• Journal of Sericultural and Entomological Science
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• v.36 no.1
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• pp.62-68
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• 1994

Isolation, indentification, bioassay and distribution of ice nucleation active(INA) bacteria were done on branch rot of mulberry which was severely developed after harvest of mulberry branches in autumn. Twelve isolates and two isolates out of thirty-six isolates had ice nucleation activity from -5$^{\circ}C$ to -1$0^{\circ}C$, and over -5$^{\circ}C$, respercively. Isolates which formed ice nucleation from -5$^{\circ}C$ to -1$0^{\circ}C$ were not inclined to injure tomato and corn seedlings. However, Two isolates, SE9316 and SE9338 which formed ice nucleation over -5$^{\circ}C$ injured mulberry, tomato and corn seedlings. SE9316 and SE9338 were identified as Pseudomonas syringae based on the morphological, cultural and biochemical characteristics. Populations of ice nucleation active bacteria, fluorecent pseudomonads, were higer in February and April, but they decreased in May. Populations decreased as they become distant from the center of the symptom. The bacterial populations of all sampling times and sites were higer than 105 cfu/g which was enough to induce frost injury.

• Korean Journal of Food Science and Technology
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• v.29 no.2
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• pp.326-336
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• 1997

Four ice nucleation-active bacteria (INA-bacteria), Pseudomonas syringae, Xanthomonas campestris, Escherichia coli JM109/pEIN229 and Gluconobacter oxydans/pKIN230, were treated with heat, pressure and gamma-irradiation to compare viability and their ice nucleation activity (INA) after sterilization. Gamma-irradiated INA-bacteria showed the least decrease in T90 value (the temperature at which the 90% of drops are frozen). According to cumulative INA spectra, gamma-irradiated INA-bacteria showed little decrease in class A ice nuclei $(nucleate\;H_{2}O\;at\;higher\;than\;-5^{\circ}C)$, pressurized INA-bacteria showed more than 90% decrease in class A ice nuclei, and heat-treated INA-bacteria barely showed class A ice nuclei. Differential scanning calorimetry (DSC) was used to examine the effect of INA-bacteria on the thermophysical properties of water at freezing temperature. Freezing peaks were appeared at about $11{\sim}15^{\circ}C$ higher on thermograms and enthalpies of phase change were decreased for the water containing INA-bacteria compared with the pure water, while melting peaks were not shifted. INA measured by DSC method were significantly correlated with INA measured by drop freezing method $(R^{2}>0.993,\;p<0.0001)$, indicating that DSC can be used as a new, simple and precise method for measuring INA.

• Plant Disease and Agriculture
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• v.5 no.1
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• pp.27-33
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• 1999

Frost injury of crops is closely related to the epiphytic population dynamics of ice nucleation-active (INA) bacteria, and the injury can be reduced by decreasing the INA bacterial population. In order to predict the epiphytic population of INA bacteria on crops, a rapid and accurate detection method has to be developed. In the previous report, we produced some antibodies against INA proteins purified from the outer membrane of INA bacteria. However it was difficult to produce the antibodies because the purification procedures of the INA proteins were complicated, and the final yield was too low. We designed a specific peptide from the N-terminal region of INA protein by computer analysis and synthesized the peptide in vitro in this experiment. The peptide sequence was Asp-Ser-Por-Leu-Ser-Leu-His-Ala-Asp, that is corresponding to the highly conserved region in several INA proteins, with predicted beta turn, coiling, and hydrophilic region. A polyclonal anti-INA peptide antiserum produced specifically recognized INA bacteria as few as 10 colony-forming units (CFU) in the ELISA reactions and did not respond to other non-INA bacteria. Serological specificity of the anti-INA peptide antiserum will facilitate the forecasting of the INA bacterial population dynamics on crops.

• Korean Journal Plant Pathology
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• v.12 no.1
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• pp.99-108
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• 1996

3종의 빙핵세균 Peudomonas syringae 8401, Pseudomonas fuorescens 8701, Erwinia herbicola 8701의 세포 외막으로부터 아무런 변성제도 사용치 않고 sucrose density gradient centrifugation, Sephacryl gel filtration chromatography, DEAE-cellulose ion exchange chromatography, non-denaturing buffer를 이용한 PAGE, electroelution, SDS-PAGE를 통해 빙핵활성 단백질을 고도로 정제할 수 있었다. P. suringae와 P. fluorescens에서는 각각 3종류(155 kD, 75 kD, 50 kD)의 빙핵활성 단백질이, E. herbicola에서는 155 kD를 제외한 2종류(75 kD, 50 kD)의 빙핵활성 단백질은 이 연구를 통해 처음 밝혀진 것으로 , 지금까지 보고된 빙핵활성 단백질(150 KD 이상)보다는 훨씬 작은 것이다. 이는 빙핵활성을 나타내는 단백질의 기본단위는 이 실험의 결과만에 의하면 최대 50 kD임을 시사한다. 이들 단백질은 그 유래된 세균의 종류나 또는 단백질 분자량의 크기에 관계없이 모두 -5.5~7.5$^{\circ}C$에서 물을 동결시키는 높은 빙핵활성을 갖고 있었다. 이는 지금까지 보고된 어느 정제단백질의 빙핵활성보다 높은 것이다. 정제된 단백질의 빙핵활성은 trypsin 처리에 의해 상실되었고, pH6~8범위에서는 안정하였으며, pH5이하, pH9이상에서는 활성을 상실하였다. 보존온도에 대한 영향은 3$0^{\circ}C$이상이 되면 점차 활성이 감소하는 경향을 보이다 37$^{\circ}C$이상에서는 활성이 완전히 상실되었다. 금속이온으로서 Hg\ulcorner이온과 SDS에 의해 활성이 상실되었으나 phosphatidylinositol의 첨가에 의해서는 활성이 약간 증가(-1$^{\circ}C$)하였다.

• Korean Journal Plant Pathology
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• v.3 no.3
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• pp.180-186
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• 1987

Cell suspensions of two isolates of Pseudomonas syringae. PS8401 from sweet persimon and PS8402 from tea plant, were active in ice nucleation at -2.5 and $-3.8^{\circ}C$, respectively. Ice nucleation at those temperature was, using micropipette method, detected in suspensions ($10^8$ olony forming unit/ml of distilled water) of cells that had been grown on nutrient agar supplemented with $2.5\%$ glycerol. Using the same method, on the other hand, the freezing temperature of distilled water only was approx. $-21.8^{\circ}C$, and those of various plant saps including corn were lower than $-11.6^{\circ}C$. Corn seedlings sprayed with cell suspensions $(10^8\;cfu/ml)$ of nutrient broth) of PS8401 began to be damaged at $-2^{\circ}C$ and were almost completely damaged at $-4^{\circ}C$, whereas seedlings sprayed with nutrient broth only were not injured until the temperature down to $-9^{\circ}C$. Amounts of frost damage measured 48 hr after application of PS8401 suspensions increased as applied bacterial cell densities were increased. Ice-nucleation activity of the cell suspensions in vitro increased with increasing the number of cells in suspension. The activity also affected by growth-medium composition or growth-temperature. Ice nucleation thus occured at -4.0, -4.4 and $-7.2^{\circ}C$ in suspensions $(10^2\;cfu/ml)$ of PS8401 that had been grown at$25\%$ nutrient agar with $2.5\%$ glycerol, nutrient agar with $2.5\%$ glucose and nutrient agar only, respectively, and occured at -4.0 and $-7.6^{\circ}C$ in suspensions $(10^2\;cfu/ml)$ of PS8401 hat had been grown on nutrient agar with $2.5\%$ glycerol at $15\~25^{\circ}C$ and $30^{\circ}C$, respectively.