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 121o 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.