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This study was conducted to elucidate the most appropriate method to obtain auxotrophic mutants from Valsa ceratosperma, the causal fungus of apple canker, which may be used as a gene marker in detecting the transfer of the factors of avirulent strains to virulent strains. Among the 3 kinds of synthetic media tested, each have two formula for minimal and complete, the medium which has been used in study if Endothia parasitica (E.P medium) was turned out to be most appropriate for the growth of V. ceratosperma. A medium for single colony formation from pycnidiospore of this fungus was developed by adding 0.5% L- sorbose to the E. P minimal medium. The period of incubation in dark for preventing the photoreactivation after U. V irradiation was estimated as about 60hrs at which most of the spores become binucleate. Largest number of putative auxotrophs were obtained at about 50second of irradiation to the spores smeared on the medium for single colony formation, at which the survival rate of spores was 5 to 6 percent. With these method developed in this experiment, 161 isolates of putative auxotrophs were detected among which the nutrient requirement for 10 isolates were determind. Five out of 10 mutants were still virulent to apple tree and all but one could not sporulate.
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A summer annual weed of monochoria (Monochoria vaginalis) grows in the edges of rice paddies, ditches,and moist upland throughout Korea. It is very difficult to control with herbicide because of its sulfonylurea resistance. It is very competitive with fast growing pattern, that can cause reducing yields of rice. Brown leaf blight of monochoria (Monochoria vaginalis) occurred naturally in rice paddy, is first reported in Korea. The fungal isolate BWC01-54 was successfully isolated from the diseased leaves of monochoria. The fungus BWC 01-54 was grown well at 25-28°C, conidia of the greysh black brown mycelia were abundant produced on PDA at 15 days. The fungus was grown well in potato dextrose broth at 28°C and fully grown within 10 days in 250 ml of flask. In host and pathogenicity test, conidia suspension of BWC01-54 was the most effective to control of monochoria compare to others isolates. Typical symptoms having pin point brown lesions were formed on stem and leaf and which severely affected the whole plants ware blighted within two weeks, respectively. Under paddies field condition, conidial suspension of the fungus BWC01-54 gave around 90% control. Therefore, we conclude that the fungus may have a potential as a biological control agent against sulfonylurea resistance weed in rice paddy.
White root rot of wild white clover (Trifoliorum repens) caused by isolate BWC98-105 has been first reported in Korea. Typical symptoms on root include water-soaked and dark-brown rot, resulting in complete blight of the whole plant. The fungus grew well at 20-28oC and produced abundant sclerotia at 10-15 days after full mycelial growth on potato dextrose agar. Sclerotia were brown to dark-brown in color and 1-3 mm in length. When white clover plants were inoculated with mycelial suspension (105 cfu/ml) of isolate BWC98-105, the plant shoots were killed within 4-6 days and the roots were completely blighted. Sclerotia were also formed on the surface of the root covered with whitish mycelia within 10-15 days in the field. All nine isolates developed high incidences of white root rot disease on white clover seedlings, of which the symptoms were similar to those observed in the fields. Hence, their pathogenicity was confirmed on white clover. The infection rate of the fungal isolates varied from 78.5% to 95.2%, among which BWC98-105 was the most virulent isolate. The weeding efficacy of the fungus was maintained until the following year, leading to a significant reduction of reshooting. The fungus was specifically parasitic to white clover, but not to four lawn species including zoysiagrass (Zoysia japonica) under greenhouse test. The fungus also had no response to some Gramineae species including rice, but caused little damage to five species of Leguminosae.
Sclerotinia sp. (isolate BWC98-105) causes stem blight and root rot in Leghum sp., and is presently being evaluated as a potential mycoherbicide for the control of Trifoliorium repens. Bioassays have shown that Sclerotinia sp. produces phytotoxic substance which is biologically active against T. repens. Two biologically active compounds, designated as compounds I and II, were produced in vitro from the culture filtrate of BWC98- 105 isolate Sclerotium sp. Compounds I and II were purified by means of liquid-liquid extraction and C18 open column chromatography (300 × 30 mm, i.d). To determine the purity, the purified compounds were analyzed by RP-HPLC. The analytical RP-HPLC column was a TOSOH ODS-120T (150 × 4.6 mm i.d, Japan), of which the flow rate was set at 0.7 mL/min using the linear gradient solvent system initiated with 15% methanol to 85% methanol for 50 min with monitoring at 254 nm. Under these RP-HPLC conditions, compounds I and II eluted at 3.49 and 4.13 min, respectively. Compound II was found to be most potent and host specific. However, compound I had a unique antibiotic activity against phytopathogenic bacteria like bacterial leaf blight (Xanthomonas oryzae) on rice, where it played a less important role in producing toxicity on T. repens. No toxin activity was detected in the water fraction after partitioning with several organic solvents. However, toxin activity was detected in the ethyl acetate and butanol fractions. In the leaf bioassay using compound II, the disease first appeared within 4-5 h as water soaked rot, which subsequently developed into well-defined blight affecting the whole plant.
미생물제재(mycoherbicide)라는 용어는 1970년대부터 사용되기 시작하였다. 하지만 미생물 제재에 대한 관심은 화학제재의 비용증가로 인해 최근 주목을 받게 되었다. 고전적인 생물학적방제재(Biological Control Agent, BCA)는 생태적으로 안전하다는 이유로 생태학적인 관점에서 관심을 끌고 있다. 반면, 현대적인 의미의 생물학적방제재는 인공적으로 배양이 가능하고, 또한 그 잡초방제 효과도 화학제초제와 같은 수준이 요구된다. 현재 미생물제재는 7개국에서 곰팡이로부터 만들어지는 26종이있다. 이들은 재배작물에는 안전한 기주특이성을 갖는다. 그러나 대부분의 약제전달체계는 살아있는 미생물을 활력을 유지한 상태로 장기간 보존할 수 있는 능력을 가지고 있어야 함에도 불구하고 대부분이 조건을 충족시키질 못하였다. 또한, 실험실 수준에서는 방제효과가 잘 나타나지만, 실무적으로 사용할 경우 사용년도 및 포장에 따라 방제효과의 발현성과 지속성이 다르게 나타났다. 이 밖에 미생물제재 사용시 습도, 이슬, 온도 및 이들 요인이 잡초방제에 미치는 효과에 대한 연구결과도 미비한 상태이다. 따라서 잡초방제효과가 뛰어난 미 생물제재를 선별해야한다. 선행된 연구에서 제안된 효과적인 미생물제재의 조건은 첫째, 인공 배양법으로 내구성이 있는 접종원(inoculum)의 대량 생산이 가능하고, 둘째, 유전적으로 안정하면서 기주 특이성이 있고, 셋째, 다양한 범위의 잡초방제효과 있어야 한다. 미생물제재의 생산과 보급은 잡초의 생물학적인 방제에 지대한 역할을 할 수 있다. 액상보다는 입상 형태의 미생물제재가 가벼워서 사용이 간편하며, 젤(gel) 타입의 미생물제재도 사용되는 것으로 알려져 있다. The term mycoherbicide started in 1970, but its interest heightened due to increase costs of chemical herbicides. A classical biocontrol agent is expected to become a permanent part of its new environment and do no harm to it. Contemporary biological control agent(BCA) must be produced by artificial culture and could be applied like chemical herbicides over weeds. BCA is different from the classical approach in that it released through natural spread. To date 26 species of fungi are used as classical BCA against 26 species of weeds in seven countries. There are a number of examples of pathogens attacking non-target plants. But through risk assessments which include understanding the taxonomy, biology and ecology, the target and non-target species, it will be safe to introduce of exotic pathogens to control weeds. But pathogens have not been successfully used in practice. Many mycoherbicides show potential in laboratories, but are ineffective in the field and not consistent from year to year or field to field. There is also a lack of understanding humidity, dew formation and temperature and their effects on suppression of weeds by plant pathogens. Potential pathogen must be selected as a BCA. Previous studies suggest that these pathogens must (1) produce abundant and durable inoculum in artifical culture, (2) be genetically stable and weed specific and (3) kill weeds in control. A granular preparation of mycoherbicide into sodium alginate is lighter than liquids and less bulky than organic matter. Gel forms have also been used.
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Effects of temperature and dew period on sporulation of a mycoherbicide, Epicoccosorus nematosporus, on the lesion of its host, Eleocharis kuroguwai were determined. Conidia formation was first observed after 10 days on plants incubated for either 12 or 16 h in a dew chamber at 28oC; 16 h dew period resulted in more conidia formation. As the dew period was decreased to less than 8 h, fewer conidia formed. Conidial production was most abundant at 28oC and produced as much as 3.3´104 conidia per lesion, while 0.1´103 and 2.3´103 conidia per lesion were produced at 16oC and 36oC, respectively. Alternating temperature regimes, i.e., 30/ 15, 30/20, 28/20, and 28/15oC (day/night) were much better than constant temperature, i.e., 30/30, 28/28/, and 20/20oC for sporulation. In the second sporulation, there were as much as 3.1´104 conidia per lesion (ca. <50% of the first sporulation). Then, sporulation dropped sharply to 6.2´102 conidia per lesion in the third sporulation. Results of this study suggest that temperature combined with dew period is the primary limiting factor in the use of E. nematosporus as a mycoherbicide of E. kuroguwai
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The study was conducted to determine the cultural conditions and the effect of inert fillers for melanization and sporulation abilities of sodium alginate pellets, and the weeding efficacy of the formula in the field. Melanin production of E. nematosporus was affected by striking frequency. Percentage of melanized beads was increased to 80.6% at higher rpm up to 180. The melanized pellets produced more conidia with abundant mucilage than unmelanized pellets. Shaker culture of Epicoccosorus nematosporus with sodium alginate yielded a total of 55 mg per 100 pellets. Percentage of melanized pellets was highest, with 81.0% and 83.3% of melanization, when wheat bran and rice polish were amended and produced the conidia with 65.4 and 68.4 mg per 100 pellets, respectively. When 1 L of conidial suspension of 6.0 ´ 105 conidia per ml was applied on 30-day-old plants in a plot, 74.5% of the plants were killed within 20 days, whereas, its melanized sodium alginate pellets killed 57.8% of the plants in the same period. The number of tuber formation of Eleocharis kuroguwai in the untreated control plots was 128.5 per plot, but those of the plots treated with conidial suspension and melanized pellets were 22.1 and 39.7, respectively, at the end of the season. Results of this study showed that melanization of mycelia-mixed sodium alginate are an important sporulation factor in E. namatosporus as a mycoherbicide.
Effects of temperature and dew period on sporulation of a mycoherbicide, Epicoccosorus nematosporus, on the lesion of its host, Eleocharis kuroguwai were determined. Conidia formation was first observed after 10 days on plants incubated for either 12 or 16 h in a dew chamber at 28oC; 16 h dew period resulted in more conidia formation. As the dew period was decreased to less than 8 h, fewer conidia formed. Conidial production was most abundant at 28oC and produced as much as 3.3´104 conidia per lesion, while 0.1´103 and 2.3´103 conidia per lesion were produced at 16oC and 36oC, respectively. Alternating temperature regimes, i.e., 30/ 15, 30/20, 28/20, and 28/15oC (day/night) were much better than constant temperature, i.e., 30/30, 28/28/, and 20/20oC for sporulation. In the second sporulation, there were as much as 3.1´104 conidia per lesion (ca. <50% of the first sporulation). Then, sporulation dropped sharply to 6.2´102 conidia per lesion in the third sporulation. Results of this study suggest that temperature combined with dew period is the primary limiting factor in the use of E. nematosporus as a mycoherbicide of E. kuroguwai.