Isolation Of Nitrogen-Fixing Bacteria From The Sugarcane Rhizosphere And Interaction Between Pseudomonas Stutzeri And Host Rice

Plant-bacteria interactions occur in rhizosphere. In the rhizosphere, plants excrete root exudates that are abundant sources of carbon and energy for the associative and endophytic bacteria. Bacteria were found in high number in that area. Associative nitrogen-fixing bacteria colonize the root surface of non-leguminous plants, without formation of differentiated structures. These diazotrophs have considerable potential in meeting future demand for crops and fertilizer. However, molecular mechanisms by which nitrogen-fixing bacteria interact with host plants are poorly understood. In this study, we conducted these experiments to isolate and characterize of nitrogen-fixing bacteria, identify the location of mutation exhibited in phynotypic variation and, study the expression of genes potentially for the interaction between bacteria and host crops.Seventeen bacterial strains were isolated from rhizosphere soil and surface sterilized roots of sugarcane, respectively, and were found to represent12species distributed in the6genera Klebsiella, Burkholderia, Erwinia, Enterobacter, Bacillus and Ralstonia based on16S rDNA sequences. The numbers of bacteria in the rhizosphere soil were of1.3-2.2×103CFU/g of dry weight and then the values found in sugarcane roots sample were of0.1-1.5×103CFU/g of dry weight. PCR amplification of nifH gene showed that of the17isolates, seven produced the expected360-bp amplification product. The results from16S rDNA analyses of these bacteria were also corroborated by morphological and biochemical data. The ntfH gene sequence analysis from the strains belonging to Klebsiella showed that the strain clustered with Klebsiella sp. Ability to fix nitrogen was verified by the acetylene reduction assay and the variation of nitrogenase activities were1.29+0.4to29.63+0.3nmol C2H4/h/Mg protein. The highest nitrogenase activity was found in Gp47, the type strain Klebsiella pneumoniae strain sctcc295which was29.63+0.3nmolC2H4/h/mg protein. Diazotrophic strains were assessed for plant-growth-promoting trait such as indole acetic acid production. The highest indole acetic acid production was found in Gp10, the type strain Klebsiella sp. strain zmmo which was99.0+7ug/mL.The rpoB gene encodes the β subunit of RNA polymerase, which is a central macromolecular machine controlling the flow of information from genotype to phenotype. Mutations in the RNA polymerase gene rpoB causing resistance to rifampicin (Rif) in some soil bacteria. To better understand the effects of rpoB mutations on metabolic capabilities of Pseudomonas stutzeri A1501, the rpoB Rif mutants Q518R, D521Y, D521V, H531R and I614T were characterized. The Rif phenotype of P. stutzeri was accompanied only with base substitutions of the amino residues. A wild-type and5distinct Rif mutant strains of P. stutzeri were tested for utilization of95substrates by use of Biolog GN2MicroPlates. A number of oxidation of substrate utilization patterns were observed in the Rifr mutants that utilization of carboxylic acids and amino acids significantly increased in several Rif mutants than that of wild type. The assimilation of carbon and nitrogen sources of Rif mutants reveals that the mutations might activate new metabolic capabilities that enhance the adaptation in nutritionally complex environments. Acetylene reduction assay carried out at different time revealed that the considerable variability in the nitrogen fixation ability among the studied mutant strains was found after 4h and5h. Except for Rif mutant D521V, which showed higher nitrogenase activity than that of wild type strain A1501, all Rif mutants display a decrease in nitrogenase activity compared to A1501. But role of rpoB in plant-microbe interaction needs further investigation.P. stutzeri A1501displays the ability to move towards root exudates, colonize on the root surface, and promote rice growth. To investigate plant-microbe interaction mechanism, a set of20genes responsible for chemotactic motility and root surface colonization was selected and their expression was determined in the presence of rice root exudate. After induction of1h, it was found that pilK, metE and rpoN transcription increased5.7,6.4and3.4-fold, respectively, whereas in the fdhE gene has no expression. Consequently, after inductions of4h, the pilk,fdhE, metE and rpoN genes were decreased-1.9,-4.4,-0.2and-0.8-fold, respectively. These results suggest that plant-microbe interaction is controlled by complex regulatory systems.