Sample Collection: Soil samples were collected by composite sampling technique from the sorghum cultivated field from North Maharashtra region (Jalgaon, Nandurbar, Dhule). Plants uprooted from the field with adherent soil samples were distributed in four different parts, bulk soil, rhizospheric soil, ectorhizosphere and endorhizosphere
(Raval et al., 2011).
Isolation of indigenous bacteria
Isolation of various rhizobacteria by using selective and differential media. Nutrient Agar was used for isolating heterotrophic bacteria. For primary isolation yeast extract mannitol agar, Ashbey’s mannitol agar, Kings B medium, Pikovskaya medium,
etc. were used. Determination of Indole Acetic Acid production: Indole acetic acid (IAA) production was determined by the modification quantitative analysis described by
Gordon and Paleg (1961). The fresh bacterial cultures were inoculated in 50ml Nutrient broth containing L-tryptophan (5μg/ml).The broth cultures were incubated at room temperature (30+2°C) for 5 days. Cultures were centrifuged at 3000 rpm for 30 min and 2 ml supernatant was transferred to the empty clean tube, then 2 drops of O-phosphoric acid and 4 ml of Salkowaski reagent were added. Red colour indicates the test positive and the absorbance of the reaction mixture were measured at 535 nm.
Determination of gibberellic acid production
Gibberellic acid (GA) production was determined following the method of
Borrow et al., (1955). The fresh bacterial cultures were inoculated in 50 ml Nutrient broth and incubated at room temperature (30±2°C) for 7 days. After incubation broth cultures were centrifuged at 8000 rpm for 5 min. Then 15 mL of supernatant was transfer in to another tube and 2 ml of zinc acetate solution was added. After 2 min add 2 ml of potassium ferrocyanide solution was added. Solution was mixed properly and then centrifuged at 8000 rpm for 10 min. Take the 2 ml supernatant 2 ml of 30% HCl was added and incubated for 75 min at room temperature. Absorbance was measured at 254 nm.
ACC deaminase production
Penrose and Glick (2001) described method was use for the ACC deaminase production The fresh bacterial cultures were inoculated in Nfb medium containing 1-aminocyclopropane-1-carboxylate (ACC). Tubes were incubated at room temperature (30±2°C) for 5-7 days. Growth appearance with change in the colour of the medium.
Nitrogen fixation
Nitrogen fixing ability the bacterial cultures were qualitatively checked as bacterial cultures were inoculated on nitrogen free malate medium containing Bromothymol blue as an indicator. The plates were then incubated at room temperature for 3-4 days and were observed for the growth and blue colour zone around the growth.
Okon et al., (1994)
Potassium solubilization
Potassium solubilization was checked by inoculating bacterial cultures on Aleksandrove agar medium
Aleksandrov et al., (1967) and incubating the plates at room temperature for 5 days. Appearance of zone of clearance around the bacterial growth was indicative of potassium solubilisation ability of an isolate.
Phosphate solubilization
Phosphate solublizing ability of the isolate was checked on Pikovskaya (PVK) medium incorporated with tricalcium phosphate Ca
3(PO
4)
2 by
Fiske and Subbarow method (1925). Formation of transparent halo zone around the developing colonies indicated phosphate solublizing ability of the bacteria.
Zn solubilization
The isolates were inoculated into modified PKV medium, (
Pikovskaya, 1948) containing 0.1% insoluble zinc compounds (ZnO, ZnCO
3 and ZnS). The test organisms were inoculated on these media and incubated at 28°C for 48 hours. Formation of transparent halo zone around the developing colonies indicated zinc solublizing ability of the bacteria.
Formulation of isolates for consortium preparation
Bacterial isolates were selected on the basis of nitrogen-fixation capability, nutrient solubilization, high IAA and GA production,
etc. Table 1 shows the list Twelve bacterial isolates out 320 were selected to formulate biostimulators 1 and biostimulators 2 to check growth promoting abilities (Table 2)
(Alam et al., 2020).
In vitro studies of seed germination by plate assay
Initially jowar seeds (
Sorghum bicolor) were surface sterilized by exposing to 95% ethanol and immersed thrice in 0.2% HgCl
2 solution for 3 min (
Stewart and Kane, 2006). The seeds were then subjected to 5 successive washings with sterile distilled water
(Benmati et al., 2020). An aliquot of biostimulator organisms overnight grown bacterial suspensions of each in proportion were (106 cells /ml, 0.5 OD at 540 nm) applied on each seed total 25 seeds were used for 30 min. Seeds were then transferred to filter papers were soaked with sterile distilled place in empty sterile Petri plate water for allowing seeds to germinate. The results of plate assay were reported as Percent germination, seedling length, seed vigor index at an interval of 24 hrs ((
Jha and Subramanian, 2014). Number of germinated seeds was recorded per plate after three days post incubation and percentage germination calculated (
Mishra and Sundari, 2015) using formula given below:
Further seedling length of all germinated seeds was recorded at same time for calculating Seed Vigor Index (SVI). Seed vigor index was calculated using following formula:
SVI= Mean seedling length (cm)×Percentage germination (%)
Effect of various treatments on germination percentage and seed vigor index was analyzed by calculating percentage increase or decrease observed in specific treatment/s as compared to uninoculated control and tabulated (Tables 5 and 6).
Pot assay
The pot experiment was conducted in pots for total seven treatments (three replications for each treatment) were filled with sterile field soil and watered a day before transferring total 10 seed per pot to it, after giving treatment to each seed same as seed bacterization experiment. After 15
th day of sowing 1
st treatment was given by adding to each pot 50 ml of the diluted inoculums of biostimulators consortium as per labels given (50 ml of 10
8 CFU/ml density culture was diluted to 2 lit) (
Bashir et al., 2013).Similar treatment of consortium was given after 30
th by adding consortium to the appropriate pot. Negative control/treatment means addition of only water to that pot.15 days old seedlings were carefully removed from the pots for studying plant vigour and growth parameters. The plants were then taken to the laboratory and shoot length, root length, fresh shoot weight, fresh root weight were recorded. Similar readings were taken after 45 and 60 days. The plants and roots were then kept in an oven at 55°C for 3 days to attain constant dry weight. After which, the dry weight of shoots and roots were recorded
(Bashir et al., 2013).
