia) were obtained from First Fungal Culture Bank (FFCB), Institute of Agricultural Sciences, University of the Punjab, Lahore. Luria Bertani (LB) agar medium was prepared for culturing by using 10g of tryptone, 10g of NaCl, 5g of yeast extract, 16g of agar, and 1000 mL of distilled water. The homogenized solution prepared from chemicals was autoclaved for 15 min at 121^o C [24] and transferred to petri plates afterwards. Both bacterial strains were cultured again, in sterilized conditions. Plates inoculated with B. japonicum and B. cepacia were transferred in two different incubators set on 20°C and 35°C [25, 26], respectively. LB broth was prepared and both bacterial strains were inoculated into falcon tubes filled with liquid medium, separately. After 36 hours at optimum temperature tubes started showing turbidity, were centrifuged at 4˚C for 20 min with 10,000g [27]. The supernatant was removed and bacteria present at the bottom was collected and the suspensions were prepared [28, 29, 30]. Total three bacterial treatments (a) B. japonicum (B1) treatment (b) B. cepacia (B2) treatment and (c) combined treatment of B1 and B2 were prepared and then directly inoculated to the plant’s rhizobium. Synthetic fertilizer was used as standard. The inoculations of all the treatments were made directly into the plant’s rhizobium. The first bacterial inoculation was made at two leaf stage and the second inoculation was made after 4 weeks of first inoculation.

Morphological Attributes

A single plant out from each pot was uprooted, and dried out with tissue paper after washing under running water. Plant growth attributes including plant height, root and shoot lengths, root and shoot fresh and dry weights, leaf area and yield were observed. At the end of crop season, the yield attributes were recorded including pods length, pod and seed number and seed weight. The growth parameters were measured according to method used by [21].

Root Colonization Test

Root colonization test was performed in vitro. 14 Days old seedlings grown in half strength MS medium prepared in 1% agar, were taken in 1.5 mL sterile tubes. 1 mL of bacteria in 0.45% NaCl solution was added in these tubes and incubated the tubes overnight on a shaker (220 rpm, 26°C). Discarded the bacterial solution and placed the seedlings on sterile aluminum foil. Cut the roots with sterile razor and kept them in 1.5 ml tubes. Root surface was sterile by adding 1 mL of 75% ethanol, through agitation using spin wheel. Ethanol was removed and roots were washed using ddH₂O for three times. Roots were grinded in sterile pistils. Following that, 1 mL of 0.45% NaCl solution was added in the tubes. Vortexed the mixture and serial dilutions were prepared. These dilutions were plated drop by drop in LB-agar plates, dried for 2 min and then incubated at 30°C for 24 h. Centrifuged the samples at 10,000 rpm till the root debris is pelleted. Roots pellets were oven dried at 60°C for 48 h and recorded their dried weight. Bacterial colonies and colony frame units (CFUs) were counted by taking in account the dilutions and plated volume. CFU values were normalized with root dry weight to obtain colonization rates [31].

$$\:Colonization\:rate\:=\:(CFUs\:\times\:\:dilution\:factor)/\:mg\:of\:root\:DW$$

Determination of Chlorophyll & Carotenoid Content

Arnon method [32] was used to determine the chlorophyll content in plants. Wisely cut fresh leaves (1g) were ground in 20 mL of 80% acetone and filtered through Whatman’s filter papers. The falcon tubes with extracts were then placed in centrifuged at 10000g for 5 min at cold. The supernatant was collected and absorbance were taken under UV/VIS Spectrophotometer on set wavelengths of 645nm, and 663nm. Chlorophyll a and chlorophyll b and total chlorophyll values were determined by using the following formula:

$$\:Chl.\:a\:\left(mg\:{g}^{-1}\:FW\right)=\frac{[12.7\:\left(OD663\right)-2.69\:\left(OD645\right)]\times\:V}{1000\times\:W}$$

$$\:Chl.\:b\:\left(mg\:{g}^{-1}\:FW\right)=\frac{[22.9\:\left(OD645\right)-4.68\:\left(OD663\right)]\times\:V}{1000\times\:W}$$

$$\:Total\:chlorophyl\:\left(mg\:{g}^{-1}\:FW\right)=\:\frac{[20.2\:\left(OD645\right)+8.02\:\left(OD663\right)]\times\:V}{1000\times\:W}\:$$

Here, Volume of Extract (mL) = V, Weight of Fresh Leaves (g) = W, Optical Density (Specific Wavelength) = OD

Carotenoid content was calculated using following formula [33]:

$$\:Carotenoids=\:\frac{\left(1000\times\:OD480\right)-\:(1.9\times\:Chl\:a\:-\:63.14\times\:Chl\:b)}{214}$$

Determination of Relative Water Content

Uniform sized fresh leaves were weighed, and placed in petri dish filled with distilled water. After 3 h, at room temperature, in dark, leaves were weighed again and then into an oven at a temperature of 80°C. After 24 h, dry weights were measured for every leaf.

By using Jones and Turner method [34], the relative water content was calculated with following formula:

$$\:RWC\:\left(\%\right)\:=\frac{(FW\:-\:DW)}{(TW\:-\:DW)}\:\times\:100\:$$

Here, FW = Fresh Weight, DW = Dry Weight, TW = Turgid Weight

Assay of Total Soluble Protein

Complete homogenized (0.25 g) fresh leaves in 10 mL of 50 mM cooled phosphate buffer (pH 7.8) were centrifuged at 4°C for 20 min at 6000g. The supernatant was collected and deep freeze. Bradford method [35] was used to measure the total soluble proteins in extracts. Bradford solution (100 mg Coomassie Brilliant Blue mixed with 50 mL of 95% Ethanol) and 100 mL of phosphoric acid (85%), added and volume of solution was raised to 1L using distilled water. Extract (0.1 mL), mixed with 5 mL Bradford solution and UV/VIS Spectrophotometer readings were taken at 595 nm.

Assay of Total Phenolics

Freshly leaves (0.05g) homogenized into 5 mL of acetone (80%), and extract was centrifuged in cold for 10 min at 10,000g. Firstly, 0.1 mL extract, 2mL of distilled water, and Folin-Ciocalteu’s Phenol reagent was properly mixed. Secondly, 5 mL sodium carbonate solution was added and the volume was raised to 10 mL using distilled water. UV/VIS Spectrophotometer readings were taken on 750 nm wavelength [36].

Assay of Catalase (CAT) Activity

The reaction solution for CAT (3 mL) was prepared by adding 1.9 mL H₂O₂ (5.9 mM), 1 mL of phosphate buffer (50 mM) with a pH maintained at 7.0, and the 100 µL of enzyme extract (which was already prepared and deep freeze for later use). Different absorbance variations, at 240 nm, for CAT solution were observed for every 30s till the time limit of 120s, using spectrophotometer [37].

Assay of Peroxidase (POD) Activity

The reaction solution for POD (2 mL) was prepared by adding 0.6 mL of Guaiacol (20 mM), 0.7 mL of phosphate buffer (pH was maintained at 5.0), 0.6 mL of H₂O₂ (40 mM) and the 100 µL of the enzyme extract. The absorbance readings were taken at 470 nm for the every 30s at total time limit of 150s, using spectrophotometer [37].

Assay of Ascorbate Peroxidase Activity (APX)

By using Nakano and Asada method [38] APX activity was assessed. The reaction mixture of APX (3 mL) was prepared by adding 2.7 mL of potassium phosphate (50 mM), 100 µL of ascorbic acid (7.5 mM), 100 µL of H₂O₂ (300 mM), and 100 µL of enzyme extract. The absorbance variations were examined at 290 nm for every 30s from 0-60s. The solution placed in blank of spectrophotometer was consisted of 100 µL of distilled water instead of enzyme extract.

Assay for superoxide dismutase (SOD) determination

SOD activity was measured using a photochemical reduction of nitroblue tetrazolium chloride (NBT) at 560 nm. The reaction mixture included 1.5 mL of 0.05 M sodium phosphate buffer (pH 7.8), 750 µM NBT, 130 mM L-methionine, 0.1 mM EDTA-Na2, 20 µM riboflavin, 4% polyvinylpyrrolidone, supernatant, and deionized water. After adding riboflavin to the reaction mixture in the dark, it was incubated for 10 minutes under 15-W fluorescent light. U mg-1 FW was used to measure SOD activity [39].

N and P nutrients determination in plant sample

Leaf powder (0.5 g) from each treatment was dry-ashed in a muffle furnace at 515°C for 5 hours. The ash was dissolved in 3 mL of 6 N HCl, then diluted with 50 mL of double distilled water. Phosphorus concentrations were measured by ICP (Perkin Elmer-Optical Emission Spectrometer, OPTIMA 2100 DV) [40]. Nitrogen was assessed using the Kjeldahl method [41]. N and P were represented as percentages of dry weight.

Data Analysis

COSTAT version 6.303, Copyright(c) 1998–2004 CoHort Software, 798 Lighthouse Ave. PMB 320, Monterey, CA, 93940, USA, was used for analysis of variance (ANOVA) with 5% level of significance. Microsoft Excel was used to analyze, calculate, evaluate the raw data collected through experiment. All the graphs were created on Microsoft Excel.

The growth of Mustard plants under the effect of rhizospheric bacteria (B. japonicum and B. cepacia) was assessed by collecting data for different morphological and physiological and biochemical attributes. The data from the samples were recorded and analyzed via analysis of variance (ANOVA). All four mustard varieties showed positive results for growth attributes under the treatments of B. japonicum and B. cepacia. The detailed results of morphological, physiological and biochemical attributes are following:

Morphological Attributes

The analysis of variance showed significance of all the observed morphological attributes of mustard varieties grown under various treatments (Table S1). In comparison to control, maximum plant height (21% increase) was represented by Tail Mustard variety with the treatment of B. japonicum. Leaf area of the same variety was observed to be increased up to 11% with all bacterial treatments. Shoot length showed the same response as that of plant height. Root length of AARI Canola was increased up to 37% under the influence of B. japonicum. Shoot and root fresh weights were positively influenced (24% and 57%) by the combined bacterial treatment in Super Raya and Tail Mustard respectively. Dry weights of shoot and root found to be increased in Tail Mustard (30–40%) with all treatments and AARI Canola (20%) with B. japonicum. Number of leaves and leaf yield were increased (up to 24%) in AARI Canola with both bacterial treatments. Pod size and number was improved (15–25%) in Super Raya and Tail Mustard while in case of seed number and seed size, positive response (11–29%) was given by all the varieties (except RBF17008) with all bacterial treatments, respectively (Fig. 1–4).

Bacterial colonization rate

Both bacterial species B. japonicum and B. cepacian successfully colonized B. juncea roots in vitro conditions. High values for colony forming units (CFUs) depicts high and efficient colonization rate for both tested bacterial species, as shown in Fig. 5.

Physiological Attributes

The analysis of variance showed significance of all the observed physiological attributes of mustard varieties grown under various treatments (Table 1).

Table 1

Physiological attributes of *Brassica juncea* (L.) Czern. cultivated under different rhizospheric bacterial treatments
SOURCE	DF	Relative Water Content (%)	P	Chlorophyll a (mg/g FW)	P	Chlorophyll b (mg/g FW)	P	Total Chlorophyll (mg/g FW)	P	Carotenoid Content (mg/g FW)	P
Variety	3	15.944693	0.0125^*	1.2808e-5	0.2697^ns	0.0023012	0.0001^***	80.285554	0.0001^***	2.9526102	0.0010^***
Treatments	4	8.3540583	0.0925^ns	3.1832e-5	0.0181^*	0.0050542	0.0000^***	167.56826	0.0000^***	1.4505766	0.0209^*
Variety × Treatments	12	6.5830014	0.1051^ns	1.3299e-5	0.2020^ns	6.6392e-4	0.0128^*	24.656188	0.0084^**	1.077601	0.0180^*
Error	40
ns = non-significant, Level of significance: , , ** at 0.05 level, LSD 0.05

Determination of Relative Water Content (%)

The mustard variety RBF17008 recorded a 2% and 1% increase in relative water content for B. japonicum and B. cepacia treatments, respectively. But the combined treatment of both bacteria caused no effect on the water content of RBF17008 plants. Tail Mustard showed a 4% increase in relative water content for B. japonicum treatment and a 2% increase for B. cepacia treatment, while it showed a 2% decrease against the combined treatment of bacteria. AARI Canola resulted in a 3% increase in relative water content against the combined treatment of bacteria, and 1% for B. japonicum treatment, while it showed a 1% decrease for B. cepacia treatment. Super Raya expressed a 2% decrease in relative water content for B. cepacia treatment and combined treatment of both bacteria. B. japonicum imposed no effect on the relative water content in Super Raya (Fig. 6).

Determination of Chlorophyll Content

Chlorophyll a

AARI Canola responded positively and showed a noticeable increase in chlorophyll a content for all three bacterial treatments. The mustard variety (AARI Canola) recorded an improvement of 3% in chlorophyll a with the treatment of B. cepacia, 1% increase with B. japonicum treatment, and 2% increase with combined treatment of B. japonicum and B. cepacia. Tail mustard showed no changes in chlorophyll content for separate treatment of B. japonicum and combined treatment of B. japonicum with B. cepacia, but there was a 4% increase in chlorophyll a with B. cepacia treatment. RBF17008 recorded a decrease in primary chlorophyll by 1% and 2% for the separate treatments of B. japonicum and B. cepacia, but for the combined treatment no change in chlorophyll a was recorded. The variety Super Raya resulted in a 2% increase in primary chlorophyll with nitrogen-fixing bacteria, and a 1% increase for combined bacterial treatment, while there was 1% decrease was recorded for phosphate solubilizing bacteria (Fig. 7).

Chlorophyll b

The treatment of nitrogen-fixing bacteria (B. japonicum) improved the chlorophyll b content in RBF17008 by 6% and in Super Raya by 10%, but there was a decrease in Tail Mustard by 13% and in AARI Canola by 10%. The treatment of phosphate solubilizing bacteria (B. cepacia) improved the chlorophyll b content by 46% in Tail Mustard and 9% in AARI Canola. B. cepacia treatment imposed a decrease in chlorophyll b by 14% in RBF17008 but caused no change in the Super Raya variety. The combined treatment of B. cepacia and B. japonicum increased the chlorophyll content by 34% in Super Raya and 26% increase in RBF17008 varieties. Tail Mustard showed a 10% and AARI Canola 1% decrease in chlorophyll b content for the combined bacterial treatment (Fig. 8).

Total Chlorophyll

Tail Mustard (32%) and AARI Canola (8%) showed an increase in total chlorophyll for the B. cepacia treatment, but RBF17008 showed a decrease by 9% and the Super Raya variety was unchanged. The B. japonicum treatment imposed an increase in chlorophyll in RBF17008 by 4% and in Super Raya by 7%, yet Tail Mustard and AARI Canola varieties showed a decrease by 8% and 5% respectively. The combined treatment of B. japonicum and B. cepacia showed an increase in total chlorophyll in Super Raya, RBF17008, and AARI Canola varieties by 32%, 17%, and 1%, respectively. Tail Mustard on the other hand showed a decrease in chlorophyll by 9% under combined bacterial treatment (Fig. 9).

Determination of Carotenoid Content

The treatment of B. japonicum showed an increase of carotenoid content in RBF17008 by 8% and 3% in Super Raya, but Tail Mustard and AARI Canola showed a decrease by 5% and 7% respectively. The treatment of B. cepacia showed an increase of carotenoids by 9% in Tail Mustard, but RBF17008 resulted in a decrease of carotenoid content by 5%. B. cepacia treatment imposed no change on AARI Canola and Super Raya varieties. The combined treatment of both bacteria showed an increase of carotenoid content in RBF17008 by 13% and in Super Raya by 4%. The other varieties showed a decrease of carotenoid content by 5% in Tail Mustad and 8% in AARI Canola (Fig. 10).

Biochemical Attributes

The analysis of variance showed significance of all the observed biochemical attributes of mustard varieties grown under various treatments (Table 2).

Table 2

Biochemical attributes of *Brassica juncea* (L.) Czern. cultivated under different rhizospheric bacterial treatments
SOURCE	DF	Total Phenolic content (µg/g FW)	P	Total Soluble Protein (mg/ml)	P	Catalase (units/mg protein)	P	Peroxidase (units/mg protein)	P	Ascorbate Peroxidase (units/mg protein)	P
Variety	3	0.1994322	0.0000^***	9.823702	0.0000^***	4.0255765	0.0000^***	1.8145446	0.0000^***	0.0839665	0.0000^***
Treatments	4	0.2913214	0.0000^***	15.769382	0.0000^***	4.8172161	0.0000^***	1.3361506	0.0000^***	0.0036489	0.0000^***
Variety × Treatments	12	0.2579203	0.0000^***	15.403379	0.0000^***	7.4427146	0.0000^***	1.0509175	0.0000^***	5.715e-4	0.0000^***
Error	40
ns = non-significant, Level of significance: , , ** at 0.05 level, LSD 0.05

Total Phenolic Contents

There were huge improvements were recorded in the RBF17008 variety as compared to the control, while the Tail Mustard variety showed the opposite response. RBF17008 showed a 224% increase in phenolics for the combined treatment of B. cepacia and B. japonicum, a 69% increase for the B. cepacia treatment, and 93% for the B. japonicum treatment. The Tail Mustard variety showed a reduction in phenolic content by 37% against B. cepacia treatment, 13% for B. japonicum treatment and 6% for B. cepacia and B. japonicum combined treatment. The B. japonicum imposed no change in AARI Canola, but the results were improved by 10% for the B. cepacia treatment and 82% for the combined treatment of both bacteria. The fourth variety Super Raya showed a 131% phenolic content improvement for B. japonicum treatment, and 25% for its collective treatment with B. cepacia. The Super Raya showed a decrease in phenols by 6% for the separate B. cepacia treatment (Fig. 11).

Total Soluble Proteins

B. japonicum treatment improved the protein content by 25% in Tail Mustard, and 12% in Super Raya, while protein content was decreased due to this treatment by 10% in RBF17008 and 12% in AARI Canola varieties. The treatment of B. cepacia increased the soluble protein in Tail mustard by 14%, in AARI Canola by 17%, and in Super Raya by 2%, but RBF17008 showed a decrease up to 11%. The combined treatment of both rhizospheric bacteria recorded a 25% increase in protein for Tail Mustard, while for other varieties there was a notable decrease in protein. There was an 18% decrease in RBF17008, 4% in AARI Canola, and 2% in Super Raya for collective treatment (Fig. 12).

Catalase Activity

RBF17008 showed a negative response when given the bacterial treatments, and the catalase quantity was reduced as compared to control. It showed an 11%, 20%, and 25% decrease in catalase for B. japonicum, B. cepacia, and combined treatment, respectively. While in other varieties, the B. japonicum treatment increased the catalase activity by 12% in Super Raya and 24% in Tail Mustard, but AARI Canola had a 12% decrease. The B. cepacia showed an increase of 1% in Super Raya, 11% in Tail Mustard, and 16% in AARI Canola. The combined treatment imposed negative effects as the Super Raya had recorded a 3% decrease and AARI Canola 5%. On the other hand, the B. japonicum and B. cepacia collectively improved the catalase by 21% in Tail Mustard (Fig. 13).

Peroxidase Activity

Tail Mustard variety showed a very positive response against both the rhizospheric bacteria and the peroxidase (units/mg protein) was improved as compared to the control plants. There was a 25% increase against B. japonicum treatment, 14% increase against B. cepacia treatment and a 25% increase against the collective treatment of both bacteria recorded in Tail Mustard. RBF17008 variety showed a notable reduction in peroxidase (POD) activity by 9% for B. japonicum, 10% for B. cepacia, and 17% for combined treatment. AARI Canola had recorded an 11% decrease in POD activity for B. japonicum, while a 19% increase for B. cepacia and 2% for combined treatment. Super Raya showed a decrease in POD by 1% for combined treatment, but there was a notable increase in total soluble protein content by 13% for B. japonicum treatment and 3% for B. cepacia treatment (Fig. 14).

Ascorbate Peroxidase Activity

The bacterial treatment of B. japonicum and B. cepacia imposed no changes in any Mustard variety, separately. The combined treatment of both the rhizospheric bacteria showed a decrease in ascorbate peroxidase quantity in Tail Mustard and RBF17008 by 1%, each. The other two varieties Super Raya and AARI Canola showed no changes in the collective treatment (Fig. 15).

Superoxide Dismutase Activity

The bacterial treatment of B. japonicum and B. cepacia imposed a slight change in SOD activity of all studied mustard varieties. B. japonicum and B. cepacia inoculation showed maximum SOD activity in Super Raya (32.3 and 28 U mg^− 1 FW) followed by BRF17008 (28.3 and 24 U mg^− 1 FW) and Tail Mustard (19 and 23.3 U mg^− 1 FW). AAR1 Canola showed the minimum SOD activity in response to bacterial inoculation as compared to other varieties. However, bacterial inoculation enhanced SOD activity in all four varieties as compared to control and fertilizer treatment (Fig. 16).

N and P uptake

Nitrogen and phosphorus uptake were significantly enhanced in all mustard varieties inoculated with bacterial colonization (Fig. 17–18). This increase in N and P uptake is also depicted in improved growth, physiological and biochemical attributes of mustard verities.

The study aimed to determine the efficiency of rhizospheric bacteria (B. japonicum & B. cepacia) on the growth of four Mustard varieties (RBF17008, AARI Canola, Tail Mustard & Super Raya). The ever-increasing population makes it difficult for the agricultural field to cope with daily demand. The land requires a continuous supply of macro and micronutrients for better crops. Plants express an ultimate decrease in growth if there is impaired nitrogen-fixing going on, in the soils which are already nutrient deficient [42]. One of the reasons for diminished nitrogen fixation is the deficiency of phosphate in soil, which reduces the nitrogenase activity; an enzyme, associated with atmospheric nitrogen fixation [43]. B. japonicum is a natural nitrogen-fixing bacteria that improves the nitrogen-fixing ability of plants [44]. It was reported that the bacterium B. cepacia owns the phosphate solubilizing activity, and it can be used as a plant growth promoter [29]. Using such microorganisms is an effective way to provide much-needed nutrients to plants [45].

The outcome observed from this experiment showed a notable increase in the plant height as well as in the total number of leaves in mustard varieties, with B. japonicum treatment compared to control. Similar results were observed when the cowpea plant was grown under the B. japonicum treatments. There was an increase in the number of leaves and the plant heights [46]. In the present results, the increase in chlorophyll content and AARI Canola, while the notable increase for RBF17008 and Super Raya varieties in chlorophyll b, and carotenoids was observed under the influence of the given bacterial treatments. It was also studied that the increase in chlorophyll content in leaves of the cowpea plants with B. japonicum treatment. It was also referred that B. japonicum could be used with inorganic fertilizer and it might show greater results when combined with phosphorus treatment [46].

In this experimentation, bacterial inoculation had very minute effect on carotenoid pigments in all four mustard varieties. A very slight increase or decrease could be observed which can be neglected. Carotenoids are very essential pigments for plants, play crucial role as secondary messenger and also act as antioxidant under stress conditions. No change in these pigments in response to bacterial inoculation can be attributed to efficient bacterial inoculation that resulted in enhanced growth of plants instead of imposing any stress over plants. Similar findings were also reported in a study on C. annuum plants inoculated with growth promoting bacteria [47]. The present study also revealed the increase in chlorophyll content in mustard plants for certain phosphate solubilizing bacteria. The RBF17008, AARI Canola and Super Raya varieties showed a huge increase in phenolic content for all three bacterial treatments. Similar kind of results were also observed for the phosphate solubilizing rhizospheric bacteria, which elevated the phenolic content in B. juncea L. [48].

In this experiment, the Tail Mustard variety showed an increase in ascorbic acid, peroxide, and catalase activities for all the three bacterial treatments. But the RBF17008 variety showed a decrease in ascorbic acid, peroxide, and catalase activities for all the three bacterial treatments. Similar findings were assessed, decrease in APX, POD, and CAT activity was respectively, when the plants were treated with B. cepacia along with glyphosate [49]. Since it postpones planned cell death, plants' antioxidant defense system is essential for them in stressful situations. Antioxidant enzymes scavenge excess reactive oxygen species, which prevents cellular organelles from carrying out their functions as intended. This leads to a number of inhibitions in enzymes, lipid peroxidation, oxidative protein damage, and breakdown of DNA and nucleic acids [50].

The amount of soluble protein in the leaves and the activity of antioxidant enzymes were both impacted by irrigation schedules and biofertilizers. With the exception of CAT, all the assessed variables showed significant effects of the irrigation regimes at p < 0.01. The activity of GPX, GR, APX (p < 0.01), and CAT enzymes (p < 0.05) may be stimulated by biofertilizers [51]. Furthermore, a noteworthy correlation was discovered between the irrigation schedules and the bio fertilizers concerning antioxidant enzymes and the amount of soluble protein [52].

The present study resulted a notable increase in shoot and root dry weights for all the three bacterial inoculations given to all mustard varieties, except RBF17008 which recorded the decrease in dry weights. PGPR have a specific mechanism for the growth of plants, it effects the plant growth in two different ways. First is direct way in which rhizobacteria enhance the growth of plant by providing the materials to plant, plants cannot take these substances from environment due their unavailable forms. Nitrogen and phosphorus are the nutrients in unavailable forms in environment. Second is indirect method in this method bacteria secret chemicals in soil with work against the disease causing agents in soil [53]. It was also reported the increase in shoot and root dry weights of the soybean plant, when given the combined treatments of B. japonicum with actinomycetes strain (Nocardia alba) [54]. The phosphorus and nitrogen level were also elevated in plants. The application of Burkholderia cepacia with a certain phosphorus dose improved the shoot dry weight in the potato plants [55].

The present experiment showed a notable increase in shoot and root fresh weights for all the mustard varieties when these were inoculated with B. japonicum and B. cepacia. It was reported that increase in the shoot’s fresh and dry weights, root’s fresh and dry weight, shoot height, root length, and leaf lengths, when the maize plants were inoculated with Alcaligenes aquatilis and B. cepacia. They also reported the increase in grain numbers and weight [28]. In the present findings, two inoculations of rhizospheric bacteria on all mustard varieties (RBF17008, AARI Canola, Super Raya, Tail Mustard) resulted in a significant increase in lengths of root and shoot. Inoculation of different plant growth-promoting bacteria enhanced the root and shoot lengths and fresh and dry weights for canola plants [56]. Shoot lengths, root lengths, and shoot dry weight were elevated in soybean against the B. japonicum treatments [57]. Here in present study, all four mustard varieties resulted in an increase in the number of seeds per plant, and the total leaf yield of the plant (g) was found to be improved under the bacterial treatments. The presence of nitrogen-fixing bacteria might be the reason for the increase in leaf yield and seeds in mustard varieties as also reported in literature. The leaf yield and seed yield were found to be increased with the elevated nitrogen doses in the coriander crop [58].

In this experiment, the pod length and the number of seeds per pod were elevated in mustard varieties under the treatment of nitrogen-fixing bacteria (B. japonicum) and phosphate solubilizing bacteria (B. cepacia). Similar results were found in the fenugreek plant when it was inoculated with different rhizospheric bacterial species [59]. This study showed that the overall yield of mustard plants was elevated as compared to the control plants in this experiment. Similar findings were observed the increase in yield of soybean by 20% with B. japonicum inoculations. The increase or decrease in the plant's morphological, or physiological attributes is possible due to the change in the microenvironment of the plant. The inoculations of phosphate solubilizing bacteria and nitrogen-fixing bacteria in the roots of mustard plants might have proposed the changes in the nutrient availability in the soil [60]. As reported by PGPR actively participate in geochemical nutrition cycle and can influence nutrient availability to plants and microbes. Also the use of such bacteria as bio-inoculant can increase nutrient availability in soil. Consequently, the change occurred with the presence of both the bacteria might have been the reason for the increase or decrease in growth and yield of mustard plants. As previous reported that the change in the bacterial environment in soil could be caused by root exudates [61].

The extensive use of chemical fertilizers continuously damaging the biotic and abiotic parts of environment. The hazardous effects of fertilizers can be reduced by using beneficial bacteria. From this research it was observed that Tail Mustard and Super Raya showed the maximum increase in morphological and physiochemical attributes for separate and combined treatments of B. japonicum and B. cepacia. These bacteria help the mustard plants in availability of nutrients which are crucial. It was found that these bacteria can be used as bio fertilizer without effecting the environment and living organisms. Bio fertilizers can be used as alternative to chemical fertilizers, so in this way good yield can be obtained without any environmental issue. In future field experiments are also needed for more growth and yield at large scale.

Availability of data and materials

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethical approval and consent to participate

We declare that the manuscript reporting studies do not involve any human participants, human data or human tissues. So, it is not applicable.

Our experiment follows the relevant institutional, national, and international guidelines and legislation.

Consent for publication

Not applicable

Clinical trial number: Not applicable

Competing interests

The authors have no relevant financial or non-financial interests to disclose.

Funding

Researchers Supporting Project number (RSP2024R393), King Saud University, Riyadh, Saudi Arabia.

Author Contributions

NB; Experimentation and Methodology, MR, ZN & MA; Conceptualization, Supervision and Validation, SU; Statistical analysis, AAS; Resource acquisition and Investigation, MS; writing-original draft preparation, and SS & MKG; Data curation and Formal analysis. All authors read and approved the final manuscript.

Acknowledgments: Authors are thankful to Researchers Supporting Project number (RSP2024R393), King Saud University, Riyadh, Saudi Arabia.

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Improving Growth and Nutrient Quality of Mustard Varieties Through Rhizobacterial Inoculation

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Abstract

Figures

Introduction

Materials and methods

Experimental design and layout

Inoculum Preparation

Morphological Attributes

Root Colonization Test

Determination of Chlorophyll & Carotenoid Content

Determination of Relative Water Content

Assay of Total Soluble Protein

Assay of Total Phenolics

Assay of Catalase (CAT) Activity

Assay of Peroxidase (POD) Activity

Assay of Ascorbate Peroxidase Activity (APX)

Assay for superoxide dismutase (SOD) determination

N and P nutrients determination in plant sample

Data Analysis

Results

Morphological Attributes

Bacterial colonization rate

Physiological Attributes

Determination of Relative Water Content (%)

Determination of Chlorophyll Content

Chlorophyll a

Chlorophyll b

Total Chlorophyll

Determination of Carotenoid Content

Biochemical Attributes

Total Phenolic Contents

Total Soluble Proteins

Catalase Activity

Peroxidase Activity

Ascorbate Peroxidase Activity

Superoxide Dismutase Activity

N and P uptake

Discussion

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

Status:

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