The root nodule bacteria were isolated from Cicer arietinum (chick pea) plants growing in different regions of Dehradun. Microscopic examination showed that the isolates were gram negative rods. Biochemical characterization revealed that the selected isolates were oxidase, catalase and urease positive. Isolates were able to utilize citrate, which complements with the findings of Lupwayi and Hague (1994). None of the isolate showed growth on medium containing dyes i.e methylene blue and gentian violet at 1% concentration, which correlates with the earlier studies by Wei et.al. (2003), who indicated that Rhizobial cells were unable to grow in the presence of these two dyes. All the isolates were able to grow on lactose peptone agar and growth was absent on glucose peptone agar. The isolates were not producing gelatinase enzyme and it is shown by Hunter (2007) that negative gelatinase activity is a feature of Rhizobium. Positive results were obtained when the isolates were subjected to medium containing starch. De Oliveria ef. al (2007) also observed that Rhizobium strains obtained from different sources can utilize starch.
Growth of the rhizobial strains were not much affected as the concentration of the salt increases from 2%- 4%. High concentration of NaCl solution can give high competitive value in the rhizosphere to survive and nodulate the host plants in harsh environmental conditions particularly at high concentrations of salt in the soil. This finding is in line with the report of Saraf and Dhandhukia (2005), who found that Sinorhizobium meliloti growth was not completely inhibited by 4% of NaCl concentration. Rabie and Alamadini (2005) also stated that the growth of rhizobium was not affected by low and moderate levels of salinity. Rhizobium has been reported to grow the best at neutral pH i.e. 7.0. All the isolates grown very well on pH 4.0-8.5. No growth was observed in pH 9.0. Our results were in agreement with previous studies (Kucuk et al, 2006; Baoling et al, 2007).
The results on the resistance patterns of the isolates to ten antibiotics reported high level of resistance against Ampicillin, Amoxycilline, Clindamycine and Streptomycin and least with Meropenem, Netillin, Amikacine, and Ceftriaxone .Several studies (Maatallah et al., 2002;Kufuk and Kivanf, 2008) also observed great variation among chickpea rhizobia with respect to their intrinsic antibiotics resistance pattern (IAR). Sensitivity of isolates to antibiotics may be due to the fact that these bacteria have not been exposed to these antibiotics in natural environments. Depending on the differences in antibiotic resistance pattern, this technique could be successfully employed in ecological studies particularly in the recovery and enumeration of rhizobia introduced into soil.
It has been a well-established fact that Rhizobium can utilize a wide variety of carbon sources for growth, an effective tool to characterize the isolates. Results of the present study ion utilization of different C sources showed that the strains isolated from Chickpea nodules were able to utilize Mannitol, Lactose, Sucrose, Sorbitol, Arabinose, Galactose, Mannose, Maltose, and Raffinose as carbon sources. Similar results were also recorded by Sadowsky et al., 1983 and Stowers, 1985 in case of the Rhizobium strains. Fast-growing rhizobia were able to grow on a large variety of carbon substrates whereas slow-growing rhizobia were more limited in their ability to use diverse carbon sources. However, our result shows that the majority of tested slow-growing chickpea rhizobia were able to use a broad range of carbohydrates. This is in line with the result of other studies (Matalah et al., 2002; L’taief et al., 2007 Mulissa Jida and Fassil Assefa., 2012). It is very interesting to notice that the types of carbohydrates utilized also varied among chickpea rhizobia. Such characteristics are usually used as diagnostic features for root nodule bacteria (Kufuk and Kivanf, 2008; Hungria et al., 2001).
Nitrogen fixation by root nodulating isolate of Rhizobium contributes a significant input into the many types of farming systems. For instance, even 80% of total N in pasture legume plants can be supplied by root nodule bacteria (Goring and Laskowski, 1982). Fungicides applied as seed dressings protect seeds against fungal pathogens and pests (Martyniuk et al., 1999b). In the case of leguminous plants, treatment of seeds with Rhizobium inoculants is also very important (Martyniuk et al., 2002). When Rhizobium bacteria are inoculated on chemically treated seeds of crop, their survival and capacity to induce symbiosis can be markedly reduced due to possible toxic effects of fungicides on these bacteria (Rennie and Dubetz, 1984).
Therefore, the effect of fungicides on legume nitrogen fixation is of great importance In present study, all the isolates were tested for their tolerance to Mancozeb, Carbendazim, Thiram and Zienb fungicides. Most of the isolates showed sensitivity to Thiram and resistance to Carbendazim. Many studies have indicated differential effect of fungicides on Rhizobium, nodulation and nitrogen fixation (Fox et al., 2007; Ramadoss and Sivaprakasam 1991).
The results obtained are a part of our successful efforts to contribute to screen bacteria from root nodules which can be future candidates for increasing productivity of agriculture crops which is at decline presently. The results on screening of Mesorhizobium strains resistant to various antibiotics and also to determine optimum level of tolerance for fungicides and other stress conditions may also help to grow crops in highly polluted soils.
Table 2 Mesorhizobium isolates obtained from different sites of Dehradun.
|BCR 114||ECR 1- 4||144|
|CHICKPEAPLANTS||TCR1-5||SCR1-8||DCR 15||5 8 5|