한국에서 분리한 Pseudomonas syringae의 빙핵활성

Ice-Nucleation Activity of Pseudomonas syringae Isolated in Korea

단감나무와 차나무의 표면에서 분리한 Pseudomonas syringae 2계통 PS8401, PS8402를 $2.5\%$ glycerol이 첨가된 nutrient agar에서 배양한 다음, 증류수로 세포현탁액($10^8$ colony forming unit/ml)을 조제하여 마이크로피펫법으로 동결온도를 측정하여 본 결과, 각각 -2.5와 $-3.8^{\circ}C$에서 동결하여 빙핵활성이 인정되었다. 한편 동일한 방법에 의한 증류수의 동결온도는 $-21.8^{\circ}C$였고 옥수수를 비롯한 8가지 작물즙액의 동결온도는 $-11.6^{\circ}C$ 이하였다. PS 8401의 nutrient broth 현탁액$(10^8\;cfu/ml)$을 살포한 옥수수유묘는 $-2^{\circ}C$에서부터 얼기 시작하여 $-4^{\circ}C$가 되면, 거의 전체 식물이 상해를 입었으나 nutrient broth만 살포한 대조구의 유묘는 온도가 $-9^{\circ}C$로 내려가기 전까지는 피해를 입지 않았다. PS8401 현탁액을 살포한 후 48시간에 측정한 유묘의 피해는 살포한 세균의 량이 증가함에 따라 심해지는 경향이었다. 상기 PS 8401의 빙핵활성은 현탁액내 세포의 수가 많아짐에 따라 증가하였으며 세균을 배양한 배지의 조성이 배양온도에 따라서도 빙핵활성이 변하였는데 $2.5\%$ glycerol 혹은 $2.5\%$ glucose를 첨가한 nutrient agar와 탄소원을 별도로 첨가하지 않은 nutrient agar에서 자란 세균현탁액$(10^2\;cfu/ml)$의 동결온도는 각각 -4.0, -4.4, $-7.2^{\circ}C$였고, 배지를 $2.5\%$ glycerol을 함유한 nutrient agar로 고정하고 온도를 달리하여 생육시킨 세균현탁액 $(10^2\;cfu/ml)$의 동결온도는 $-4.0^{\circ}C$(생육온도가 $15\~25^{\circ}C$인 경우)와 $-7.6^{\circ}C$(생육온도가 $30^{\circ}C$인 경우)였다.

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.