Evaluation of phenotypic and growth promotion characteristics of rhizobia isolated from soybean root nodules

Background and objectives: Nitrogen fixation is a biological process, which provides atmospheric nitrogen in usable form for plants, and satisfies the plants N requirement and can replace chemical fertilizers in high proportion. On the other hand, calcareous and high pH soils have usually small amounts of nutrients in available forms and use of plant growth promoting rhizobacteria can increase the availability of nutrients. Soybean nodulating rhizobia and establishment of their symbiosis is affected by different factors in soil. The objective of this research was to isolate and screen the root nodule rhizobia of soybean and evaluation of their resistance to some stresses and capability production of some growth promotion metabolites and biocontrol agents in selected bacteria.
Materials and methods:
Soybean root nodules have been collected from 21 different fields in various zones of Golestan province, Iran. Root nodules surface sterilization was performed by immersing intact nodules for 30 seconds in 95% alcohol and immediately transferred into 2.5% sodium hypochlorite for 2-3 minutes and finally were rinsed seven times by sterile distilled water. The sterilized nodules were crushed using 1 ml of sterile water in test tube and the slurry was streaked on Yeast Extract Mannitol Agar (YMA) containing Congo red. The inoculated petri plates were incubated at 28°C for 3-6 days. Plant nodulation test was done with 21 rhizobial isolates under sand condition in pot experiment. The different rhizobial isolates were screened for stresses tolerance (salinity, drought, temperature,), herbicide and antibiotics resistance, production of hydrolytic enzymes, siderophore and hydrogen cyanid (HCN).
Results: Nodulation assay showed only 11 of 21 isolated strains formed nodule on soybean roots. Only three of the isolates produced siderophore and none of them showed HCN production capability. The results showed that all the strains tolerated up to 5% NaCl. Evaluation of tolerance to temperatures showed that only isolates No. 2 and 16 were sensitive to 42 °C and the other strains grew well at high temperature. All the isolates were sensitive to tetracycline antibiotic while no inhibitory effect of the chloramphenicol and streptomycin was observed on growth of rhizobial isolates. Between two herbicides Trifluralin showed more inhibitory effect on rhizobial growth than Gallant super. Drought stress imposed by polyethylene glycol affect negatively the rhizobial growth and the severity effect depends on rhzobial isolates and drought levels. Capability of hydrolytic enzymes secretion showed that 14.2, 42.8 and 47.6 % of the isolstes produced α-amlyase, cellulose and protease, respectively.

Conclusion: Plant growth promotion characteristics of bacterial isolates, in this research, showed that some of the isolates had the potential to use as PGPB. Those isolates capable to produce siderophore and protease, amylase and cellulase secretion as well as have the ability to grow under various stresses conditions, could use as potential isolates in inoculum production to evaluate their nitrogen fixation in soybean –rhizobium symbiosis under pot and field culture.