Congo red-YEMA (Yeast Extract Mannitol Agar) medium (10.0 g mannitol, 0.5 g dipotassium hydrogen phosphate, 0.2 g magnesium sulphate, 15.0 g agar-agar, 15 ml congo red (0.47%), distilled water (DW) 1 L, pH 6.8 – 7.0) was used for isolation of Rhizobium. Pink multilobed nodules of Pongamia pinnata situated on the tap root were carefully collected and surface sterilized by HgCl2 with 1% for 3 min and 70% C2H50H for 3 min. The sterilized root nodules were trampled and diluted suspensions 10-5 were chosen and 0.1 ml of suspension was spread and incubated at 30±2°C for 2-3 days. Small, round, colourless, translucent colonies with entire margin were picked out and streaked on YEMA medium. The rhizobial isolates were purified, subcultured and confirmed by congo red test, growth on Hoofer’s alkaline medium, glucose peptone medium and ketolactose test (Vincent, 1970).
1.1. Nodulation Test: Healthy sterilized Pongamia seeds collected from University Campus at Kakatiya University, Warangal were mixed with bacterial (rhizobial) coating solution (100 ml 1% CMC + 10 ml log phase rhizobial broth culture) and allowed to dry for 30 min. Sets were placed in green house and strict aseptic and moist conditions were sustained. The nodulation was observed after 90 days of sowing and assessed the shoot length, root length and numbers of nodules were counted. Uninoculated seeds were maintained for comparison.
1.2. Germination Percent of Seeds: The percent germination of seeds was calculated by the following formula:
1.3. Root and Shoot Length: The length of roots and shoot was measured with a cm scale.
1.4. Determination of Dry Weight: The plant materials cut into bits were dried in an oven at 90°C for 3 days and dry weight was determined.
1.5. Nodule Number: Plants were removed carefully from the bags with their root system and nodules intact, after 90 days of growth. The nodules with roots were washed, separated and counted.
1.6. Seedling Vigour Index: The vigour index was calculated by using the formula:
Seedling Vigour Index = Seedling length × percent germination.
1.7. Statistical Analysis
All data were analyzed using Analysis of Variance (ANOVA). Significant differences between treatments were analyzed using SPSS 17 (Duncan’s Multiple Range Test).
RESULTS AND DISCUSSIONS
Isolation of Rhizobia
A total 30 potential Rhizobia were successfully isolated from the nodules of P. pinnata was enumerated by an indirect method and the results are precised in table-1. Data presented in table reveals that sixty percent isolates were fast growers and acid producers. Earlier reports of rhizobia associated with woody legumes described them as either of slow-growing type or cowpea miscellany (Lange, 1961), but more recent reports have shown that this population includes very diverse type of rhizobia including fast, intermediate and slow-growing bacteria (Shetta et al.,2011). Appearance of yellow colonies on YEMA plates incorporated with indicator bromothymol blue would indicates acid producer is a fast growing strain (s) whereas a blue colony would indicate alkaline producer is a slow growing strain(s). In longer incubation Rhizobia isolated from nodules of Huzurabad (HBPP), Karimnagar (KNPP) Illandu (IDPP), Khammam (KMPP), Kesamudram (KSPP), Aleru (ALPP) and Mylaram (MLPP) were shown white but later observed to have red center. No correlation could be observed between the red color in the white slimy colony and slow grower or fast grower (Sharma et al., 2010).
Screening and Selection of Efficient Rhizobial Isolates
Besides N2 fixation, Rhizobia can promote growth affecting, directly or indirectly by the production of various metabolites (Pavan Kumar Pindi, 2011; Vigya Kesari et al., 2013). A total of 30 rhizobial isolates were screened for expression of plant growth promoting characters (PGP traits). Isolates with high relative efficacy of PGP characters were selected for further study. Details regarding the production of selected biochemical traits by the isolates of three host species were presented in the tables 2. A critical perusal of the table-2 reveals that out of thirty P. pinnata isolates, seventeen isolates showed significantly positive for protease (RZETPP-1, RZBPPP-2, RZHBPP-3, RZHPPP-6, RZKNPP-7, RZMGPP-8, RZSKPP-11, RZGKPP-14, RZNBPP-15, RZMKPP-16, RZSCPP-18, RZCPPP-19, RZAMPP-21, RZRPPP-24, RZALPP-25, RZMDPP-26 and RZBGPP-28). Six and five isolates were positive for chitinase (RZBPPP-2, RZVVPP-4, RZIDPP-9, RZCPPP-19, RZKSPP-20 and RZMLPP-30) and ?-1,3-glucanase (RZBPPP-2, RZIDPP-9, RZNBPP-15, RZALPP-25 and RZMLPP-30) production respectively. Gelatin, IAA production and starch hydrolysis were shown by 56.6, 50 and 53.3 percent of positive isolates. On subjecting inoculated plates to iodine test, clear zones around the colonies were seen. Thirteen, twelve, Nineteen and Seventeen isolates were found positive for HCN, gibberellic acid, ammonia production and phosphate solubilization respectively. Based on the maximum number of positive traits eight isolates (RZETPP-1, RZBPPP-2, RZVVPP-4, RZIDPP-9, RZCPPP-19, RZKSPP-20, RZALPP-25 and RZMLPP-30) were selected for further studies.
Evaluation of Tolerance of Selected Rhizobial Isolates Towards Stress Conditions
Many abiotic factors such as temperature, pH, salinity, alkalinity, antibiotics, heavy metals, osmotic pressure, moisture content, soil physico-chemical characters and its pollution due to organic and inorganic pollutants interact with the organisms affecting plant growth and yield production. (Ali et al., 2009). Hence a microorganism possessing tolerance traits along with PGP traits can be a potential bioinoculants (Dardanelli et al., 2009). Resistance of different isolates of rhizobia from P. pinnata towards pH, sodium chloride and temperature are presented in Table-3.
As indicated in Table-3, pH is an important parameters for the growth of the Rhizobium. In the present observation, pH 7.0 was found to be ideal for all rhizobial isolates. Similar findings were made by Singh et al. (2008). With regard to salt tolerance all isolates showed growth at 0.2 percent of NaCl2. However, only 75 percent, 50 percent and 25 percent of the isolates were found to grow on media supplied with 0.4 percent, 0.8 percent and 1 percent NaCl2 respectively. Isolates RZVVPP-4 could resist NaCl2 of 2 percent and can be regarded as osmotolerant strains (Bouhmouch et al., 2005). Reports of previous workers (Keneni et al., 2010) showed that fast growing Rhizobium, in general, grew well at NaCl2 concentration between 3-5 percent. Temperature is known to influence survival, growth, and nitrogen fixation of Rhizobium (Graham et al., 1991). Three isolates, RZBPPP-2, RZVVPP-4, and RZCPPP-19 showed growth at temperatures of 450C.
Influence of Inoculations of Rhizobial Isolates on Seed Germination and Seedling Growth
A critical perusal of the table-4 reveals that the percentage of seed germination and radical length varied from plant to plant and with also different rhizobial treatments. At 90 days (DAS) root length, shoot length and dry weight were higher in the Rhizobium inoculated plants when compared to control plants. In case of P. pinnata rhizobial isolates, RZVVPP-4 isolate enhanced high percentage of seed germination (92 percent), seedling length (88 mm) and vigour index (8360) followed by isolate RZALPP-25 with seed germination (90 percent), seedling length (81 mm) and vigour index (7290). Maximum root length (15.61 cm), shoot length (33.67 cm) and dry weight (5.03 gm ) was shown by isolate RZIDPP-9. Recently, several workers have attempted the role of rhizobial inoculation on improvement in growth and yield of agroforestry tree species (Uddin et al., 2008; Kumudha, 2006). All isolates were also found to enhance root length and shoot length than control. Nodulation ability is the typical character of the genus Rhizobium and also essential for their symbiotic relationship with legumes. The nodulation ability of rhizobial isolates on P. pinnata when grown in pots varied with different Rhizobium isolates. Inoculation of isolate RZVVPP-4 significantly increased the nodule number (22) followed by RZALPP-25 (21). Nodules were assessed as effective based on their size and presence of legheamoglobin. Bigger nodules with legheamoglobin were considered as effective. Most of nodules were in spherical form and determinate type (Rasul et al., 2012).
This research work concluded that eight isolates (RZETPP-1, RZBPPP-2, RZVVPP-4, RZIDPP-9, RZCPPP-19, RZKSPP-20, RZALPP-25 and RZMLPP-30) were positive for maximum number of plant growth promoting traits. Among eight isolates three isolates, RZBPPP-2, RZVVPP-4, and RZCPPP-19 showed growth at temperatures of 450C. RZVVPP-4 isolate enhanced high percentage of seed germination (92 percent), seedling length (88 mm) and vigour index (8360) followed by isolate RZALPP-25 with seed germination (90 percent), seedling length (81 mm) and vigour index (7290). Maximum root length (15.61 cm), shoot length (33.67 cm) and dry weight (5.03 gm) was shown by isolate RZIDPP-9. Production of plant growth promoting compounds indicated that these species have ability to increase the plant growth thereby these Rhizobium species can be used for the development of bioinoculants to generate potent biofertilizers.