本 硏究는 木蓮에서 分離한 흰비단病菌 Sclerotimu rolfsii Sacc.의 分化型을 밝히고 菌?生長 및 菌核形成에 對한 榮養生理를 究明코저 vitarnin, 窒素源, 炭素源의 ?果를 檢討했으며 또 本 菌과 Penicillium sp.와의 生態的 關係를 解明하기 爲한 基礎的인 硏究로서 木菌의 菌?生長 및 菌核形成에 對한 Penicillium 培養濾液의 促進?果와 그 要因을 밝히려고 試圖하였다.
本 硏究結果를 綜合해서 摘要하면 다음과 같다.
1. 木蓮에서 分離한 흰비단病菌 第1型, 第2型은 培地上의 狀態이나 生理的 性質 및 病原性이 相異하였다. 特히 木蓮 아카시아에 對한 病原性은 兩者 同一하나 콩이나 오이에 對해서는 第2型菌이 第1型菌보다 더 强하였다.
2. 供試된 14種의 窒素源中 KNO₂와 glycine을 除外하고는 모두 thiamine hydrochloride 10r/ℓ가 添加 되었을때 비로소 供試菌의 菌?生長 및 菌核形成에 利用되었다. 窒素의 形態別로 보면 菌?生長에 있어서는 NO₃-N보다는 NH₄-N이 훨씬 더 ?果的이며 organic N은 化合物에 따라 相異하였다. 그러나 菌核形成에 있어서는 이와 反對로 NO₃-N이 ?果的이었다. NO₂-N은 菌?生長이나, 菌核形成에 全혀 ?果가 없었다.
3. 供試 炭素源 7種도 大體的으로 thiamine이 存在하지 않는 限 菌?生長이나 菌核形成에 아무런 ?果가 없었다. thiamine이 添加될 경우 菌?生長에 있어서 glucose와 saccharose가 가장 ?果的이고 maltose와 soluble starch는 ?果가 적었으며 xylose, lactose, glyceline은 全혀 ?果가 없었다. 菌核形成에 있어서는 loctose를 除外하고는 全區에서 菌核形成을 보였으며 모두 비슷한 ?果를 나타냈다.
4. 培地中의 窒素源이 同一水準이면 炭素源이 增加함에 따라 菌?生長量이 增加하였다. 그러나 窒素源의 量에는 限度가 있는 것으로 窒素 0.5g/ℓ以上에서는 오히려 菌?生長이 抑制되었다. 그러나 菌核形成에 있어서는 炭素源의 增加에 따라 菌核形成量이 低下하였다.
5. 供試菌은 thiamine 缺乏菌으로서 菌?生長 最適 thiamine 濃度는 20r/ℓ이고 이 濃度를 超過하면 오히려 菌?生長이 抑制되는데 150r/ℓ에서는 無添加區와 거의 같은 程度로 抑制되었다.
6. 供試菌의 生長에 있어서 thiamine의 添加에 따른 窒素源利用度는 NH₄NO₃＞(NH₄)₂SO₄＞asparagine＞KNO₃의 順位이며 窒素源別 thiamine 最適要求量은 KNO₃인 境遇 12r/ℓ, asparagine인 境遇 16r/ℓ 程度였다. 菌核形成量에 있어서는 KNO₃＞NH₄NO₃＞asparagine＞(NH₄)₂SO₄의 順位로서 thiamine 最適量은 KNO₃, NH₄NO₃인 境遇 8r/ℓ에서 全菌核生?量의 大部分이 形成되나 asparagine인 境遇에는 16r/ℓ 程度였다.
7. 培養液의 pH는 供試菌이 生長을 開始하자마자 3.5 程度로 急激히 떨어지나 그 以後부터는 生長量이 增加함에 따라 緩慢하게 떨어졌다. 그러나 pH2.2 以下로는 더 내려가지 않았다.
8. 供試菌의 菌?生長에 對한 各種 vitamin의 相互?果는 thiamine, biotin, pyridoxine, inositol의 4가지 組合에 있어서도 thiamine이 添加되지 않은 곳에서는 거의 ?果가 나타나지 않았다. 그러나 thiamine+pyridoxine, thiamine+inosital, thiamine+biotin+pyridoxine, thiamine+pyridoxine+inositol區에 있어서는 thiamine 單獨 添加區와 同等 혹은 그 以上의 ?果를 나타내지만 thiamine+biotin과 thiamine+biotin+inosital區는 오히려 떨어졌다. 菌核形成에 있어서는 thiamine 單獨區에 比하여 各區 모두 약간씩 增加하였다.
9. Penicillium 培養濾液中에는 供試菌의 菌?生長을 促進하는 物質이 存在하며 培養濾液 6~15㎖/50㎖培養液의 濃度에서 거의 最高菌?生長量에 達하였다.
10. 窒素源으로서 添加한 NH₄NO₃ 혹은 asparagine은 菌?生長에 있어서 培養濾液濃度 如何에 關係없이 NH₄NO₃가 더 有?하였다.
11. 培養濾液에 對한 一聯의 處理에 있어서 揮發性物質分劃, 非揮發性物質分劃, 揮發酸分劃, ether 可溶性有機酸分劃, ether不溶性物質分劃, cation 吸着物質分劃, cation非吸着物質分劃의 9分劃中 非揮發性物質分劃, ether不溶物質分劃, cation吸着物質分劃 및 anion 非吸着物質分劃에서만 菌?가 잘 자랐다. 그러나 菌核은 오직 cation 吸着物質分劃에서만 形成되었다.
12. 이 結果는 培養濾液中에 菌?生長物質, 菌核形成物質 및 菌核形成抑制物質이 存在하며 이들 物質은 各各 別個의 物質로서 前2者는 非揮發性이고 ether不溶性이며 cation交換樹脂에는 吸着되지 않는 物質이며 後者는 非揮發性이고 ether不溶性이며 cation 및 anion 交換樹脂에 吸着되지 않는 物質임을 暗示한다.
13. DNP-amino acids paper chromatography에 依하여 cation 交換樹脂吸着分劃中에서 aspartic acid, cystine, glycine, histidine, lycine, tyrosine 및 dinitroaniline 7種의 아미노酸이 檢出되었다.
14. S. rolfsii의 菌?生長 및 菌核形成은 glutaminc acid, aspartic acid, cystine, histidine 및 glycine의 單獨添加나 混合添加에 依해서 促進되지 않았고 다만 tyrosine에 依해서 약간 促進되었다.
15. 菌核의 濕熱에 對한 抵抗性은 菌核의 水分含有量에 따라 다르며 水分含有量이 적은 것이 보다 더 强하였다.
培地에서 採取한 菌核은 第 1, 2 型菌 모두 52℃에서 5分에 死滅하나 155日間 26℃에서 乾燥시킨 것은 52℃에 있어서 第1型菌은 15分, 第2型菌은 10分, 57℃에 있어서는 第1型菌은 5分, 第2型菌은 10分處理에 死滅하였다.
16. 培養菌核을 132日間 26℃에서 乾燥시킨것은 第1, 2 型菌 모두 全部 發芽하였고 氣乾狀態에서 283日間 放置한 天然菌核도 第1, 2 型菌 모두 發芽하였으며 443日間處理한 것도 아직 第1型菌 20%, 第2型菌 16.9%의 發芽率을 保持하고 있었다.
17. 低溫에 對한 抵抗性은 菌?, 菌?塊, 菌核의 順으로 强하였는데 菌?는 -7~-8℃ 1週間 處理에서 完全 死滅하였으나 菌?塊는 -17~-20℃ 3週에서도 아직 死滅치 않은 것이 있었으며 菌核은 -17~-20℃ 3週에서 大部分 生存하였다.
18. 藥劑抵抗力은 昇汞水 0.05%에 있어서 第1型菌은 180分, 第2型菌은 240分, 0.1%에 있어서는 第1型菌 60分, 第2型菌 30分에 各各 死滅하였고 Uspulun 800倍에 있어서는 第1型均은 120分에 死滅하나 第2型菌은 180分에도 死滅치 않으며 500倍에 있어서는 第 1, 2 型菌 모두 90分에 비로소 死滅하였다. 그러나 硫酸銅 5% 240分, Ceresan 石灰, Mercuron 各 500倍 80分處理에도 아무런 影響이 없었다.
19. Benlate와 Tachigaren의 濃度가 增加함에 따라 菌?生長 抑制?果도 增加하였다. 處理 6째에는 Benlate 0.5ppm을 除外하고는 全濃度에서 뚜렷한 抑制?果를 나타내었으나 12日째에는 濃度에 따라 顯著한 差異를 나타냈다. Benlate 0.5ppm處理는 對照區에 比하여 66%, 2.0ppm은 92%의 抑制?果를, Tachigaren은 1ppm 54%, 1.5ppm과 2.0ppm은 77%의 抑制?果를 나타냈다. 兩者 모두 500ppm에서는 거의 完全히 菌?生長을 抑制시켰다. 菌核形成은 Benlate 500ppm과 Tachigaren 500ppm 및 1000ppm에서 100% 抑制되었다.
20. 一般的으로 菌?生長量이 增加함에 따라 培地中의 glucose나 NH₄-N의 消費量도 增加하였으나 Benlate나 Tachigaren을 處理할 境遇 그 濃度의 增加에 따라 이들의 消費量이 抑制되었다. 그러나 Benlate 低濃度 (0.5ppm 및 1ppm)에 있어서는 NH₄-N의 消費가 無處理區보다 많았다.
21. glucose와 NH₄-N의 吸收利用?果 ? glucose나 NH₄-N 1㎎을 消費하여 生?된 菌?量은 Benlate나 Tachigaren의 處理로 말미암아 크게 低下되었다. 그 程度는 濃度에 關係없이 處理 3日째에 가장 甚했고 以後 時日이 經過함에 따라 높아졌다. Benlate 處理의 glucose를 除外하고는 大體로 濃度가 增加함에 따라 吸收利用?果가 低下되었다.
22. 土壤培養에 있어서 CO₂ 排出量으로 測定한 菌?生長은 어느 濃度에서나 阻止되지 않았고 다만 Tachigaren 100㎎/g 土壤에서만 顯著하게 抑制되었다. 菌核形成은 Benlate나 Tachigaren 10㎎/g 土壤에서 完全히 억제되었다.
23. Benlate와 Tachigaren 0.1, 1.0, 10, 100, 1000ppm에 10分 및 20分間 浸漬處理한 結果 菌核의 發芽抑制?果를 認定할 수 없었다.

The present study is an attempt to solve the basic problems involved in the control of the Sclerotium disease. The biologic stranis of Sclerotium rolfsii Sacc., pathogen of Sclerotium disease of Magnolia kobus, were differentiated, and the effects of vitamins, various nitrogen and carbon sources on its mycelial growth and sclerotial production have been investigated. In addition the relationship between the cultural filtrate of Penicillium sp, and the growth of Sclerotium rolfsii, the tolerance of its mycelia or sclerotia to moist heat or drought and to Benlate(methyl-(butylcarbamoyl)-2-benzimidazole carbamate), Tachigaren(3-hydroxy-5-methylisoxazole) and other chemicals were also clarified.
The results are summarizee as follows :
1. There were two biologic strains, Type-1 and Type-2 among isolates. They differed from each other in the mode of growth and colonial appearance on the media, aversion phenomenon and in their pathogenicity. These two types had similar pathogenicity to the Magnolia kobus and Robinia pscudoacasia, but behaved somewhat differently to the soybaen and cucumber, the Type-1 being more virulent.
2. Except potassium nitrite, sodium nitrite and glycine, all of the 12 nitrogen sources tested were utilized for the mycelial growth and sclerotial production of this fungus when 10r/ℓ of thiamine hydrochloride was added in the culture solution. Considering the forms of nitrogen, ammonium nitrogen was more available than nitrate nitrogen for the growth of mycelia, but nitrate nitrogen was better for sclerotia formation. Organic nitrogen showed different availabilities according to compounds used. While nitrite nitrogen was unavailable for both mycelial growth and sclerotial formation whether thiamine hydrochlioride was added or not.
3. Seven kinds of carbon sources examined were not effective in general, as long as thiamine hydrochloride was not added. When thiamine hydrochloride was added, glucose and saccharose exhibited mycelial growth, while maltose and soluble starch gave lesser, and xylose, lactose, and glycine showed no effect at all,. In the sclerotial production, all the tested carbon sources, except lactose, were effective, and glucose, maltose, saccharose, and soluble starch gave better results.
4. At the same level of nitrogen, the amount of mycelial growth increased as more carbon sources were applied but decreased with the increase of nitrogen above 0.5g/ℓ. The amount of sclerotial production decreased with the increase of carbon sources.
5. Sclerotium rolfsii was thiamine-defficient and required thiamine 20r/l ℓfor maximun growth of mycelia. At a higher concentration of more than 20r/ℓ, however, mycelial growth decreased as the concentration increased, and was inhibited at 150r/ℓ to such a degree of thiamine-free.
6. The effect of the nitrogen sources on the mycelial growth under the presence of thiamine were recognized in the decreasing order of NH₄NO₃(NH₄)₂SO₄ asparagine, KNO₃, and their effects on the sclerotial production in the order of KNO₃, NH₄NO₃ asparagine, (NH₄)₂SO₄. The optimum concentration of thiamine was about 12r/ℓ in KNO₃ and about 16r/ℓ in asparagine for the growth of mycelia ; about 8r/ℓ in KNO₃ and NH₄NO₃, and 16r/ℓ in asparagine for the production of sclerotia.
7. After the fungus started to grow, the pH value of cultural filtrate rapidly dropped to about 3.5. Hereafter, its rate slowed down as the growth amount increased and did not depreciated below pH2.2.
8. The role of thiamine in the growth of the organism was vital. If thiamine was not added, the combination of biotin, pyridoxine, and inositol did not show any effects on the growth of the organism at all. Equivalent or better mycelial growth was recognized in the combination of thiamine+pyridoxine, thiamine+inositol, thiamine +biotin+pyridoxine, and thiamine+biotin+pyridoxine+inositol, as compared with thiamine alone. In the combinations of thiamine+biotin and thiamine+biotin+inositol, mycelial growth was inhibited.
Sclerotial production in dry weight increased more in these combinations than in the medium of thiamine alone.
9. The stimulating effects of the Penicillium cultural filtrate on the mycelial growth was noticed. It increased linearly with the increase of filtrate concentration up to 6-15 ㎖/50㎖ basal medium solution.
10. NH₄NO₃ as a nitrogen source for mycelial growth was more effective than asparasine regardless of the concentration of cultural filtrate.
11. In the series of fractionations of the cultural filtrate, mycelial growth occured in unvolatile, ether insoluble, cation-adsorbed or anion-unadsorbed substance fractions among the fractions of volatile, unvolatile acids, ether soluble organic acids, ether insoluble, cation-adsorbed, cation-unadsorbed, anion-adsorbed and anion-unadsorbed and anion-un-adsorbed substance tested. Sclerotia were produced only in cation-adsorbed fraction.
12. According to the above results, it was assumed that substances for the mycelial growth and sclerotial formation and inhibitor of sclerotial formation were included in cultural filtrate and they were quite different from each other. It was further assumed that the former two substances are unvolatile, ether insotuble, and adsorbed to cation-exchange resin, but not adsorbed to anion, whereas the latter is unvolatile, ether insoluble, and not adsorbed to cation or anion-exchange resin.
13. Seven amino acids-aspartic acid, cystine, glysine, histidine, lycine, tyrosine and dinitroaniline-were detected in the fractions adsorbed to cation-exchange resin by applying the paper chromatography improved with DNP-amino acids.
14. Mycelial growth or sclerotial production was not stimulated significantly by separate or combined application of glutamic acid, aspartic acid, cystine, histidine, and glysine. Tyrosine gave the stimulating effect when applied alone and when combined with other amino acids in some cases.
15. The tolerance of sclerotia to moist heat varied according to their water content, that was, the dried sclerotia are more tolerant than wet ones. The sclerotia harvested directly from the media, both Type-1 and Type-2, lost viability within 5 minutes at 52℃. Sclerotia dried for 155 days at 26℃ had more tolerance : sclerotia of Type-1 were killed in 15 mins. at 52℃ and in 5 mins. at 57℃, and sclerotia of Type-2 were killed in 10 mins. both at 52℃ or 57℃.
16. Cultural sclerotia of both strains maintained good ger-minability for 132 days at 26℃. Natural sclerotia -of them stored for 283 days under air dry condition still had good germinability, even for 443 days : type-1 and type-2 maintained 20% and 16.9% germinability, respectively.
17. The tolerance to low temperature increased in the order of mycelia, felts and sclerotia. Mycelia completely lost the ability to grow within 1 week at 7-8℃ below zero, while mycelial felts still maintained the viability after 3 weeks at 7-20℃ below zero, and sclerotia were even more tolerant.
18. Sclerotia of type-1 and type-2 were killed when dipped into the 0.05% solution of mercury chloride for 180 mins, and 240 mins, respectively : and in the 0.1% solution, Type-1 for 60 mins, and Type-2 for 30 mins. In the 0.125% uspulun solution, Type-1 sclerotia were killed in 180 mins., and those of Type-2 were killed for 90 mins. in the 0.125% solution. Dipping into the 5% copper sulphate solution or 0.2% solution of Ceresan lime or Mercron for 240 mins. failed to kill sclerotia of either Type-1 or Type-2.
19. Inhibitory effect on mycelial growth of Benlate or Tachi-garen in the liquid culture increased as the concentration increased. 6 days after application, obvious inhibitory effects were found in all treatments except Benlate 0.5ppm ; but after 12 days, distingushed diflerences were shown among the different concentrations. As compared with the control, mycelial growth was inhibited by 66?6 at 0.5ppm and by 92% at 2.0ppm of Benlate, and by 54% at 1ppm and about 77% at 1.5ppm or 2.0ppm of Tachigaren. The mycelial growth was inhibited completely at 500ppm of both fungicides, and the formation of sclerotia was checked at 1,000ppm of Benlare ant at 500ppm or 1,000ppm of Tachigaren.
20. Consumptions of glucose or ammonium nitrogen in the culture solution usually increased with the increment of mycelial growth, but when Benlate or Tachigaren were applied, consumptions of glucose or ammonium nitrogen were inhibited with the increment of concentration of the fungicides. At the low concentrations of Benlate (0.5ppm or 1ppm), however, ammonium nitrogen consumption was higher than that of the antral.
21. The amount of mycelia produced by consuming 1㎎ of glucose or ammonium nitrogen in the culture solution was lowered markedly by Benlate or Tachigaren. Such effects were the severest on the third day after their treatment in all concentrations, and then gradually recovered with the progress of time.
22. In the sand culture, mycelial growth was not inhibited. It was indirectly estimated by the amount of CO₂ evolved at any concentrations, except in the Tachigaren 100㎎/g sand in which mycelial growth was inhibited significantly. Sclerotial production was completely depressed in the 10㎎/g sand of Benlate or Tachigaren.
23. There was no visible inhibitory effect on the germination of sclerotia when the sclerotia were dipped in the solution 0.1, 1.0, 100, 1.000ppm of Benlate or Tachigare n for 10 minutes or even 20 minutes.