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Anti-listeria Activity of Lactococcus lactis Strains Isolated from Kimchi and Characteristics of Partially Purified Bacteriocins (김치에서 분리한 Lactococcus lactis 균주의 항리스테리아 활성 및 부분 정제된 박테리오신의 특성)

  • Son, Na-Yeon;Kim, Tae-Woon;Yuk, Hyun-Gyun
    • Journal of Food Hygiene and Safety
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    • v.37 no.2
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    • pp.97-106
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    • 2022
  • Listeria monocytogenes (L. monocytogenes) is one of gram-positive foodborne pathogens with a very high fatality rate. Unlike most foodborne pathogens, L. monocytogenes is capable of growing at low temperatures, such as in refrigerated foods. Thus, various physical and chemical prevention methods are used in the manufacturing, processing and distribution of food. However, there are limitations to the methods such as possible changes to the food quality and the consumer awareness of synthetic preservatives. Thus, the aim of this study was to evaluate the anti-listeria activity of lactic acid bacteria (LAB) isolated from kimchi and characterize the bacteriocin produced by Lactococcuslactis which is one of isolated strains from kimchi. The analysis on the anti-listeria activity of a total of 36 species (Lactobacillus, Weissella, Lactobacillus, and Lactococcus) isolated from kimchi by the agar overlay method revealed that L. lactis NJ 1-10 and NJ 1-16 had the highest anti-listeria activity. For quantitatively analysis on the anti-listeria activity, NJ 1-10 and NJ 1-16 were co-cultured with L. monocytogenes in Brain Heat Infusion (BHI) broth, respectively. As a result, L. monocytogenes was reduced by 3.0 log CFU/mL in 20 h, lowering the number of bacteria to below the detection limit. Both LAB strains showed anti-listeria activity against 24 serotypes of L. monocytogenes, although the sizes of clear zone was slightly different. No clear zone was observed when the supernatants of both LAB cultures were treated with proteinase-K, indicating that their anti-listerial activities might be due to the production of bacteriocins. Heat stability of the partially purified bacteriocins of NJ 1-10 and NJ 1-16 was relatively stable at 60℃ and 80℃. Yet, their anti-listeria activities were completely lost by 60 min of treatment at 100℃ and 15 min of treatment at 121℃. The analysis on the pH stability showed that their anti-listeria activities were the most stable at pH 4.01, and decreased with the increasing pH value, yet, was not completely lost. Partially purified bacteriocins showed relatively stable anti-listeria activities in acetone, ethanol, and methanol, but their activities were reduced after chloroform treatment, yet was not completely lost. Conclusively, this study revealed that the bacteriocins produced by NJ 1-10 and NJ 1-16 effectively reduced L. monocytogenes, and that they were relatively stable against heat, pH, and organic solvents, therefore implying their potential as a natural antibacterial substance for controlling L. monocytogenes in food.

Effects of Gypsum on Dry Matter Yield and Chemical Composition of Alfalfa in Reclaimed Tidal Land with Soil Dressing (객토 간척지에서 석고처리가 알팔파 건물수량 및 사료성분에 미치는 영향)

  • Kim, Ji Yung;Jo, Hyun Wook;Lee, Bae Hun;Jo, Mu Hwan;Kim, Byong Wan;Sung, Kyung Il
    • Journal of The Korean Society of Grassland and Forage Science
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    • v.41 no.4
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    • pp.223-233
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    • 2021
  • The objective of this study was to investigate the effect of gypsum on the dry matter yield and the chemical composition of alfalfa in reclaimed tideland with soil dressing. The experimental site was Sukmoon reclaimed tideland. The tideland was reclaimed approximately 17 to 33 years ago and the 70 cm of soil was top-dressed. The soil that covers the reclaimed tideland brought from the island did not treat di-salinized. Treatments were consisted of three groups; control group where no gypsum (G0) was applied and two experimental groups where 2 ton/ha (G2) and 4 ton/ha (G4) of gypsum were applied, respectively. The first harvest was conducted when the alfalfa reached early flowering (open the flower 10%), and after that subsequent harvest was conducted at approximately 35 days intervals. The dry matter yield of the alfalfa showed that G2 was significantly higher in the first year than G0 and G4, and G2 tended to be higher in the second year than G0 and G4, although there were no significant differences between treatments. The reason for the high dry matter yield in G2 was that the soil pH and EC of the soil were at marginal and ideal levels and the coverage and alfalfa botanical composition were also high. In both years, there were no differences in the crude protein, neutral detergent fiber and acid detergent fiber contents and relative feed value between gypsum treatments. Meanwhile, the results in the first and second years showed that the alfalfa dry matter yield were negatively affected by droughts stress in spring and concentrated precipitation in summer. Therefore, this study suggests gypsum treatment in reclaimed tidal land could increase the dry matter yield of alfalfa, and 2 ton/ha of gypsum was the optimum rate.

Effects of Mixed Application of Chemical Fertilizer and Liquid Swine Manure on Agronomic Characteristics, Yield and Feed Value of Rye (Secale cereale L.) (화학비료와 발효 돈분 액비 혼용 시용이 호밀(Secale cereale L.)의 생육특성 및 영양성분에 미치는 영향)

  • Sang Moo Lee
    • Journal of The Korean Society of Grassland and Forage Science
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    • v.42 no.4
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    • pp.215-221
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    • 2022
  • This study was carried out to investigate the growth characteristics, yield and chemical compositions of rye according to mixing ratio of chemical fertilize r(CF) and liquid swine manure (LSM) in paddy field cultivation. The experimental design was arranged in a randomized block design with three replications. The manure fertilizer ratio of five treatments were CF 100% + LSM 0% (C), CF 70% + LSM 30% (T1), CF 50% + LSM 50% (T2), CF 30% + LSM 70% (T3), and CF 0% + LSM 100% (T4) of rye. At this time, the application of liquid swine manure was based solely on nitrogen. Plant length did not show significant differences among treatments. Ear length, leaf length and leaf width were the longest in C, T3, and T2, respectively (p<0.05). The stem diameter showed in the order of T4 > T3 > T2 > T1 > C, which was thicker as the LSM application rate increased (p<0.05). Fresh, dry matter and total digestible nutrient (TDN) yield were the highest in T4 (p<0.05), whereas the lowest in C treatment. Crude protein, neutral detergent fiber (NDF), acid detergent fiber (ADF) and crude fiber content were did not show significant difference among treatments. However, compared to C, crude fat and crude ash were significantly higher in T2 and T3, respectively (p<0.05). Total mineral content decreased significantly as the LSM application rate increased (p<0.05). Total free sugar showed high in T3 and T4 with a high LSM ratio, but showed significantly lower in chemical fertilizer treatment (C) and low LSM treatment (T1) (p<0.05). The analysis of all the above results suggests that the application of LSM is very effective, considering the dry matter yield and the contents of free sugar. In addition, LSM may be possible to grow rye without chemical fertilizer.

Study on Forage Cropping System Using Summer Forage Crops with Italian Ryegrass at Paddy Land of Gangjin Region in South Korea (남부지역 논에서 이탈리안 라이그라스와 하계 사료작물을 연계한 작부체계에 관한 연구)

  • Jeong Sung Jung;Se Young Lee;Mirae Oh;Hyung Soo Park;Ana Yun;Ki Choon Choi
    • Journal of The Korean Society of Grassland and Forage Science
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    • v.43 no.1
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    • pp.35-41
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    • 2023
  • This experiment was carried out to examine double cropping system using Italian ryegrass (IRG) 'Kowinearly', whole crop rice 'Yeongwoo' and barnyard millet 'Jeju native' during the Oct. 2020 to Oct. 2022. Three kinds of forage crops were cultivated at paddy field in Livestock Institute, Jeollanamdo Agricultural Research and Extension Services, Jeollanamdo, South Korea. Whole crop rice (WCR) was sown in late May and barnyard millet (BM) sown early June each year after harvesting IRG. We examined dry matter yield and feed value of forage crops depending on harvest time of forage crops during the experimental period. The plant height in heading stage of IRG ranged from 108 to 112cm and dry matter yield ranged from 6,783 to 11,530 kg ha-1. The crude protein (CP) of IRG ranged from 6.0 to 8.44%, acid detergent fiber (ADF) ranged from 55.6 to 60.2% and neutral detergent fiber (NDF) ranged from 32.58 to 36.7%, The dry matter yield of WCR increased as the harvest stage was delayed (14,310 kg ha-1 in milk, 16,167 kg ha-1 in yellow ripen, and 18,891 kg ha-1 in mature). Similar to results of dry matter yield of WCR, dry matter yield of BM increased as the harvest stage was delayed (11,194 kg ha-1 in late heading. and 14,308 kg ha-1 in mature), However nutrient content of WCR and BM showed a decreasing trend. As shown in above results, the productivity of WCR after harvesting IRG was shown to be high at paddy field in the southern region. However, BM also was appeared to have potential as summer forage crops.

Isolation and Characterization of the Indigenous Microalgae Chlamydomonas reinhardtii K01 as a Potential Resource for Lipid Production and Genetic Modification (지질생산 및 유전자 조작의 잠재적 자원으로서의 토착 미세조류 Chlamydomonas reinhardtii K01의 분리 및 특성)

  • Kim, Eun-Kyung;Cho, Dae Hyun;Suh, Sang-Ik;Lee, Chang-Jun;Kim, Hee-Sik;Suh, Hyun-Hyo
    • Journal of Life Science
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    • v.32 no.3
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    • pp.202-209
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    • 2022
  • The green alga Chlamydomonas reinhardtii, a unicellular haploid eukaryote, has long been used by researchers and industries as a cell factory to produce high value-added microalgae substances using genetic modification. Microalga K01, presumed to be Chlamydomonas, was isolated from 12 freshwater samples from the Chungcheong and Jeolla regions to replace C. reinhardtii, an introduced species currently used in most basic and industrial research. The isolated K01 strain was identified as C. reinhardtii through morphological and phylogenetic studies of the 18S rDNA gene sequence (NCBI accession number KC166137). The growth and lipid content of the isolated C. reinhardtii K01 were compared with three wild and four mutant strains in TAP medium, and it was found that the K01 strain could produce 1.74×107 cells/ml by the third day of culture. The growth rate of C. reinhardtii K01 was 1.5 times faster than UTEX2244, which showed the highest number of cells (1.20×107 cells/ml) among the compared strains. The lipid content of the isolated C. reinhardtii K01 (20.67%) was similar to those of the wild strains, although the fatty acid oleate C18:1 was not detected in the isolated strain but was identified in the seven others. The cell density of the isolated strain increased to 0.87 g/l during a six-day culture in BG11 medium, where nitrate (NaNO3) was introduced as a nitrogen source, while the seven acquired strains showed almost no cell proliferation.

Effects of Mixed Application of Chemical Fertilizer and Liquid Swine Manure on Dry Matter Yield and Feed Value of Whole Crop Barley (화학비료와 발효 돈분 액비 혼용 시용이 총체보리의 생산성 및 영양성분에 미치는 영향)

  • Sang Moo Lee
    • Journal of The Korean Society of Grassland and Forage Science
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    • v.43 no.4
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    • pp.225-231
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    • 2023
  • This study was carried out to investigate the growth characteristics, yield and chemical compositions of whole crop barley (Hordeum vulgare L.) according to mixing ratio of chemical fertilizer (CF) and liquid swine manure (LSM) in the paddy field cultivation. The experimental design was arranged in a randomized block design with five treatments and three replications. The manure fertilizer ratio of five treatments were CF 100% (T1), CF 70% + LSM 30% (T2), CF 50% + LSM 50% (T3), CF 30% + LSM 70% (T4), and LSM 100% (T5) of whole crop barley. At this time, the application of liquid swine manure was based solely on the nitrogen. Plant length was higher at T1 as compared to other treatments (T2, T3, T4 and T5). Fresh yield, dry matter yield and total digestive nutrients (TDN) yield were the highest in T1, whereas the lowest in T5 treatment (p<0.05). Chemical compositions (crude protein, crude fat, neutral detergent fiber, acid detergent fiber and TDN) did not show significant difference among treatments. Ca and Na contents were significantly lower at T1 as compared to other treatments (T2, T3, T4 and T5). However, Mg and P contents were significantly higher at T1 as compared to other treatments(p<0.05). There was no significant difference in total free sugar content among T2, T3, T4 and T5 treatments, but the chemical fertilizer (T1) was significantly lower than the other treatments (p<0.01). Considering the above results, liquid swine manure application showed lower dry matter yield and TDN yield than chemical fertilizer, but higher free sugar content. Therefore, in order to increase the productivity of whole crop barley, it is considered desirable to mix liquid fertilizer with chemical fertilizer, taking into account the decomposition rate and insufficient components (P, K) of the liquid swine manure.

Effects of silage storage period of grass clippings on methane production by anaerobic digestion (잔디 예지물의 혐기소화에서 사일리지 저장기간이 메탄 생산에 미치는 영향)

  • Jin Yeo;Tae-Hee Kim;Chang-Gyu Kim;Seo-Yeong Lee;Young-Man Yoon
    • Journal of the Korea Organic Resources Recycling Association
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    • v.31 no.4
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    • pp.13-28
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    • 2023
  • This study assessed the biochemical methane potential (Bu-P) of three grass species-Poa pratensis (PP), Zoysia japonica (ZJ), and Agrostis stolonifera (AS). Bu-P values were determined as 0.330 Nm3/kg-VSadded for PP, 0.297 Nm3/kg-VSadded for ZJ, and 0.261 Nm3/kg-VSadded for AS. Notably, PP exhibited superior suitability for methane production. The investigation also examined the impact of silage storage duration on PP grass clippings, revealing a 19% decline in Bu-P from an initial value of 0.269 Nm3/kg-VSadded on day 0 to 0.217 Nm3/kg-VSadded on day 180. Throughout the storage period, there were significant increases in neutral detergent fiber (NDF), acid detergent fiber (ADF), and crude protein (CP) contents, rising from 67.59%, 39.68%, and 3.02% on day 0 to 77.12%, 54.65%, and 6.24% on day 180, respectively. These findings highlight the influence of storage duration on the anaerobic digestibility of PP grass clippings. To effectively utilize grass clippings as a renewable resource for methane production, further studies considering factors such as initial moisture content, pretreatment methods, and potential effects of residual pesticides are necessary to optimize anaerobic digestion efficiency for herbaceous biomass.

Investigation on the Safety of Biodegradable Synthetic Resins for Food (식품용 생분해성 합성수지제의 안전성 조사)

  • Hee-Jeong Yun;Jong-Sup Jeon;Young-Su Kim;Sung-Hee Park;Seo-Hyeon Song;Eun-Jung Ku;Sun-Young Chae;Myung-Jin Lee
    • Journal of Food Hygiene and Safety
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    • v.39 no.2
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    • pp.95-101
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    • 2024
  • In this study, we investigated the migration level of items specified in the Korean Standards and Specifications for Utensils, Containers, and Packages (Ministry of Food and Drug Safety Notification) for 50 utensils and hygiene products made of biodegradable resins. Our results revealed that one Polylactide (PLA) baby tableware contained 20 mg/L in consumption of potassium permanganate, exceeding the standard of 10 mg/L or less. In all other samples, formaldehyde, lead (Pb), and arsenic (As) levels could be considered very safe and remained below the standard. Moreover, we tested the PLA baby tablewares (n = 21) for migration into a food simulant (4% v/v acetic acid) upon repeated elution at 100℃ for 30 min or UV irradiation for 2 h. We detected increased formaldehyde and As amounts at the repeated 100℃ treatment for 30 min compared to those upon repeated UV irradiation. However, the migration level was markedly low under both conditions. Furthermore, the Estimated Daily Intake (EDI) calculated on an infant-to-child basis from the formaldehyde and As migration at 100℃ for 30 min in the PLA sample was at the maximum value, i.e., 6.0×10-4 mg/kg b.w./day and 1.3×10-1 ㎍/kg b.w./day, corresponding to 0.40% and 10.42% of the Tolerable Daily Intake (TDI, 0.15 mg/kg b.w./day) and Provisional Tolerable Weekly Intake (PTWI, 9.0 ㎍/kg b.w./week), respectively. Therefore, in this study, we confirmed that biodegradable synthetic resins are safe to use for food.

Changes in Fermentation Characteristics and Bacterial Communities of Whole Crop Rice Silage during Ensiling Period (저장기간에 따른 사료용 벼 사일리지의 발효특성 및 미생물상 변화)

  • Mirae Oh;Hyung Soo Park;Bo Ram Choi;Jae Hoon Woo;Seung Min Jeong;Ji Hye Kim;Bae Hun Lee
    • Journal of The Korean Society of Grassland and Forage Science
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    • v.44 no.1
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    • pp.1-5
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    • 2024
  • Understanding changes in fermentation characteristics and microbial populations of forage silage during ensiling is of interest for improving the nutrient value of the feed for ruminants. This study was conducted to investigate the changes in fermentation characteristics and bacterial communities of whole crop rice (WCR) silage during the ensiling period. The chemical compositions, pH, organic acids and bacterial communities were evaluated at 0, 3, 6, and 12 months after ensiling. The bacterial communities were classified at both the genus and species levels. The dry matter content of WCR silage decreased with the length of storage (p<0.05), but there was no significant difference in crude protein and NDF contents. Following fermentation, the pH level of WCR silage was lower than the initial level. The lactic acid content remained at high levels for 3 to 6 months after ensiling, followed by a sharp decline at 12 months (p<0.05). Before fermentation, the WCR was dominated by Weissella (30.8%) and Pantoea (20.2%). Growth of Lactiplantibacillus plantarum (31.4%) was observed at 3 months after ensiling. At 6 months, there was a decrease in Lactiplantibacillus plantarum (10.2%) and an increase in Levilactobacillus brevis (12.8%), resulting in increased bacteria diversity until that period. The WCR silage was dominated by Lentilactobacillus buchneri (71.2%) and Lacticaseibacillus casei (27.0%) with a sharp reduction in diversity at 12 months. Overall, the WCR silage maintained satisfactory fermentation quality over a 12-month ensiling period. Furthermore, the fermentation characteristics of silage were found to be correlated to bacterial microbiome.

Studies on Sclerotium rolfsii Sacc. isolated from Magnolia kobus DC. in Korea (목련(Magnolia kobus DC.)에서 분리한 흰비단병균(Sclerotium rolfsii Sacc.)에 관한 연구)

  • Kim Kichung
    • Korean journal of applied entomology
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    • v.13 no.3 s.20
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    • pp.105-133
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    • 1974
  • 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-(butylcarbamoy 1)-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-l 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 pseudoacasia, but behaved somewhat differently to the soybaen and cucumber, the Type-l 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 rnaltose 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/1. The amount of sclerotial production decreased wi th the increase of carbon sources. 5. Sclerotium rolfsii was thiamine-defficient and required thiamine 20r/l for 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 l50r/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 $NH_4NO_3,\;(NH_4)_2SO_4,\;asparagine,\;KNO_3$, and their effects on the sclerotial production in the order of $KNO_3,\;NH_4NO_3,\;asparagine,\;(NH_4)_2SO_4$. The optimum concentration of thiamine was about 12r/l in $KNO_3$ and about 16r/l in asparagine for the growth of mycelia; about 8r/l in $KNO_3$ and $NH_4NO_3$, 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 ml/50ml basal medium solution. 10. $NH_4NO_3$. 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 include::! in cultural filtrate and they were quite different from each other. I was further assumed that the former two substances are un volatile, 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, Iycine, 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 $52^{\circ}C$. Sclerotia dried for 155 days at$26^{\circ}C$ had more tolerance: sclerotia of Type-l were killed in 15 mins. at $52^{\circ}C$ and in 5 mins. at $57^{\circ}C$, and sclerotia of Type-2 were killed in 10 mins. both at $52^{\circ}C$ or $57^{\circ}C$. 16. Cultural sclerotia of both strains maintained good germinability for 132 days at$26^{\circ}C$. Natural sclerotia of them stored for 283 days under air dry condition still had good germinability, even for 443 days: type-l and type-2 maintained $20\%$ and $26.9\%$ 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 $7-8^{\circ}C$> below zero, while mycelial felts still maintained the viability after .3 weeks at $7-20^{\circ}C$ below zero, and sclerotia were even more tolerant. 18. Sclerotia of type-l 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-l for 60 mins. and Type-2 for 30 mins. In the $0.125\%$ uspulun solution, Type-l 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-l 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 $66\%$ 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 Benlate 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 ontrol. 21. The amount of mycelia produced by consuming 1mg 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 $CO_2$ evolved at any concentrations, except in the Tachigaren 100mg/g sand in which mycelial growth was inhibited significantly. Sclerotial production was completely depressed in the 10mg/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 Tachigaren for 10 minutes or even 20 minutes.

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