Collection of rhizospheric soil
Rhizospheric soil adhering to root of rice plants was collected in sterile polythene bags from different rice cultivating fields of Uttar Dinajpur, West Bengal in the year 2018. The samples were brought to Mycology and Plant Pathology Laboratory, Raiganj University.
Isolation of the fungal isolates
Isolation of indigenous soil borne fungi were executed through serial dilution method as demonstrated by Johnson and Curl (1972). In this method, 1 gm soil was dissolved in 10 ml of sterile double distilled water aseptically to make soil concentration 0.1 g/ml (10
-1). A serial dilution in the range of 10
-3 to 10
-5 was prepared with sterilized double distilled water. 20 ml of potato dextrose agar (PDA) amended with Monocef antibiotic (2.5 mg/ml) was poured aseptically in each sterile Petridish and allowed to solidify for 1 hr. Then 1.0 ml of soil suspension from dilution 10
-4 and 10
-5 were poured into the Petridishes separately under aseptic condition and spread uniformly, followed by sealing the Petridishes with parafilm. The Petridishes were incubated at 28°C for 7 days. Different fungal colonies started to appear into the Petridishes after 3-7 days of incubation period. These colonies were picked up carefully and transferred on freshly prepared PDA slants and PDA plates, marked with proper labelling and incubated at 28±2°C for 7 days. The PDA slants were then stored at -4°C for future use.
Preparation of fungal inoculum
Spore suspension
Fungal isolates were multiplied in 250 ml Erlenmeyer flask containing 200 ml Potato dextrose broth (PDB) at 23±2°C for 10 days. Then the culture broth was centrifuged at 10,000 rpm for 10 mins. The pellets were suspended in sterile distilled water and washed repeatedly for 3-4 times. The washed fungal pellet was made into a turbid solution with sterile distilled water. The OD of the solution was adjusted to 0.45 at 610 nm to obtain 1×10
8 cfu ml
-1 (Niranjana,
Umesha et al., 2006).
Maize granules inocula (MGI)
One kilogram of maize grains was crushed into granules using mixer grinder for 2-3 minutes
. Then the granules were semi-boiled for 5 minutes and air dried. The air-dried maize granules were amended with calcium carbonate (2%) and calcium sulphate (1%) at pH 6.48. Then mixed granules were filled (1/4
th) in 500 ml Erlenmeyer flask, plugged with non-absorbent cotton, wrapped with paper and autoclaved. The autoclaved flasks were inoculated with mycelia discs (5 mm) obtained from the actively growing margins of 7 days-old culture of fungal isolates and incubated at 25°C. After 12-15 days of incubation period, completely colonized maize granules were used as maize granules inocula (MGI) and stored at -4°C until further studies.
Priming of seed with mycoflora isolates
Rice seeds were surface sterilized with 0.1% mercuric chloride for 2-3 minutes and then rinsed 3-4 times with sterile distilled water. Then average 50 seeds were treated with 10 ml of conidial suspension (10
8 cfu ml
-1) of fungal isolates. Three replicates were maintained for each treatment. In another set of experiment, surface sterilized 50 rice seeds were coated with MGI of different fungal isolates in sterile potting soil in the ratio of 5:95 (5%), 10:90 (10%) and 20:80 (20%). Treated seeds were kept at normal temperature for germination and growth observation. Seeds treated with sterile distilled water St rikeout served as untreated control. After 10 days, germination percentage root length and shoot length were recorded and vigor index was calculated following the method of Abdul-Baki and Anderson (1973).
Vigor index (VI) = Seed germination (%) × [Mean of root length + Mean of shoot length]
In vitro assay of indole acetic acid (IAA) production
Indole acetic acid (IAA) production was measured following standard methods with some modification
(Bric et al., 1991). Fungal isolates were cultured on PDA media and incubated at 37°C for 7 days. After 7 days of incubation, fungal discs (5 mm) from culture media of each isolate were transferred to PDA broth amended with 500 µg/ml tryptophan as the precursor of IAA production and incubated in a shaker incubator at 28±2°C with rotation of 120 rpm for 3-5 days. Control set was maintained without tryptophan. Fully grown cultures were centrifuged at 10,000 rpm for 10 minutes. Then 1 ml of the supernatant was mixed with 4 ml of the Salkowski reagent (50 ml of 35% of HClO
4, 1 ml of 0.5 m FeCl
3 solution) followed by addition of 2-3 drops of 10 mM orthophosphoric acid and kept in dark for colour formation. Three replicates for each isolate were maintained. Appearance of pink color in test tubes indicated the production of IAA.
IAA production was expressed by + and - sign.
In vitro assay of phosphate solubilization
The phosphate (P) solubilizing activity of fungal isolates was performed on Pikovskaya’s solid agar medium amended with Rose Bengal at 10 ml/L as described by Johnson (1959) with some modifications. After incubation for seven days at 28°C, the formation of clear zone around the fungal hyphae indicated the ability of the fungus to solubilize inorganic phosphorous. Each treatment was replicated three times. The performance of each fungus was marked by assigning - and + sign. The - sign indicates no P solubilisation; + sign indicates small amount of P dissolved; ++ denotes medium amount of P dissolved; +++ indicates high amount of P dissolved. Percent solubilization efficiency and phosphate solubilization index was calculated as:
Where,
SI = Solubilization index.
SE = Solubilization efficiency.
Z = Clear zone diameter (mm).
R = Colony diameter (mm).
