Sample Collection and isolation of nitrogen-fixing bacterial epiphytes and endophytes
Sample Collection-In the current investigation, eight rhizospheric epiphytes and two endophytic bacteria were isolated on nitrogen-free medium and the source of isolation is tabulated in (Table 1). Nitrogen-free Rennie media used for isolation of diazotrophs is a combination of two solutions with the composition given in Annexure I.
Isolation of diazotrophs- Soil from the rhizosphere region at a depth of 5 cm of differently N fertilized (organic and inorganic nitrogen) rice plants (Genotypes- MTU1010, CR Dhan 310, Pusa 44 and IR 64) of Indian council of agricultural research (ICAR)- Indian institute of rice research (IIRR) farm, Rajendranagar, Hyderabad, Telangana, India were collected in sterile polythene bags and stored at 4 ℃ until further analysis. For isolating diazotrophic rhizobacteria, serial dilution spread plate technique was carried out on nitrogen- free media. For endophytic diazotroph isolation, rice roots were harvested from the rice plants that were variedly (organic and inorganic nitrogen) fertilized and surface sterilized according to Hou et al. (2022) with slight modifications. Briefly, root samples of >5 cm was cut into 1/10th size using sterile blades followed by multiple sterile water washes. The roots were then rinsed with 70% ethyl alcohol (C2H5OH) for 2 min there after 3- min dip in 2% sodium hypochlorite (NaOCl) solution, 70% ethanol for 1 min and intermediate multiple water washes. The final wash solution was inoculated onto nutrient and N free agar plates for surface sterility confirmation. The surface sterilized root samples were then ground in sterilized mortar pestles and 100 µL of ground paste was inoculated on N free media. Inoculated plates were incubated for 48 h at 28℃, morphologically dissimilar single colonies were picked after incubation and sub cultured three to four times for purification. Purified epiphytes and endophytes were stored in 30% glycerol stock for further analysis.
In-vitro illustration of plant growth promotion traits and hydrolytic enzyme synthesis
For the studies mentioned below, active culture of bacteria was prepared by inoculation of pure bacterial isolate in 10 ml N free media and used
Indole-3-acetic acid (IAA) synthesis- Qualitative and quantitative estimation of IAA was carried out in nutrient agar and nutrient broth (supplemented with 5 mM tryptophan) respectively. The active culture of each bacterial isolates was inoculated and incubated at 30 ℃ for 72 h, after incubation in sterile filter paper disks presoaked in Salkowski reagent (1 mL of 0.5 M FeCl3, 49 mL HClO4) were overlaid on the inoculated spots and observed for pink coloration. Positive bacterial isolates in plate culture conditions were assessed quantitatively by inoculation of 50 µL of active culture in 5 mL nutrient broth amended with 5 mM tryptophan and incubated on an orbital shaker at 30 ℃, 200 rpm for 48 h. After incubation, cultures were centrifuged at 10,000 rpm for 10 min. Next to this, 1 mL supernatant was added to 4 mL of Salkowski reagent, then incubated for 30 min at 30 ℃ for the development of pink color and absorbance was measured at 530 nm against standard IAA by UV- spectrophotometer (Shimadzu UV-VIS) (Gordon SA and Weber 1951).
Phosphate solubilization- Phosphate-solubilizing ability of the bacterial isolates was assessed by the spot technique, by inoculation of actively grown culture on National Botanical Research Institute's Phosphate Growth (NBRIP) medium in plate culture conditions (Annexure I). A 0.1 mL of active culture prepared as mentioned above was inoculated separately in NBRIP broth, incubated on an orbital shaker at 200 rpm, 30 °C for 5 days along with uninoculated control. Broth was centrifuged after incubation at 10,000 rpm for 10 min, and ANSA reagent (0.5 mL sol A + 0.2 mL sol B) was added to 0.1 mL of supernatant, final volume was made up to 5 mL with distilled water, incubated for 10 min at room temperature, OD was measured at 660 nm using a UV spectrophotometer (Shimadzu UV-VIS) (Murphy J and Riley 1962).
Zinc solubilization- Zinc-solubilizing ability of the bacterial isolates was evaluated by spot inoculation of actively grown culture on tris mineral salt agar medium supplemented with zinc oxide at 0.1% concentration (ZSB medium- Annexure I). Inoculated plates were incubated for 3-5 days at 30 ℃ and halo zone around each colony was measured using scale (Goteti et al. 2013).
Siderophore production- Chrome azurol S (CAS) agar was used for detection of siderophore synthesis by the isolated bacteria (Annexure I). Spot-inoculated plates with actively grown culture were incubated at 30 ℃ for 72 h in dark and observed for the development of orange halo zone around the bacterial colonies (Schwyn and Neilands 1987).
Hydrocyanic acid (HCN) synthesis- Synthesis of HCN by bacterial isolates was assessed by the Bakker and Schippers method (1987). Briefly, active bacterial isolates were streaked on King’s B agar (Annexure I) and incubated at 30 ℃ for 72 h. After incubation, sterile filter paper presoaked in 2% sodium carbonate (Na2CO3) and 0.5% picric acid ((O₂N)₃C₆H₂OH) solution was placed in the inoculated plate’s lid. Plates were sealed with parafilm, then incubated upright for 3-5 days at 28 ± 2 ℃ and observed for the color change of filter paper from yellow to brown.
Synthesis of ammonia- Ammonia synthesis by the bacterial isolates was assessed in peptone water medium (Annexure I). Ten microliters (µL) of actively grown bacterial culture were used as inoculant in 10 mL peptone broth, incubated for 72 h at 30 ℃. Nessler’s reagent was added to the incubated tubes and color change (yellow-brown-deep brown) was observed for ammonia synthesis (Cappucino and Sherman 1992).
ACC (1-aminocyclopropane-1-carboxylate) deaminase activity- Actively grown bacterial isolates were assessed for ACC deaminase by spot inoculation on Dworkin and Foster (DF) mineral salt medium supplemented with 3 mM ACC and DF medium with 0.2 % ammonium sulfate served as test and positive controls, respectively (Annexure I). The growth of the bacterial isolates on DF-ACC plates was compared with DF-NH4(SO4)2 plates after incubation of 4-5 days at 30 ℃ and considered possessing ACC deaminase activity (Penrose and Glick 2003).
Amylase activity- Amylase production by the isolated bacteria was evaluated according to Gupta et al. (2003). In brief, actively grown bacterial isolates were spot inoculated on starch agar medium plates (Annexure I) and incubated at 28 ± 2 ℃ for 72 h. Plates were then flooded with iodine (I2) solution for a minute, excess solution was drained off and observed for the zone of hydrolysis around the colonies.
Urease activity- Christensen’s urea agar medium was used to detect the urease activity of the bacterial isolates as described by Brink (Annexure I) (Ding et al. 2005). Actively grown bacterial isolates were spot inoculated on urea agar plates and incubated for 125 h at 35 ℃ and observed for color change, yellow or pink.
Cellulase activity- Actively grown bacterial isolates were spot inoculated on carboxymethyl cellulose (CMC)- Congo red medium plates (Annexure I), incubated for 72 h at 28 ± 2 ℃ and observed for clear zone around the colonies (Hendricks et al. 1995).
Determination of fungal phytopathogenic activity invitro- Antifungal activities of Rhizoctonia solani and Macrophomina phaseolina was demonstrated with the cultures procured from Osmania University, Hyderabad, India. Actively grown fungal culture was cut into 5 mm mats and inoculated onto the centre of potato dextrose agar (PDA) plate (Annexure I). While the actively grown test bacterial culture was streaked at a 10-mm distance from the edge of the plate that has a 90mm diameter and incubated at 28 ± 2 ℃ for 125 h while control plate had only a fungal mat (Mir et al. 2021). After incubation, the percent inhibition was calculated over the control using the formula given below.
I = (C-T/C) × 100
Where, I = inhibition % of mycelial growth, C = radial growth of the pathogen without antagonists, T= radial growth of the pathogen with antagonists.
Antagonistic activity of bacteria using biomass reduction- Bacterial isolates that demonstrated antagonism on plates were assessed for fungal biomass reduction in potato dextrose broth (PDB) medium against above said fungi according to Hameeda et al. (2010) Then, to 50 mL of PDB 1 mL of actively grown test bacterial isolate was inoculated into actively grown, homogenized fungal culture in each 100 mL conical flask, a control was maintained without bacterial isolate incubated on an orbital shaker at 200 rpm, 30 ± 1 ℃ for 24-48 h. After the incubation period, fungal mycelium was harvested by filtration on a sterile pre-weighed Whatman No.1 filter paper, dried for 24 h at 65 ℃ and dry weight was recorded. Morphological changes in inhibited fungi were demonstrated under a light microscope using a drop of culture from each flask on a clean glass slide with lactophenol cotton blue.
Scanning Electron Microscopy (SEM)- Mycelium near the interaction region of the dual culture was processed for SEM analysis. Fixation was carried out in Karnosvsky buffer (1.7% glutaraldehyde, 1.5% paraformaldehyde, pH-7.2) for 24 h, followed by consecutive solutions of 30, 50, 70, 90, 100 % ethanol dehydration for 10 min, acetone (100%) washing twice for 10 min and finally dehydrated in the final wash solution. Before visualization, samples were ion sputter coated with gold and SEM analyzed under a ZEISS microscope (Ana et al. 2021) at Department of Physics, Osmania University, Hyderabad.
Evaluation of nitrogen fixing ability (diazotrophy) and nitrogen content in the isolated bacteria
Dobereiner’s nitrogen-free solid malate (NFM) medium- Nitrogen fixing ability was assessed in Dobereiner’s nitrogen-free solid malate (NFM) medium supplemented with malic acid (Annexure I). Active bacterial culture was streaked on NFM media, incubated at 30 ℃ for 48-72 h and observed for media color change green-blue (Döbereiner 1988).
Evaluation of the nitrogen fixing ability by nifH gene- Diazotrophy of the bacterial isolates were further figured out at the molecular level via the existence of nifH gene. Genomic DNA of the isolated bacteria were amplified using PolF and PolR as forward and reverse primers, respectively, from the nifH gene fragment for nitrogen fixation. Briefly, genomic DNA of bacteria grown in liquid medium was extracted using a microbial DNA isolation kit manufactured by MACHEREY‑NAGEL. Two microliters (µL) of extracted genomic DNA of each bacterial isolate was used for polymerase chain reaction (PCR) amplification following the profile described by Poly et al. (2001). The rest of the DNA was stored at 4 ℃ for future use. The amplified PCR product was documented by UV based gel document system in 3% agarose gel with ethidium bromide (EtBr) against 100 bp marker.
Evaluation of nitrogen fixing ability by ARA assay- The ability of bacterial isolates to fix nitrogen was quantitatively evaluated by acetylene reduction assay (ARA). Bacterial isolates were inoculated onto N-free medium slants, covered by suba-seals and incubated at 30 ℃ for 72 hrs. After the bacterial culture grewdensely, 1 mL of the gas in the inoculated vial was replaced by high purity acetylene gas using a sterile microsyringe and incubated. After incubation at 30 ℃, 1 mL of the gas was extracted and injected into a gas chromatography (GC) (Thermo Scientific Trace 1011,USA) equipped with a flame ionization detector (FID) to determine the ethylene production (μmol C2H4/ mg protein/hour) against standard curve and peak-area percentage to determine nitrogenase activity (Hardy et al. 1968).
Characterization of bacterial isolates
Molecular characterization of the isolated bacteria was performed using 2 µL of extracted genomic DNA, FGPS6 and FGPS1509 as forward and reverse primers respectively by 16S rRNA gene amplification (Normand et al. 1992). PCR profile used for amplification had initial denaturation at 94 ℃ - 5′; 35 cycles of denaturation at 94 ℃ - 1′, annealing at 55 ℃ - 1′, extension at 72 ℃ - 2′ and single cycle of final extension at 72 ℃ - 7′. The amplified PCR product was documented by UV based gel document system in ethidium bromide (EtBr) based 1% agarose gel against 1 kb marker. Obtained nucleotide sequences ofbacterial isolates were identified using the NCBI BLAST tool and aligned using CLUSTAL W. Phylogenetic trees were constructed by Neighbor-Joining method using consensus sequence in MEGA X software and sequences were submitted to NCBI GenBank to obtain accession number and percentage similarity.
Seedling vigour and root analysis
Plant growth promotion and germination efficiency by isolated bacteria- Rice seeds of the BPT 5204 cultivar were used for the seedling vigour test, seeds were surface sterilized with sterile water, 70% ethanol and 0.2% HgCl2 (mercuric chloride) with intermittent multiple sterile water washes, each step for a minute. All the bacterial isolates were grown for 72 h, active culture pelleted down at 5,000 rpm for 15 min. Sterilized seeds were soaked in a cell pellet amended with sterile 0.5% carboxy methyl cellulose (CMC) for 30 min laminar air flow cabinet. Bacterized seeds were then transferred into sterilized tubes containing 0.7% water agar using sterile forceps in three replications of each bacterial isolate along with a control treatment soaked in sterile distilled water. The tubes with rice seeds were kept for a fortnight for germination and growth under 16 h light and 8 h dark at 28 ℃. After the growth period, root length, shoot length, total biomass and seed vigour index were recorded according to International seed testing authority (ISTA) formula as given below.
Seed vigor index (SVI) = % germination x mean length (shoot +root)
WinRHIZO root analysis- Roots were carefully removed from the agar tubes after the growth period, then detached from their upper/shoot parts and rinsed with distilled water to remove any attached agar particles. Washed roots were then soaked overnight in 0.5% methylene blue dye for better contrast as the roots were young and hairlike. Dyed roots were immersed in 0.5% NaOH solution for a few seconds and then washed in water multiple times till no more dye was visible in the washed (final) water. Dyed root samples were spread carefully in a thin water layer (2-3 mm) on a transparent root scanning tray and scanned, acquired images were analyzed using WinRHIZO software (Himmelbauer et al. 2004).
Statistical analysis
The experimental data were statistically analysed using ANOVA at P<0.05 significance followed by comparative analysis by Duncan’s multiple range test (DMRT) using SAS 9.3 version.