New strains of drechslera monoceras and control compositions containing the same

[0031] Embodiment 1: Isolation of Desseria monoceratum of the present invention

[0032] Naturally infected barnyardgrass is collected, and the pathogen is isolated from diseased tissues of the blades and sheaths of the leaves. Specifically, cut off a tissue part of 1 square centimeter with damage in the center, soak the cut part in 70% ethanol aqueous solution for 1-2 seconds, and then soak it in an aqueous solution of sodium hypochlorite with an effective chlorine concentration of about 2% for 10 seconds. minutes to sterilize the surface of the excised part. The sterilized diseased tissue portion was washed three times with sterile water, placed on an agar medium supplemented with antibiotics, and then incubated statically in an incubator. The top of the growing reticular mycelium was picked under a light microscope and transferred to nutrient medium for pure culture.

[0033] Test Example 1: Examination of Selective Herbicidal Activity and Identification of Isolated Mold

[0034] (1) Test of selective herbicidal activity

[0035] Different species of barnyard weeds and rice crops were cultured in test tubes under aseptic conditions to prepare test materials. Specifically, seeds of E. crus-galli var. crus-galli and rice crops, namely Nipponbare Koshihikari and Sasanishiki, were sterilized in the same manner as described above. Seeds of these crops were sown in test tubes filled with sterile distilled water and allowed to grow.

[0036] On the other hand, at 25°C, the completely isolated mold was cultured on a plate containing potato-dextrose agar medium (Difco) for 1 week, and then the outer edge of the obtained colony was excised using a cork borer to obtain Mecerial dishes for inoculation.

[0037] The mecerial discs were inoculated into the test tubes in which barnyard weeds or rice crops had been grown, and then the plants were grown by incubating in an incubator. Two weeks after the inoculation, the effect of the test fungus on barnyard weeds and rice crops was calculated into 4 grades, namely - (no effect), +, ++ and +++ (wilting). The results are shown in Table 1-1 and Table 1-2.

[0038] strain

[0039] *Known strains

[0040] strain

[0041] *Known strains

[0042] (2) Identification of mold

[0043] A mold with significant herbicidal activity against barnyard weeds and no effect on rice crops was identified. MH-1901 and MH-2001 showed gray-black colonies and irregular growth on malt agar medium (Difco). Conidia had an apparently smooth and slightly curved shape measuring 87.5-127.5 μm x 15-17.5 μm, most with 5-7, with a maximum of about 9 septa. Conidia are straight. From these characteristics, the molds MH-1901 and MH-2001 were identified as Demariaceus Hyphomycetes. Commonwealth Mycological Institute, Kew, England and Eilis, M.B. 1976: More Demariaceus Hyphomycetes. Institute, Kew, England). As described above, these new strains, MH-1901 and MH-2001, were deposited at the National Institute of Bioscience and Human-Technology Agency of Industrial Science and Technology under the accession numbers FERM BP-6091 and FERM BP-6092, respectively.

[0044] Test Example 2: Differences between Desseria monoceratum strains according to the present invention and known bacterial strains

[0045] Desseria unicacerata MH-1901 and MH-2001 strains and known strains of Dessia unicacerata MH-9011 according to the present invention (according to the Chordapest Treaty for the International Recognition of Deposits of Microorganisms for Patent Processes, deposited as No. FERM BP-3416 deposited in the National Institute of Bioscience and Human-Technology Agency of Industrial Science and Technology) with DNA amplified by arbitrary primers using the RAPD method (Arbitrarily Amplified Polymorphic DNA, Williams, J.G.K, etc. (1990) Polymorphic forms can be used as group markers, Nucleic Acid Research, Vol.18) The electrophoretic patterns of PCR products obtained were compared.

[0046] DNA was extracted and purified from MH-1901, MH-2001 and MH-9011 according to conventional methods. The purified DNA and a commercially available PCR reagent (Takara shuzo Co. Ltd) were mixed in the ratio as shown in Table 2 to prepare a PCR reaction solution.

[0047] The composition of table 2PCR reaction solution

[0048] PCR buffer (×10) 1.5μl

[0049] 25mM magnesium chloride 0.3μl

[0050] 2.5mM dNTP 0.6μl

[0051] Taq polymerase 0.06μl

[0052] 0.1μM primer 0.6μl

[0053] DNA (1ng/μl) 0.5μl

[0054] Sterilized water 11.34μl

[0055] OPE-1 (5'-CCCAAGGTCC-3' (SEQ ID NO: 1), product of Operon Technology) of RAPD was used as a primer. Use DNA thermal cycler (Gene Amplification PCR System 9600, product of Perkin Elmer Company) to carry out PCR reaction, start to be 94 ℃, 2 minutes, one cycle, then carry out 45 cycles as follows: 94 ℃, 10 seconds, 40 ℃ 1 minute at 72°C, 1 minute at 72°C, and 10 minutes at 72°C.

[0056] The DNA obtained by the PCR reaction was subjected to underwater electrophoresis (50V, 50 minutes) on 1.5% agarose gel, and the gel was soaked in ethidium bromide aqueous solution (0.5um/ml) for 20 minutes, and then stained in ultraviolet light. Under irradiation, detect the band pattern of the PCR product. The results are shown in figure 1 middle.

[0057] Comparison of the electrophoretic patterns of the PCR products shows that MH-1901 and MH-2001 according to the present invention are significantly different from MH-9011.