• Korean Journal of Soil Science and Fertilizer
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• v.21 no.3
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• pp.307-315
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• 1988

This experiment was conducted to find out the effects of fertilizer-N and co-inoculation of the Rhizobium japonicum and the Azospirillum lipoferum on nodulation, $N_2$-fixation, and growth of soybean under in situ conditions. The results obtained were summarized as follows: 1. The yield of soybean dry matter was significantly greater in the R. japonicum alone, and the mixed inoculation of R. japonicum and A. lipoferum than those of un-inoculation. But inoculum applied by different strains did not significant effect on plant growth. The effects of nitrogen applied on soybean dry matter were higher in the ammonium sulfate than potassium nitrate, and decreased with increasing rates of two forms of nitrogen applied regardless of nitrogen source. 2. Acetylene redution activity was more increased in a single inoculation of R. japonicum than those of the mixed inoculation of the R. japonicum and the A. lipoferum, in cases of Danyeup cultivar, regardless of the form of combined nitrogen used. 3. Nodule mass and total nitrogenase activity per plant showed the positively significant effect in the interrelationship between dry matter of soybean and some factors related to nitrogen fixation efficiency. 4. The highest symbiotic effect in Danyeup cultivar was obtained when a single R. japonicum 84 Dy-1 strain was inoculated and fertilized with 18 mM potassium nitrate.

• Korean Journal of Soil Science and Fertilizer
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• v.37 no.1
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• pp.46-53
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• 2004

In this study, we compared the levels of methylotrophic bacterial community diversity in the leaf, stem, grain, root and rhizosphere soil sainples of four rice cultivars collected from three regions of Korea. Thirty five pigmented and five non-pigmented isolates showing characteristic growth on methanul were obtained. When phylotypes were defined by performing numerical analysis of 42 characteristics, four distinct clusters were formed. While two clusters, I and IV diverged on the basis of nitrate and nitrite reduction, other two clusters, comprising only pink pigmented colonies, diverged on the basis of cellulase activity. Out of the two reference strains used in the analysis, Methyhbacterium extorquens AM1 diverged from all the clusters and M. fujisawaense KACC 10744 grouped under cluster III. All the isolates were positive for urease, oxidase, catalase and pectinase activity and negative for indole production, MR and VP test, $H_2S$ production, starch, and casein hydrolysis. No clusters were found to possess thermotolerant isolates, as no growth of the isolates was observed at $45^{\circ}C$. Two strains in cluster I were found to possess gelatin hydrolysis and methane utilizing properties respectively. Most of the isolates in all the four clusters utilized monosaccliarides, disaccharide and polyols as carbon source. Six isolates showed considerable nitrogenase activity ranging from 86.2 to $809.9nmol\;C_2H_4\;h^{-1}\;mg^{-1}$ protein.

• Journal of Microbiology and Biotechnology
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• v.21 no.6
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• pp.545-555
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• 2011

The diversity elucidation by amplified ribosomal DNA restriction analysis and 16S rDNA sequencing of 96 associative diazotrophs, isolated from the feeder roots of tea on enriched nitrogen-free semisolid media, revealed the predominance of Gram-positive over Gram-negative bacteria within the Kangra valley in Himachal Pradesh, India. The Gram-positive bacteria observed belong to two taxonomic groupings; Firmicutes, including the genera Bacillus and Paenibacillus; and Actinobacteria, represented by the genus Microbacterium. The Gram-negative bacteria included ${\alpha}$-Proteobacteria genera Brevundimonas, Rhizobium, and Mesorhizobium; ${\gamma}$-Proteobacteria genera Pseudomonas and Stenotrophomonas; and ${\beta}$-Proteobacteria genera Azospira, Burkholderia, Delftia, Herbaspirillum and Ralstonia. The low level of similarity of two isolates, with the type strains Paenibacillus xinjiangensis and Mesorhizobium albiziae, suggests the possibility of raising species novum. The bacterial strains of different phylogenetic groups exhibited distinct carbon-source utilization patterns and fatty acid methyl ester profiles. The strains differed in their nitrogenase activities with relatively high activity seen in the Gramnegative strains exhibiting the highest similarity to Azospira oryzae, Delftia lacustris and Herbaspirillum huttiense.

• Korean Journal of Agricultural Science
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• v.16 no.2
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• pp.163-168
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• 1989

Fourty-four isolates showed acetylene-reducing(nitrogenase)activity under the atmosphere of 89% Ar, 10% $C_2H_2$ and 1 % $O_{2{\cdot}}$, these nitrogen-fixing isolates characterized chemotaxonomically and their taxonomic status was disscussed; twenty-three isolates corresponded to Azospirillum. They were curved/spiral rods, gram negative, motile by a polar flagellum, and also utilized glucose in nitrogen free medild by a polar flagellum, and also utilized glucose in nitrogen free medium. but the cellular fatty acid composition and quinone system of these isolates showed quite different characteristics with reference strains. Therefore, the taxonomic status of this nitrogen-fixing bacteria is disscussed and a new species Azospirillum. Sixty forur isolates utilized C-l compounds such as methanol and formic acid. phenotypic and chemotaxonomic characteristics of methanol utilizing isolates were investigated and their taxonomic status was discussed; Twenty-one isolates corresponded to Hyphomicrobium and for the other regular rods and irregular rods utilizing isolates showed different cellular fatty acid composition. These isolates were grouped into 8 cluster analysis and similarity values based on correlation coefficients. Among these 8 clusters, two corresponded Pseudomonas and for the other were not decided.

• Journal of Microbiology
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• v.44 no.3
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• pp.363-368
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• 2006

In this study, native Byadyrhizobium strains were isolated from the host plant, Glycine max, harvested from fields in Madhya Pradesh, India, and were typed by Iytic rhizobiophages. Eight indigenous (Soy2, ASR011, ASR031, ASR032, MSR091, ISR050, ISR076 and ISR078) and two exotic strains (USDA123 and CB1809), all of which evidenced a distinct reaction with six phages, were employed in this study. The symbiotic interaction of these strains was studied initially using soybean cultivar JS335 in a sand culture in a controlled environment, and the efficiency was assessed based on the nodule number, nodule dry weight, plant dry weight, nitrogenase activity, and total accumulation of N per plant. Symbiotic effectiveness was found to be highest with the native phage-sensitive isolate ASR011, whereas it was at a minimum with the phage-resistant isolates, ISR050 and ISR078. Additionally, the effectiveness of these strains was evaluated using six soybean cultivars belonging to different maturity groups; namely, Brags, Lee, Pusa20, PK416, JS33S and NRC37. Analysis of variance data evidenced significant differences due to both symbionts, for the majority of the tested parameters. The CB1809, USDA123, and ASR011 strains evidenced relatively superior symbiotic effectiveness with soybean cultivars Brags, Lee and JS335. Strain ISR078 evidenced no significant responses with any of the cultivars. The ASR031 strain performed moderately well with all tested cultivars. The symbiotic response of all the strains was quite poor with cultivar PK416. Our studies showed that a significant relationship existed between the phage sensitivity and symbiotic efficiency of the bacterial strains with the host-cultivars.

• Korean Journal of Microbiology
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• v.26 no.4
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• pp.305-311
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• 1988

The spectinomycin resistant strain of slow growing R. japonicum R-168 was selected to be participated in conjugation with E. coli WA803/pGS9. Tn5 was introduced from suicide vector pGS9 into R. japonicum R-168 $spr^{r}$ chromosome at the frequency of $1.0\times 10^{-5}-5.0\times 10^{-7}$ and the transconjugante were selected on the yeast extract-mannitol plate containing kanamycin ($50{\mu}$g/ml) and spectinomycin ($100{\mu}$g/ml) after 8-9 days incubation. All transconjugants we tested were found to contain Tn 5 DNA on their genome, which was confirmed by Southern hybridization experiments. R. japonicum RNa75, which had been selected through plant test, was found to be defective in symbiotic nitrogen fixing ability and the production of leghemoglobin in soybean nodules formed by the inoculation of this mutant. In addition, this mutant strain hardly developed nitrogenase activity asymbiotically in contrast with the wild type strain, indicating that some nitrogen fixing gene might be blocked in this strain and the production of leghemoglobin could be decreased by the interference in nitrogen fixing genes.

• Korean Journal of Soil Science and Fertilizer
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• v.20 no.1
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• pp.43-53
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• 1987

In order to improve effectiveness of rhizobia- legume symbiotic nitrogen fixation, ecological and physiological characteristics of indigenous rhizobia distributed in Korea, that is, symbiotic effectiveness of indigenous soybean rhizobia, nitrate reductase activities of the soybean bacteroid from five different soils, and differences of host-infection abilities among the soybean cultivars under population densities of the same indigenous soybean rhizobia, were investigated. The results were summarized as follows: 1. The number of indigenous soybean rhizobia was ranged from $9.2{\times}10^2$ cells per gram of soil in calcareous soil II to $42.4{\times}10^3$ cells per gram of soil in calcareous soil I in Danyang. 2. The symbiotic effectiveness of indigenous soybean rhizobia from five different soils was high in the case of soybean continuously cultivated, and calcareous soil I that population densities of indigenous soybean rhizobia were observed highly. 3. Inverse relationship was observed between total nitrogenase activity (TNA) and nitrate reductase activity (NRA) from the soybean bacteroids ($r=-0.502^*$), but the correlation between nitrate reductase and specific nitrogenase activities (SNA) could be devided into two groups. It was classified into group I which is high in SNA and low in NRA, and group II which is low in SNA and high in NRA. 4. The infection ability of the indigenous soybean rhizobia in the same soil conditions showed the reciprocal difference among each soybean cultivars. In Kwangkyo and Jangyeup, the symbiotic effectiveness appeared by infection of indigenous soybean rhizobia was higher than it of the other soybean cultivars.

• Korean Journal of Soil Science and Fertilizer
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• v.23 no.3
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• pp.220-226
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• 1990

This experiment was carried out to study the effects of Bradyrhizobium moculation and fertilizer N application on symbiotc $N_2$ fixation of peanut (Arachis hypogaea L. cv. Younghotangkong) at newly reclaimed soil. Treatments consisting of B. sp. (Vigna) HCR-3 and HCR-46 with control and five levels of nitrogen (0, 4, 8, 16, 32kg/10a) were arranged with split design of four repetitions. The results obtained were as follows. The number of viable Bradyrhizobium in rhizosphere was decreased to 20 days after sowing and thereafter it was remarkably increased, until full ripe stage of pods, decreased as fertilizern rates increased. Nodulation and nitrogenase activity were reduced with increasing levels of fertilizer, whereas top dry matter and nitrogen accumulation where increased. Seed yield of Bradyhizobium inoculation under the fertilizer N zero level was similar to that N : 8kg/10a under uninoculation. It suggested that Bradyrhizobium inoculation could replace the effect of nitrogen application.

• Journal of Korean Society of Forest Science
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• v.85 no.1
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• pp.96-106
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• 1996

Red alder(Alnus rubra Bong.) seedlings inoculated with Frankia only or both Frankia and spores of Alpova diplophloeus(Zeller & Dodge) Trappe & Smith were grown in a greenhouse for ten weeks. The ten-week-old seedlings were fertilized with six nitrogen(N) and phosphorus(P) fertility regimes (no fertilization, 10mM $NH_4NO_3$, 50mM $NH_4NO_3$, 5mM $KH_2PO_4$, 10mM $NH_4NO_3+5mM$ $KH_2PO_4$, and 50mM $NH_4NO_3+5mM$ $KH_2PO_4$) three times a week for ten weeks. The higher N-fertilization significantly increased mycorrhiza formation by greenhouse contaminant mycorrhizal fungi, but decreased N-fixation and P concentration in nodule tissues. P-fertilization significantly increased nodule and shoot dry weight, and P concentration in plant tissues. When N was highly fertilized, however, the P-fertilization effect disappeared in nodule P concentration but doubled in leaf P concentration. A. diplophloeus inoculation significantly increased diameter growth and $CO_2$ exchange rate, but decreased leaf dry weight. Our results suggest that the higher N- or P-fertilization affect nitrogenase activity and mycorrhizal development but the effects are changed by their interactions.

• Journal of Microbiology and Biotechnology
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• v.25 no.8
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• pp.1339-1348
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• 2015

Until now, considerable effort has been made to engineer novel nitrogen-fixing organisms through the transfer of nif genes from various diazotrophs to non-nitrogen fixers; however, regulatory coupling of the heterologous nif genes with the regulatory system of the new host is still not well understood. In this work, a 49 kb nitrogen fixation island from P. stutzeri A1501 was transferred into E. coli using a novel and efficient transformation strategy, and a series of recombinant nitrogen-fixing E. coli strains were obtained. We found that the nitrogenase activity of the recombinant E. coli strain EN-01, similar to the parent strain P. stutzeri A1501, was dependent on external ammonia concentration, oxygen tension, and temperature. We further found that there existed a regulatory coupling between the E. coli general nitrogen regulatory system and the heterologous P. stutzeri nif island in the recombinant E. coli strain. We also provided evidence that the E. coli general nitrogen regulator GlnG protein was involved in the activation of the nif-specific regulator NifA via a direct interaction with the NifA promoter. To the best of our knowledge, this work plays a groundbreaking role in increasing understanding of the regulatory coupling of the heterologous nitrogen fixation system with the regulatory system of the recipient host. Furthermore, it will shed light on the structure and functional integrity of the nif island and will be useful for the construction of novel and more robust nitrogen-fixing organisms through biosynthetic engineering.