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Characterization of Rhizosphere Soils of Foxtail Millet and Isolation of Azospirillum Strains

Soil microorganisms, like Azospirillum spp., Azotobacter Sp. and Enterobacter Sp. have shown to encourage plant growth, by promoting the outbreak of secondary roots. Azospirillum have been isolated from the rhizosphere and roots of a variety of plants including cereals and grasses. Inoculation with indigenous Azospirillum is an important procedure when studying their inherent capacity to benefit crops. In some cases, indigenous strains can perform better than introduced strains in promoting the growth of crops due to their superior adaptability to the environment.

production of plant growth promoting substances (Okon and Itzigsohn, 1995;Salomone et al., 1996). Upon Azospirillum inoculation change in root morphology was observed, which has been described to the bacterial production of plant growth regulating substances (Umalia-Garcia et al., 1980;Tien et al., 1979). An increased number of lateral roots and root hairs enlarge the root surface available for nutrients. This results in higher nutrient uptake by inoculated roots and an improved water status of the plant, which in turn could be the main factor for enhancing plant growth (Fallik and Okon, 1996). Azospirillum is not only able to fix atmospheric N (Dobereiner and Day 1976), but also to mineralize nutrients from the soil, to sequester, Fe, to survive to marsh environmental conditions, and can help plants minimize the negative effects of abiotic stresses.

Collection of soil and root samples and their characterization
A total of 40 foxtail millet root bit samples were collected from the foxtail millet fields located in the Raichur and Koppal districts of Northern Karnataka for isolation of Azospirillum strains. The details on the soil type and locations from where the samples taken are presented in Table 1. Roots of foxtail millet along with rhizosphere soils were collected from 40 different localities of Raichur and Koppal districts. The collected samples were brought in polythene bags and stored in a refrigerator at 4 o C to maintain their chemical properties and for further study.

Soil Chemical analyses
The soil samples were analyzed for pH, EC, OC, N, P and K content by following the standard procedure.

Soil pH
Soil pH was determined in 1:2.5 soil water suspension using pH meter (Piper, 1966) with a glass electrode.

Electrical conductivity (EC)
The Electrical conductivity was determined in 1:2.5 soil water extract using conductivity bridge (Jackson, 1973).

Organic carbon
The organic carbon content of the soil samples was determined by using wet oxidation method (Jackson, 1973). For this a known weight of soil was treated with excess volume of potassium dichromate solution in the presence of concentrated H 2 SO 4 . Organic carbon in the soil was oxidized to CO 2 . The excess of potassium dichromate unused is titrated back against ferrous ammonium sulphate in the presence of concentrated phosphoric acid and diphenyl amine indicator.

Available nitrogen of soil samples
For the estimation of available N content in soil, the alkaline potassium permanganate method of Subbaiah and Asija (1956) was followed. A known weight of soil was treated with excess of alkaline (0.32%) potassium permanganate (made alkaline with 25% NaOH solution). The liberated ammonia was trapped in boric acid and determined by titration against standard H 2 SO 4 . The available N content in kg ha -1 was computed using titer value.

Available phosphorus of soil samples
Available phosphorus content of the soil was extracted by using 0.5 M NaHCO 3 . The Phosphorus content was determined by chloromolybdic blue colour method using UV spectrophotometer. The intensity of blue colour was determined at 660 nm wavelength. The available Phosphorus content was computed and expressed in kg ha -1 (Jackson, 1973).

Available potassium of soil samples
Available potassium content was extracted by using neutral normal NH 4 OAc solution as described by Jackson (1973). The concentration of Potassium in the extractant was determined by flame photometer.

Isolation of Azospirillum strains from foxtail millet root samples
The Azospirillum strains from foxtail millet samples were isolated by following the enrichment culture technique as adopted by (Dobereiner and Day, 1976) and (Baldani and Dobereiner, 1980). The fresh roots of foxtail millet were collected from the farmer's fields at different locations of Raichur and Koppal districts. The plants were uprooted carefully with root system intact and brought to the laboratory in sterile propylene bags. Roots were thoroughly washed in running tap water, cut into small bits of 1 cm length and surface sterilized by dipping in 0.1 per cent HgCl 2 solution for three minutes followed by dipping in 70 per cent alcohol for one minute. The roots were finally washed in six to eight changes of sterile distilled water.
The root bits were aseptically placed in tubes containing sterilized semisolid N free malate medium (Baldani and Dobereiner, 1980). The tubes were incubated at 30 o C for a period of one week and observed for growth of Azospirillum as subsurface white undulating pellicles.
The repeated sub culturing was done to confirm the Azospirillum isolates i.e., a loopful of culture was streaked on malate agar plates containing 1 per cent NH 4 Cl. After a week of incubation, typical small, white dense single colonies were picked and transferred to culture tube containing semisolid N-free malate medium. The isolates that formed characteristic subsurface white undulating pellicle in this medium were tentatively considered as Azospirillum.
The isolates were finally purified by streaking on potato infusion agar. Typical pink often wrinkled colonies on potato infusion agar were transferred to semi-solid medium for storage and characterization.

Chemical properties of rhizosphere soil
The chemical properties of soil samples collected in foxtail millet rhizosphere from different fields of Raichur and Koppal districts are presented in Table 2.
The results of Raichur district samples indicated that the pH ranges between 7.58 and 8.85, EC ranges between 0.12 and 0.99 dS m -1 , OC ranges between 0.11 and 0.87 per cent. In UAS campus Raichur, results of soil samples indicated that the pH ranges between 8.30 and 8.60, EC ranges between 0.16 and 0.99 dS m -1 , OC ranges from 0.11 and 0.87 per cent. In Tuntapur village of Raichur, results of soil samples indicated that the pH ranges between 7.58 and 8.85, EC ranges between 0.22 and 0.51 dS m -1 , OC ranges between 0.15 and 0.55 per cent and in Yergera village of Raichur, results of soil samples indicated that the pH ranges between 7.95 and 8.20, EC ranged between 0.12 and 0.90 dS m -1 , OC ranges between 0.19 and 0.85 per cent.
The results of Koppal district samples indicated that the pH ranges between 6.30 and 8.50, EC ranges between 0.12 and 0.85 dS m -1 , OC ranges from 0.12 and 0.91per cent. In Basapur village of Koppal, results of soil samples indicated that the pH ranges between 6.90 and 8.30; EC ranges between 0.12 and 0.31 dS m -1 , OC ranges from 0.27 and 0.91 per cent. In Kutaganalli village of Koppal, results of soil samples indicated that the pH ranges between 7.31 and 8.50, EC ranges between 0.16 and 0.34 dS m -1 , OC ranges between 0.52 and 0.77 per cent and in Halarti village of Koppal, results of soil samples indicated that the pH ranges between 6.65 and 7.80, EC ranges between 0.21 and 0.85 dS m -1 , OC ranges from 0.12 and 0.56 per cent.
The overall soil samples pH ranges between 6.30 and 8.85, EC ranges between 0.12 and 0.99 dS m -1 , OC ranges between 0.11 and 0.91 per cent. The highest and the lowest value of pH, EC and OC were 8.85 and 6.30, 0.99 and 0.12 dS m -1 and 0.91 and 0.11 per cent respectively.

The major nutrients in rhizosphere soil
The major nutrient status of soil samples collected in foxtail millet rhizoplane from different villages of Raichur and Koppal districts are presented in Table 2.
The result of Raichur district samples indicated that the available nitrogen content ranged from 110.40 to 358.80 kg ha -1 , available phosphorus content ranged from 9.35 to 29.92 kg ha -1 , and available potassium content ranged from 117.60 to 362.88 kg ha -1 . In UAS campus Raichur, results of soil samples indicated that the available nitrogen content ranged from 110.40 to 305.12 kg ha -1 , available phosphorus content ranged from 9.35 to 26.78 kg ha -1 , and available potassium content ranged from 117.60 to 307.30 kg ha -1 . In Yergera village of Raichur, results of soil samples indicated that the available nitrogen content ranged from 171.40 to 190.40 kg ha -1 , available phosphorus content ranged from 15.07 to 26.78 kg ha -1 , and available potassium content ranged from 120.69 to 319.20kg ha -1 . In Tuntapur village of Raichur, results of soil samples indicated that the available nitrogen content ranged from 166.30 to 288.50 kg ha -1 , available phosphorus content ranged from 9.92 to 35.63 kg ha -1 , and available potassium content ranged from 171.36 to 362.88 kg ha -1 .
The result of Koppal district samples indicated that the available nitrogen content ranged from 188.60 to 552.10 kg ha -1 , available phosphorus content ranged from 8.66 to 35.07 kg ha -1 , and available potassium content ranged from 117.60 to 406.54 kg ha -1 . In Basapur village of Koppal, results of soil samples indicated that the available nitrogen content ranged from 219.50 to 552.10 kg ha -1 , available phosphorus content ranged from 8.66 to 24.64 kg ha -1 , and available potassium content ranged from 117.60 to 319.20 kg ha -1 . In Kutaganalli village of Koppal, results of soil samples indicated that the available nitrogen content ranged from 188.60 to 298.60 kg ha -1 , available phosphorus content ranged from 15.35 to 28.92 kg ha -1 , and available potassium content ranged from 117.60 to 406.54 kg ha -1 . In Halarti village of Koppal, results of soil samples indicated that the available nitrogen content ranged from 212.30 to 402.60 kg ha -1 , available phosphorus content ranged from 10.50 to 29.64 kg ha -1 , and available potassium content ranged from 117.60 to 376.35 kg ha -1 .
The Azospirillum isolates responded the overall available nitrogen content in soil samples ranged from 110.40 to 552.10 kg ha -1 , available phosphorus content ranged from 8.66 to 35.63 kg ha -1 and available potassium content ranged from 117.60 to 406.54 kg ha -1 . The highest and lowest value of N, P and K were 552.10 and 110.40 kg ha -1 , 35.63 and 8.66 kg ha -1 and 406.54 and 117.60 kg ha -1 respectively.  The foxtail millet rhizospheric soil samples were analyzed for pH, EC, Organic carbon, available N, available P 2 O 5 , and available K 2 O. The soil samples were low in available nitrogen, low to moderate in available phosphorus and moderate to high in available potassium.

Isolation of Azospirillum isolates
All the isolates formed subsurface pellicles in NFBTB medium including the reference strain. The formation of pellicle was below 1-2 mm from the surface of semisolid NFBTB medium. All the Azospirillum strains turned olive green colour of Bomothymal Blue (BTB) to brilliant blue. All isolates were microscopically observed for their cell shape and gram reaction. The cell shape of all the isolates was spiral; all the isolates were Gram negative and had cork screw movement when observed under microscope.