Abstract
Activities of various hydrolytic enzymes produced by three plant pathogenic fungi, Sclerotium rolfsii Sacc., Sclerotinia sclerotiorum (Lieb.) deBary and Helminthosporium sigmoideum var. irregulare Crallery et Tullius, were measured. Activties and amounts of the enzymes in mycelia, cultural filtrates, and sclerotia(except of sclerotia of H. sigmoideum var. irregulare) were estimated at various pH levels in order to find out optimal pH for their enzymatic activities. Enzymes such as cellulase (ex), invertase, xylanase, $\beta-amylase$, polymethylgalacturonase, polygalacturonase, phosphatase and protease were estimated. Culture solution for production of enzymes was prepared by adding of 10g, D-glucose, 1.3g $NH_4NO_3,\; 0.5g\; MgSO_4,\;7H_2O,\; and\; 1.0g\; KH_2PO_4$ into 1 liter of potato decoction plus 2ml of micro element solution consisting of 0.2mg. Fe, 0.2mg Zn, and 0.1mg Mn as the sulphates into 1 liter of distilled water. All tested mycelia and cultural filtrates were obtained from the cultures incubarted in previous solution for ten days at $25^{\circ}C$, and sclerotia were harvested from PDA plates of 3. days old, The crude enzyme solutions were prepared according to the method of Miyazaki etal. Ten days after incubation, activities of Cx produced by Scl. sclerotiorum were higher than those of the other fung and each of Cx from three fungi showed different pH optima, such as S. rolfsii and Scl. schlerotiorum in acid side (around pH 3.0), H. sigmoideum var. irregulare in neutral side (around pH 6.3). Invertase activities of S. rolfsii were 20 times higher than those of the other fungi in all samples. All tested fungi, however, showed no significant difference between the enzymatic activities of their cultural filtrate and mycelia and the activities in sclerotia of S. rolfsii and Scl. sclerotiorum were hardly recognized. There were multiple peaks on the xylanase activity curves of three fungi in terms of pH values. High activities of the xylanase were revealed in sclerotia of S. rolfsii and Scl. sclerotiorum, and in mycelia of H. sigmoideum var. irregulare. The highest activities of $\beta-amylase$ were shown both in mycelia and cultural filtrate of H. sigmoideum var. irregulae among the tested fungi, and their optimal pH was 6.2 in both mycelia and cultural filtrate. In the S. rofsii and Sel. sclerotiorum, however, the activities of cultural filtrates were higher than those of the other fungi, and optimal pH was 3.0 and 6.2 for cultural filtrate and both mycelia and sclerotia, respectively. Activities of PMG were high in cultural filtrates of all tested fungi, especially in Scl. sclerotiorum and H. sigmoideum var. irregulare. Mycelia of themalso showed the considerable activities. Optimal pH for enzymatic activities were variable with thekind of fungi or with the samples measured. The highest activities of PG were presented by mycelia of S. rolfsii and Scl. sclerotiorum. $9.l\mu /min.\; and\; 9.5\mu g/min.$, respectively. Optimal pH for activity of PG in mycelia was around 4.5 in S. rolfsii and around 3.0 in Scl. sclerotiorum. Phosphatase of S. rolfsii and Scl. sclerotiorum was more active in acid side (optimal PH3. 5) and that of H. sigmoideum var. irregulare showed one peak each in acid, neutral and alkaline side. But the highest peak was at pH 9.5. Protease of all tested fungi was more active at pH 10.0, especially that of the cultural filtrate of H. sigmoideum var. irregualre.
수종의 식물병원균(흰비단병균, 균핵병균, 좀검은균 핵병균)이 생산하는 가수분해산소(Cellulase Cx, Invertase, $\beta-amylase$, Xylanase, PMG, PG, Phosphatase, Protease)를 균계체내의 효소 및 배양여액내의 효소와 균핵내의 효소(좀검은균핵병균은 제외)로 나누어 그의 생산량과 pH에 따른 활성의 변화를 검토한 결과를 요약하면 다음과 같다. 1. 배양 10일후의 Cx활성은 균핵병균이 다른 균에 비하여 활성이 높았고 흰비단병균과 균핵병균은 산성측(pH 3.0 부근)에서, 좀검은균핵병균은 중성측(pH6.0 부근)에서 활성이 높았다. 2. 흰비단병균의 Invertase는 다른균에 비하여 약 20배정도 높은 활성을 보였고 3균주 모드 배양여액과 균계체간에 효소활성의 차가 인정되지 않았다. 3. Xylanase의 활성은 3균주 모두 균계체, 균핵 및 배양여액에 따라 또 pH에 따라 아주 다양한 변화를 나타냈고 균핵내에서 활성이 높았다. 4. $\beta-amylase$의 활성은 공시균중 좀검은균핵병균의 균계체, 배양여액이 가장 높았다. (약 12.0ug/min) 흰비단병균과 균핵병균ㅇ서는 균계체나 균핵에서 보다 배양여액에서 높았는데 활성최적pH는 균계체, 균핵 모두 pH 6.2였으나 배양여액에서는 흰비단 병균과 균핵병균이 pH3.0이었는데 반해 좀검은균핵병균은 pH6.2였다. 5. PMG의 활성은 공시균 모두 배양여액에서 높았고 균계체에서는 균핵병균과 좀검은균핵병균이 높았으며 활성최적 pH는 균에 따라 또는 측정부분에 따라 다양하게 나타났다. 6. PG의 활성은 흰비단 병균과 균핵병균의 균계체에서 각각 9,1ug/min. 9.5ug/min으로서 가장 높았고 활성최적 pH는 흰비단병균이 pH4.5부근 균핵병균이 pH.3.0부근이었다. 7. 흰비단병균과 균핵병균의 Phosphatase는 산성측(최적 pH3.5)에서 활성이 높았고 좀검은 균핵 병균은 산성, 중성, 알카리측에서 각각 Peak가 나타났으나 최적 pH는 9.5였다. 8. 공시균주 모두 Protease는 pH 10.0에서 최고활성을 나타냈고 특히 좀검은균핵 병균의 배양여액내 효소활성이 높았다.
