• Korean Journal of Soil Science and Fertilizer
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• v.25 no.3
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• pp.275-280
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• 1992

Inoculation responses of alfalfa[Medicago sativa(L.) Vernal] to Rhizobium meliloti with nitrogen fertilization were characterized by soil populations and plant yields from 1989 to 1991 at cultivated upland soil, which contained $3.1{\times}10$cells of R. meliloti/g.soil. The results obtained accordingly to fertilizing with 8Kg N/10a and differently inoculating the host such as initial and annual inoculation methods were as follows : 1. Soil populations of R. meliloti were increased more at no nitrogen fertilized condition compared to nitrogen fertilized condition to 2nd year experiment from 1st year that came up $1.6{\times}10^3$cells/g.soil, but at 3rd year the trend was reversed. Between inoculation methods, on the whole, the annual inoculation caused more populations, which were $2.0{\times}10^4$cells/g.soil in maximum number. And the populations declined after winter but recovered passing through summer season. 2. Alfalfa yields were mainly influenced by rhizobial populations rather than by fertilizing nitrogen showing a significant correlation(Y=0.36+0.287X, $r^2=0.58^{**}$) with the former. The increased extents of yields obtained by inoculation at no nitrogen and nitrogen fertilized conditions, respectively, were 66 and 10% in 1st : 13 and 20% in 2nd : 19 and 13% in 3rd year experiment with the initial inoculation, and were 66 and 10% in 1st ; 30 and 20% in 2nd : 35 and 36% in 3rd year experiment with the annual inoculation. 3. The results demonstrated the importance of inoculating, if possible, annual inoculating alfalfa to get much yields even at cultivated upland soil.

• Microbiology and Biotechnology Letters
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• v.23 no.3
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• pp.275-281
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• 1995

From the plasmid pYA300 carring a CMCase of Rhizobium fredii USDA193 plasmid was subcloned into pBluescript II KS(+)/pBluescript II SK(+) vectors and designated pYA500 and pYA600, respectively. Escherchia coli cells transformed with pYA500 porduced the CMCase more than with pYA600. The orientation of the cloned fragment in pBluescript vector had the effect on gene expression in E. coli background. When the 1.7 kb CMCase gene fragment of R. fredii USDA193 was hybridized to EcoRI-digested total DNA from R. meliloti and R. fredii USDA 191 the unique bands hybridized respectively, indicating that some genetic diversity exists in the EcoRI restriction enzyme site for CMCase gene in Rhizobium strains. The optimum pH of enzyme activity was 7 and the optimum temperature of that was nearly 37$\circ$C. The cellulase-minus derivatives of pYA500 were constructed by Tn5 insertional mutation. Among 6000 transconjugants, two mutant plasmids (designated pYA500::Tn5a and pYA500::Tn5b) were detected from the cellulase- negative transconjugants. The product of CMCase gene was analyzed by one dimensional SDS- PAGE of the cell extracts. About 45 kDa protein was considered to be a product of CMCase gene.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.26 no.2
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• pp.198-206
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• 1981

A field research was conducted to evaluate the effects of inoculation of Rhizobium meliloti and lime application on the early growth of alfalfa, var. Luna, in an acid soil. Alfalfa seeds were inoculated by lime-peat pelleting method with $A_3$ strain isolated from alfalfa roots. Both inoculated and noninoculated seeds were sown in acid soil (pH 5.5) with three levels of lime application (1, 3, 6 ton/ha) on mid-April. Inoculated seeds produced many effective nodules in early stage and increased nitrogen and chlorophyll contents of leaf, consequently stimulating alfalfa growth. Pseudo nodules were found in noninoculated, while they were seldom formed in inoculated. Lime application increased soil pH, plant height, root length, and shoot and root weight. Liming effect on nodulation was significant at first cut, but no longer after then. Both inoculation and liming increased the forage yield of alfalfa. Inoculation and liming of 6 ton/ha together produced the highest yield. As compared to control plot of nitrogen application, the effect of seed inoculation only could be equal to about 120 kg/ha nitrogen application. The total nitrogen contents of forage were little affected by number or weight of nodules and amounts of lime application.

• Korean Journal of Soil Science and Fertilizer
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• v.20 no.4
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• pp.369-374
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• 1987

This study was conducted to evaluate the degree of acid tolerance of indigenous Rhizobium in natural soils, and to select the superior Rhizobia for legume forage on hilly land. 153 Rhizobial strains isolated from two host genera, Medicago and Trifolium in the pastures of Yeongnam area were screened on acidic agar plates. The results obtained are summarized as follows: 1. The degrees of acid tolerance of R. meliloti and R. trifolii were ranged from 4.3 to pH 5.5 and from 4.0 to pH 5.5 respectively. 2. Among acid tolerant Rhizobia isolates, two strains of R. meliloti at pH 4.3 and three strains of R. trifolii at pH 4.0 were shown to have the activities of nodulation and nitrogenase. 3. R. trifolii strains were shown to survive about 80 to 99% at the pH range 4.8 to 5.0, while R. meliloti were about 35 to 47% at that pH range. Therefore, more acid tolerant R. meliloti should be developed for legume forage in Korean hilly land.

• Journal of Microbiology and Biotechnology
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• v.12 no.3
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• pp.522-525
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• 2002

A very rapid and efficient separation technique for cellular rhizobial cyclosophoraoses was developed based on fractional precipitation and partition chromatography. Cyclosophoraoses are known to function in the osmotic regulation and root nodule formation of legumes during the nitrogen fixation process. Cyclosophoraoses are produced as unbranched cyclic (1longrightarrow12)-${\beta}$-D-glucans in Agrobacterium or Rhizobium species. Recent research has shown that cyclosophoraoses can form inclusion complexation with various unstable or insoluble guest chemicals, thereby implying great potential for industrial application. Typical separation of pure cellular cyclosophoraoses has been so far carried out by several time-consuming steps, including size exclusion, anion exchange, and desalting liquid chromatographies, with a relatively poor recovery. However, the proposed method demonstrated that the successive application of fractional ethanol precipitation and one step of silica gel-based flash column chromatography was enough to simultaneously purify neutral or anionic forms of cyclosophoraoses. This novel technique is very rapid and provides a high recovery.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.25 no.2
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• pp.38-48
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• 1980

To evaluate Rhizobium meliloti BALSAC, a strain selected from Canada as an acid tolerant one, and ordinary lucerne inoculant in acid condition, lucerne (Medicago sativa L. cv. Wairau) was inoculated and/or pelleted in the laboratory, and grown for two months in an acid soil (Lismore silt loam, pH 5.4) with three levels of lime in the, glasshouse. The results of controlled (noninoculated), nitrogen fertilized, laboratorial and commercial inoculated seeds were compared to give the following conclusions: 1. There was no significant difference in the top and root dry matter yields between two Rhizobium strains. However, Balsac inoculant showed higher single nodule dry matter weight and relatively higher number of larger nodules than the ordinary inoculant. 2. Lime application increased dry matter yields of plants and nodules, and the number of nodules per pot and the increase of nodules on the lateral roots in both inoculants. Lime application also caused an evenly distribution of nodules on the root by showing an increase of nodules mainly on the lateral roots. 3. Fertilizer nitrogen without inoculant slightly increased the nodulation percentage, the nodule dry matter weight per nodule and the relative proportion of larger nodules. 4. Commercially inoculated and pelleted seed showed less consistent results. 5. Relatively larger variations in measuring nodule characteristics was discussed and concluded that extreme cares should be given to reduce the variation.

• Korean Journal of Soil Science and Fertilizer
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• v.8 no.1
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• pp.37-43
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• 1975

Effect of inoculation trial on plant height and dry matter yield of seven alfalfa varieties (Medicago sativa L. var. Washoe, Ranger, Lahontan, Narragensett, Atlantic, Vernal, and Moapa 69) which are considered to be well adapted in these areas were conducted during 1973-1974 in a relatively poor soil at the field station in Kyung Hee University. Root nodule bacteria: Rhizobium meliloti (Noculator Northrup Kings Co. Ltd) was applied at a rate to provide 6 oz/30l of alfalfa seeds. Seven varieties were seeded in pots on May 31, 1973 and in second year, they were transplanted in field. 1. Dry matter yield and plant height of all inoculation plots were significantly greater than those of the no inoculation plots in all varieties. 2. In the 1973, dry matter yield of inoculation plot a in pot experiment was in the order of Moapa 69> Vernal> Lahontan> Washoe> Atlantic> Narragensett> Ranger. 3. There was no difference in dry matter yield between with and without-inoculation after transplanted in a field in the second year (1974). Total dry matter yield of varieties was in the order of Washoe> Lahontan> Moapa 69> Ranger> Narragensett> Vernal> Atlantic.

• Applied Biological Chemistry
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• v.28 no.3
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• pp.149-155
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• 1985

590 strains of Rhizobia were isolated from root nodules of the legumes collected at 223 sites in Korea. According to their host specificities they were classified into R. japonicum(218 strains), R. phaseoli(101 strains), R. trifolii(97 strains), R. meliloti(4 strains), R. leguminosarium(1 strain), Rhizobium species(101 strains), and unidentified species(159 strains). 3 potent strains R-138, R-168, and R-214 of R. japonicum have been selected based on the infectivity to soybean cultivar and effeciency of nitrogen fixation. It was observed that the fast-growing strains of R. japonicum contained 1 to 4 plasmids of M.W. of 35-300 Md. However, plasmids were hardly detected for the slow-growing strains.

• Journal of Microbiology and Biotechnology
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• v.11 no.3
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• pp.463-468
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• 2001

Cyclosophoraoses are a class of unbranced cyclic-(1longrightarrow2)-${\beta}$-D-glucans found in the Rhizobium species. Their unique cyclic structures and high solubility make them potent for inclusion complexation as a host for an insoluble guest molecule. A family of neutral cyclosophoraoses (DP 17-27) isolated from Rhizobium meliloti 2011 was used as a host for inclusion complexation with an insoluble guest drug, indomethacin. A high performance liquid chromatographic analysis indicated that the inclusion complexation of cyclosophoraoses greatly ehanced the solubility of indomethacin compared with ${\beta}$-cyclodextrin. The estimated value of the association constant of the complex in water for $\beta$-cyclodextrin and cyclosophoraoses was $523M^{-1} and 17,570M^{-1}$, respectively. NMR spectroscopy showed that the inclusion complex was characterized by the interaction of the indole ring moiety of indomethacin with the cavity of cyclosophoraoses.

• Korean Journal of Agricultural Science
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• v.8 no.2
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• pp.238-243
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• 1981

This experiment was carried out to study the availability of Korean peat as a main carrier material of rhizobial inoculant, using the alfalfa strain Rhizobium meliloti M 14 which was isolated in the previous report. Modification of powdered peat with calcium carbonate and other materials was studied; inoculation of the peat with culture broth, maturation of the mixture under different conditions, and survival of the strain in the peat culture was examined. The results obtained were as follows. 1. Peat produced in Pyongtak was highly acidic, pH 3.8, and addition of calcium carbonate by 14% was required for pH adjustment to 6.4. However the amount of calcium carbonate could be reduced by 4 to 8% when carbon or charcoal was mixed with the peat. 2. Viable number of the strain reached to $7-9{\times}10^9cells/g$ after 3 days, when inoculated with the culture broth of early stationary growth phase and matured in unsteriled peat of open trays; and the number in steriled peat was $1.1-6.2{\times}10^{10}cells/g$ after 5 days, when matured in closed bottles. 3. Survival of the strain was affected markedly by storage temperature, and positive effect of D-sorbitol on the viability was recognized at elevated temperatures, when added as an additional carbon source and moistening agent. Glycerol, sorbitol, or sodium lactate was utilized by the strain as a sole source of carbon, and the decimal reduction time of viable number in the peat culture was was found to be 8 to 9 weeks at $25^{\circ}C$ when these agents were added by 0.5%.