ISOLATION OF IAA PRODUCING BACTERIA FROM SOIL AND OPTIMISATION OF CULTURE CONDITIONS FOR MAXIMUM IAA PRODUCTION

* Sadhana baggam 1 , S. B. Padal 1 , V Ravi Sankar Ummidi 2 , Ashok Paltati 2 and Nikitha Thanagala 1 . 1. Department of Botany, Andhra University, Visakhapatnam, India. Pin530 003. 2. TRIMS Lab Visakhapatnam India. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

Plant root system is a vital zone which could enhance the growth of soil microbes, the term rhizosphere first coined by Lorenz Hiltner (Anton Hartmann et al 2007). The soil bacteria were divided into three classes based on their interactions with plants; they include plant beneficial bacteria, pathogenic bacteria, plant neutral bacteria (bais et al., 2006). The bacteria rapidly colonize plant roots of different crops and promote plant growth and yield were termed as plant growth promoting rhizobacteria shortly PGPR (Joseph et al., 1980). The mechanism of plant growth promotion by the PGPR may be direct or indirect. PGPR could directly promote plant growth by producing various phytohormones or enhancing phosphate availability etc. ( Indole 3-acetic acid been characterised as the most predominant, physiologically active, naturally occurring auxin, produced in larger quantities than any other related compounds (Hariharan et al.,2014). Various microorganisms present in soil capable of producing IAA include various bacterial and fungal species. The rhizospheric bacteria utilise the root exudates to produce IAA as part of their secondary metabolism (Hariharan et al., 2014). Mostly microorganisms produce IAA through tryptophan dependent pathway (Shulamit et al.,1994, Jiahui et al 2015, tryptophan independent pathway also hypothesised by researchers (Ricardo et al.,2015).  (Hariharan et al.,2014). The production of IAA in culture media varies with culture conditions like pH, incubation period, tryptophan concentration, type of carbon and nitrogen sources etc. (Mohite 2013). In the present study efficient IAA producing bacteria were isolated from different agriculture and virgin soils and the IAA production was optimized by additional carbon, nitrogen sources and pH of the growth media.

Materials and methods:-
Sample collection and bacterial isolation:-Four soil samples collected for this study viz., virgin soil sample from forest regions of araku hilly region, agriculture field under Podu cultivation from Paderu region, agriculture fields under organic forming from Krushivignan Kendra and the agriculture field under chemical fertilizer and pesticide cultivation practices from anandhapuram. The soil samples are serially diluted and 100µl standard inoculum plated on to LB media. The media composition is as fallows, 1 litre of media contains Casein enzymic hydrolysate 10.0gms, yeast extract 5.0gms. Sodium chloride 10.0gms, Distilled water 1000ml and pH adjusted to 7.5±0.2. Plates are incubated at 28 0 C for 2 days and growth was monitored regularly. The clearly visible and morphologically different dominant colonies were selected from each plate and pure cultured on nutrient agar media.
Estimation of IAA production:-All pure isolates were inoculated into 250ml conical flasks containing 50ml LB broth media. The liquid cultures are incubated on shaker incubator at 180rpm for 2days at 28 0 ± 1 0 c. Cultures were centrifuged at 12000rpm for 10 min and 500µl of supernatant from liquid cultures are taken into 1.5ml tube, 1ml of Salkowski reagent was added. Salkowski reagent was prepared by dissolving 2% of 0.5M FeCl 3 in 35% perchloric acid. Reaction tubes were incubated for 30 min in dark at room temperature. The development of pink colour indicated the IAA production. Optical absorbance was measured at 530 nm.
Standard curve:-Auxin production was determined by using standard graph of concentrations ranged from 1.25µg/ml to 100µg. Concentration of IAA in µg/ml was taken on X-axis and the optical density on Y-axis for making standard curve. And by extrapolating the obtained OD value onto standard graph, the quantity of IAA produced by the bacterial isolates was obtained.

Optimisation of iaa production:-Effect of incubation time on IAA production:-
The bacteria inoculated into liquid broth of LB medium and incubated on shaker at 120 rpm for eight days at 28 0 C. IAA production was estimated at regular time intervals.
Effect of additional carbon and nitrogen sources IAA production:-LB media was prepared and amended with two additional carbon sources Mannital and sucrose in 1% concentration. The efficiency of additional carbon source in influencing the IAA production was observed. And the additional yeast extract at three different concentrations i.e., 0.1, 0.5 and 1% in LB media prepared. The effect of additional nitrogen source on IAA production at varying concentrations was observed.
Effect of pH on IAA production:-LB media was prepared and adjusted to 3, 5, 7 and 9 pH conditions and bacterial strains were inoculated. The effect of different pH conditions on IAA production were observed at regular time intervals.

1005
Results and discussion:-Plant growth promoting bacteria has ability to colonise in rhizospheric region of plant system exert stimulation of plant growth either directly or indirectly. In our study we have isolated 6 bacterial strains from four soil samples and tested for their ability to produce IAA. All six isolates namely I1, I2, I3, I4, I5, I6 were able to produce moderate to high amount of IAA under laboratory conditions (Table. 1). During the time of nine days of incubation period, the IAA production by all isolates was ranged from 0 to 33µg/ml. And 6 th day was the optimal day for the IAA production in all cultures (Fig. 1). Among them I4 and I6 have shown highest IAA production of 27.1µg/ml and 33.5µg/ml and were considered as potential IAA producers than the remaining cultures and continued for further optimisation studies. Production of phytohormones phosphate solubilisation nitrogen fixation and etc. were considered as direct effects of growth promotion (Rishi et al., 2015). Among other auxins IAA had been the native and well-studied for their plant growth promoting activities. Many rhizospheric bacteria have reported to produce IAA in in-vitro condition from low to high efficiency. The IAA production was generally effected by the culture conditions, growth stage and substrate availability for bacteria (Farah et

Effect of incubation time on IAA production:-
IAA concentration produced in LB media at pH 7 and 28±2 0 C observed for four times in period of 8 days includes 1 st 3 th 5 th 7 th 9 th days respectively. All six strains have shown their highest activity at 6 th day of incubation period ranged from 7µg/ml to 33µg/ml. Our findings were in agreement with other researchers that 6 th day incubation of bacterial cultures has shown maximum IAA production.  (Fig. 2). But mannitol addition have results in higher IAA production then the sucrose was observed in few reports (Jeyanthi et al., 2013). The IAA concentration reached 39.7µg/ml for I5 strain in 6 th day incubation when additional 1% sucrose present in the culture medium (Fig. 3). Yeast extract at varying concentrations of 0.1%, 0.5% and 1% on culture conditions was observed for its effect in IAA production. Effect of change in nitrogen source effected the IAA production was previously reported (Umang et al., 2013, Apine et al., 2011. The concentration of the nitrogen source present in the media also shows significant effect on IAA production. Apine et al., 2010 standardized the optimum concentration of 0.8% meat extract in the culture medium for highest IAA production. The positive effect of yeast extract added to the culture medium for optimal IAA production have observed in statistical analysis of optimisation of IAA production with changes in media composition (Sasirekha et al., 2012). Here in our study we optimised the concentration of yeast extract on IAA production. I4 strain with 0.5% yeast extract has shown highest IAA production of 35.4µg/ml than the control and 1% yeast extract concentration (Fig. 4). I5 strain has shown highest IAA production of 39.9µg/ml at 1% yeast extract concentration (Fig. 5).
Effect of pH on IAA production:-Neutral pH that is 7-8 was general optimal condition for IAA production by different bacterial strains. I4 strain was actively producing IAA only at pH7 (Fig. 6) and there was drastic decrease in IAA production when pH was higher or lower than that. The results were in agreement with other reports (Hariharan et al., 2014, Umang et al., 2013, Apine et al., 2011. I5 strain has effective IAA production at pH 5 then the pH 7 (Fig. 7). This result was not agreed with other researchers (Hariharan et al., 2011). Optimal pH for maximum IAA production was reported to be lower than neutral by some previous reports similar to our present result (Nita et al., 2011).

Conclusion:-
All six isolated bacterial strains were potent IAA producers. But two strains namely I4 and I5 were considered best among all other strains, and considered for further analysis. Media optimizations were carried for the effective incubation period for highest IAA production, effect of additional carbon source and effect of nitrogen source concentration and the optimal pH for maximum IAA production. It was concluded that IAA production was maximum at 6 th day of incubation with 1% additional sucrose, 0.5% yeast extract and pH 7 in I4 isolate. And I5 isolate have shown its effective IAA producing activity at 6 th day incubation period with 1% additional sucrose, 1% yeast extract concentration and pH 5.