Ecological Characterization of Indigenous Rhizobium Populations in Rajshahi Area

Abstract

Rhizobium spp. were isolated from root nodules of Green Gram, Black Gram, Lentil, Pigeon Pea, Sweet Pea, Chick Pea, Cow Pea and Bean on Yeast Extract Mannitol Agar (YEMA) and their morphological, cultural, physiological and biochemical characteristics were studied. It was observed that the colonies were circular, light pink, convex, entire and opaque. The bacterium was gram negative, rod shaped, aerobic, non-spore forming and motile. In this study, all isolates were showed hazy appearance in the motility media and also were positive for Catalase, Citrate Utilization Test, Urea Hydrolysis, Congo red test, Nitrification test, Oxidase Test, Triple Sugar Iron Test , Mac Conkey Agar Test and, motility Tests. The samples were found negative for Methyl Red (MR), Voges-Proskauer (VP), Indole, Starch hydrolysis test, Hydrogen Sulphide production and Hofer’s alkaline test. Utilization of different carbon sources is an effective tool to characterize the isolates. In the present study sucrose, fructose, galactose, maltose and mannitol (25 mg Hi-media, India) and 20% solution of glucose, lactose, arabinose and xylose were utilized for this test. All the isolates tested here can ferment sucrose, glucose, fructose, mannitol and produce acid. These carbon sources are generally utilized by bacteria of the genus Rhizohium. Fast growing Rhizobial strains utilized a wider variety of carbohydrates than the slow growing strains. Their ability to metabolize a broad range of carbon substrates may be advantageous for survival in soil. Isolates which were named as Lentil, Cow Pea and Bean can be utilized in all the nine sugar and the produced acid. Green Gram, Black Gram, Pigeon Pea, Sweet Pea, Chick Pea cannot ferment maltose sugar. Besides this isolates from Green gram, Black Gram, Pigeon Pea Sweet Pea and Chick Pea fermented all sugar but Green gram and Black Gram cannot fermented galactose, lactose, arabinose and xylose. Resistance patterns of the isolates to thirteen antibiotics were studied. Screening for antibiotic resistance in our study revealed that most of the strains were resistant to Ampicilin, Erythromycin, Gentamicin, Amoxycillin, Penicillin, Streptomycin and Nalidixic acid. The generalized sensitivity to Mecillinam, Ciprofloxacin, Cotrimoxazole, Pefloxacin, Ceftazidime and Tetracycline were found in our study. An optimum growth was observed at room temperature (28ºC to 30ºC). The organisms were found to be temperature sensitive as at higher and lower temperatures, a low growth was observed that might be due to a hindrance in the metabolic activity. pH is an important parameter for the growth of the organism. Best growth of Rhizobium was reported at pH i.e. 7 and very less growth was observed in the medium with pH 5.0 and 9.0. Salt stress may decrease the efficiency of the Rhizobium-legume symbiosis by reducing plant growth and photosynthesis, and hence nitrogen demand, by decreasing survival and proliferation of rhizobia in the soil and rhizosphere, or by inhibiting very early symbiotic events, such as chemotaxis and root hair colonization, thus directly interfering with root nodule function. The experiments also showed that the cells were able to grow in 1% NaCl containing medium but unable to grow on higher concentration, showing that the isolate was sensitive to the salt concentration. In general, the phenotypic study isolates showed large physiological and biochemical biodiversity. Indeed, the studied strains showed a variable resistance against stress factors, temperature, pH, salinity, resistance to antibiotics and heavy metals, which allowed the selection of good candidates for genetic studies. Several investigators have studied the genetic diversity of Rhizobium isolated from several countries around the world. However, it needs I6S rDNA sequencing experiment for further identification of the phenotypic species. These experiments will help to know the relationship between the phenotypic and genotypic identification as well as the phylogenetic position of the identified isolates. 16S rRNA sequencing has helped the description of Rhizobium spp. In the studies reported here, the genetic biodiversity of eight represented strains were examined. The genetic study carried out is a quick method that gives a better idea of the diversity of these strains. The 16S rDNA was sequenced to determine the taxonomic position of these strains, and the results revealed that there was a great genetic diversity among the eight rhizobial strains studied. Indeed, sequence analysis of 16S rDNA and subsequent BLASTn analyses indicated that six strains had 95 to 98% and other two bellow similarity with Rhizobium spp. The phylogenetic distances indicate that the relationships among this group and the Agrobacterium species are as distant as those among the genera Mesorhizobium and Sinorhizobium. This clearly showed that subgroup belonged phylogenetically to the genus Rhizobium. Although the phylogenetic position of subgroup was not found to be distinct from that of other Rhizobium sp. by analysis of the partial 16s rRNA sequences. It was distinguished clearly from the Rhizobium sp. CCNWYC119, Rhizobium sp. CCNWYC119, Rhizobium sp. SOY12, Rhizobium sp. CCNWYC119, Rhizobium sp. CCNWYC119, Rhizobium sp. CCNWYC119, Rhizobium sp. SOY7, Rhizobium sp. SOY12 position by the full sequence of its 16S rRNA genes. So it seems that the partial sequencing of 16S rDNA has limited value in determining phylogenetic relationships among rhizobial species. The BLASTn search results for the partial sequences of the 16S rRNA gene of the isolates revealed that the purity and concentration of extracted DNA were performed at 260/280 nm obtained as 1.77 for Green Gram, 1.74 for Black Gram, 1.74 for Lentil 1.77 for Pigeon Pea,1.85 for Sweet Pea,1.77 for chickPea,1.82 for Cow Pea and 1.84 for Bean and concentration as 42.0, 83.9, 28.9, 49.0, 88.5, 68.8, 86.8 and 69.7 ng/µl for Green Gram , Black Gram, lentil, Pigeon Pea Sweet Pea, Chick Pea, Cow Pea and Bean, respectively. Finally the selected eight bacterial isolates were identified up to species as Rhizobium sp. through 16S rDNA gene sequencing. There were eight experimental plants from which eight colonies of Rhizobium were isolated of which five strains showed similarity among them (Rhizobium sp. CCNWYC119 ) another two strains were also similar (Rhizobium sp. SOY12 ) and only one(Rhizobium sp. SOY7 ) was different based on the nucleotide sequence. The 16S rDNA sequence reveals that the isolated strains Green Gram , Black Gram, Lentil, Pigeon Pea Sweet Pea, Chick Pea, Cow Pea and Bean are homologous to bacterial strain Rhizobium sp.CCNWYC119, Rhizobium sp.CCNWYC119, Rhizobium sp.SOY12, Rhizobium sp.CCNWYC119, Rhizobium sp.CCNWYC119, Rhizobium sp. CCNWYC119, Rhizobium sp. SOY7, Rhizobium sp. SOY12 respectively. Also the results obtained in this study show some interesting aspects on the growth effects of Rhizobium inoculation in Green Gram, Black Gram, Lentil, Pigeon Pea Sweet Pea, Chick Pea, Cow Pea and Bean which was grown in pots under controlled environment. The results indicate the successful nature of usage of commercial inoculants of Rhizobia to improve the growth effects of the pulses studied. Successful results by using commercial inocula of Rhizobia were obtained in field grown eight isolates ( Green Gram , Black Gram, Lentil, Pigeon Pea Sweet Pea, Chick Pea, Cow Pea and Bean) showed significant difference between inoculated and control. The no of nodule, fresh weight of nodule, dry weight of nodule, plant height, pod weight and seed weight were taken. Rhizobial inoculation induced significant changes in plant growth characteristics. This study also showed that enhancement of soil fertility along with the yield of legume plants occurred due to nodulation. Soil treated with Rhizobium spp. showed higher values than control regarding total nitrogen and organic matter (%).