KR102651204B1 - Selective medium composition for Sclerotium sp. fungi and uses thereof - Google Patents
Selective medium composition for Sclerotium sp. fungi and uses thereof Download PDFInfo
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/045—Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
Abstract
본 발명은 스클레로티움 속(Sclerotium sp.) 진균에 대한 선택배지 조성물 및 이의 용도에 관한 것으로, 보다 구체적으로 항세균성분, 항진균성분 및 항미생물성 계면활성제를 포함하는 선택배지 조성물과 상기 선택배지 조성물을 이용한 스클레로티움 속 진균의 선택적 검출과 분리방법에 관한 것이다. 본 발명의 선택배지 조성물은 스클레로티움 속 이외의 미생물 생장을 효과적으로 억제시키는 효과를 가지고 있으므로, 스클레로티움 속 진균이 서식하는 공간에서 관찰되는 식물체 또는 토양으로부터 스클레로티움 속 진균을 효과적으로 검출할 수 있다.The present invention relates to a selective medium composition for Sclerotium sp. fungi and its use, and more specifically to a selective medium composition containing an antibacterial component, an antifungal component and an antimicrobial surfactant, and the selection medium composition. It relates to a method for selective detection and isolation of fungi of the genus Sclerotium using a medium composition. Since the selective medium composition of the present invention has the effect of effectively inhibiting the growth of microorganisms other than the Sclerotium genus, it effectively removes Sclerotium fungi from plants or soil observed in spaces where Sclerotium fungi inhabit. It can be detected.
Description
본 발명은 항세균, 항진균 및 항미생물 효과를 나타내는 성분을 포함하는 스클레로티움 속(Sclerotium sp.) 진균에 대한 선택배지 조성물 및 이의 용도에 관한 것이다.The present invention relates to a selective medium composition for Sclerotium sp. fungi containing components showing antibacterial, antifungal and antimicrobial effects and its use.
스클레로티움 속(Sclerotium sp.) 진균은 분류학적으로 담자균류(Basidiomycota)에 속하며, 각종 식물의 뿌리, 줄기, 잎 및 다육질의 열매에 병을 일으키는 것으로 알려져 있다. 스클레로티움 진균에 의해 나타나는 전형적인 병징(symptom)은 모잘록병, 줄기궤양병, 수관마름병, 열매썩음병, 흰비단병, 칼라병 등으로 다양하게 나타난다(Punja. 1985. Annual Review of Phytopathology. 23:97-127). 스클레로티움 진균은 전 세계적으로 분포하고, 주로 아메리카, 호주, 아프리카 및 아시아 등 온대, 열대, 및 아열대 지역에서 많이 발생한다. 이 진균은 많은 양의 균사체를 형성하고 옥살산, 펙틴 분해효소, 셀룰로스 분해효소 등의 효소들을 분비하고, 이 효소들은 기주(host)를 침입하는 과정에서 기주 조직을 분해하고 죽이는 기능을 제공한다. 스클레로티움 진균은 균핵(sclerotia)이라는 특별한 영양구조체를 형성하는데, 균사체가 서로 뭉치면서 형성되고 성숙하기 전에는 흰색이지만 점차 점점 어두운 갈색이나 검은색으로 바뀐다. Sclerotium sp. Fungi taxonomically belong to the Basidiomycota family and are known to cause diseases in the roots, stems, leaves, and fleshy fruits of various plants. Typical symptoms caused by the Sclerotium fungus appear in a variety of ways, such as blight, stem canker, crown blight, fruit rot, white silk, and collar disease (Punja. 1985. Annual Review of Phytopathology. 23:97- 127). Sclerotium fungi are distributed worldwide and occur mainly in temperate, tropical, and subtropical regions such as the Americas, Australia, Africa, and Asia. This fungus forms a large amount of mycelium and secretes enzymes such as oxalic acid, pectin decomposition enzyme, and cellulose decomposition enzyme, and these enzymes provide the function of decomposing and killing host tissue during the process of invading the host. Sclerotium fungi form special vegetative structures called sclerotia, which are formed when mycelium clumps together and are white before maturity, but gradually change to dark brown or black.
중국에서는 스클레로티움 롤프시(Sclerotium rolfsii) SC64 균주의 논잡초(특히, 사초과 및 부처꽃과)에 대한 생물학적 방제 활성이 보고되었다(Tang et al., 2011. Crop Protection. 30:1315-1320). 국내에서는 스클레로티움 델피니(S. delphinii) BWC04-107 균주를 활용한 망초류, 쑥류, 자귀풀, 클로버 그리고 붓꽃에 대한 제초 활성이 보고되었다(한국등록특허 제0769363호). 하지만 스클레로티움 롤프시는 500종 이상의 식물에 대해 병원성이 보고된 다범성 식물병원균이므로(El-Nagar et al., 2013 Journal of pure and applied microbiology. 7(3):1693-1705), 안전한 미생물 제초제로서의 활용을 위해선 균 생존성 및 전파성을 확인할 수 있는 모니터링 방법이 필요하다.In China, the biological control activity of Sclerotium rolfsii SC64 strain against rice field weeds (particularly Cyperaceae and Aquilaceae) was reported (Tang et al., 2011. Crop Protection. 30:1315-1320). . In Korea, the herbicidal activity of Sclerotium delphini ( S. delphinii ) BWC04-107 strain against sagebrush, mugwort, silkworm, clover, and iris was reported (Korean Patent No. 0769363). However, Sclerotium rolfsi is a widespread plant pathogen whose pathogenicity has been reported for more than 500 species of plants (El-Nagar et al., 2013 Journal of pure and applied microbiology. 7(3):1693-1705), so it is safe. In order to use it as a microbial herbicide, a monitoring method that can confirm the survival and spread of bacteria is required.
Elsheshtawi 등은 스클레로티움 세피보룸(S. cepivorum)을 선택적으로 분리할 수 있는 방법을 제시하였다(Elsheshtawi et al., 2014. Journal of Pure and Applied Microbiology. 8(4):2785-2790). 해당 연구에서는 양파 추출물(onion bulb-extract)을 배지에 첨가하면 스클레로티움 세피보룸이 다량의 균사체와 균핵을 생성한다고 보고하고 있다. Backman 등은 스클레로티움 롤프시 균주 분리를 위한 선택배지를 제시하였다(Backman et al., 1975. Phytopathology. 66:234-236). 해당 연구에서는 배지에 옥살레이트(oxalate)와 갈릭산(gallic acid)을 첨가하면 균핵의 활성을 선택적으로 확인할 수 있다고 보고하고 있다. 하지만 균핵 발아율은 대조구 PDA(potato dextrose agar) 배지에서 70% 이하였으며, 선택배지에서는 80% 이상으로, 스클레로티움 진균에 대한 선택성이 낮았다. 따라서 스클레로티움 진균을 효율적으로 검출하고 분리할 수 있는 선택배지의 필요성이 대두되었다.Elsheshtawi et al. presented a method to selectively isolate S. cepivorum (Elsheshtawi et al., 2014. Journal of Pure and Applied Microbiology. 8(4):2785-2790) . The study reported that when onion bulb-extract was added to the medium, Sclerotium sepiborum produced a large amount of mycelium and sclerotia. Backman et al. Sclerotium rolfsi A selective medium for strain isolation was presented (Backman et al., 1975. Phytopathology. 66:234-236). The study reports that the activity of sclerotia can be selectively confirmed by adding oxalate and gallic acid to the medium. However, the sclerotia germination rate was less than 70% in the control PDA (potato dextrose agar) medium and more than 80% in the selective medium, showing low selectivity for Sclerotium fungi. Therefore, the need for a selective medium that can efficiently detect and isolate Sclerotium fungi has emerged.
본 발명에서는 진균류의 배양에 일반적으로 사용되는 기본배지와 항세균 성분, 항진균 성분 및 항미생물성 계면활성제를 조합한 조성물을 완성하고 그 사용방법을 제시하였다.In the present invention, a composition combining a basic medium commonly used for culturing fungi, an antibacterial component, an antifungal component, and an antimicrobial surfactant was completed and a method of using the composition was presented.
한편, 한국등록특허 제0892359호에는 '감귤박을 탄소원으로 첨가한 배지에서 스클레로티움속의 배양을 통한 스클레로글루칸의 제조방법'이 개시되어 있고, 한국공개특허 제1991-0009918호에는 질소함유 영양물질, 당, 혈액 응고 방지제, 철(Fe) 소스 및 항균제를 포함하는 '선택성이 있는 진균배지'가 개시되어 있으나, 본 발명의 '스클레로티움 속 진균에 대한 선택배지 조성물 및 이의 용도'에 대해서는 기재된 바가 없다.Meanwhile, Korean Patent No. 0892359 discloses a 'method for producing scleroglucan through culturing Sclerotium in a medium containing citrus peel as a carbon source', and Korean Patent Publication No. 1991-0009918 discloses a method for producing scleroglucan. Although a 'selective fungal medium' containing nutrients, sugar, anti-coagulant, iron (Fe) source and antibacterial agent is disclosed, the 'selective medium composition for Sclerotium fungi and its use' according to the present invention 'There is nothing written about it.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명의 목적은 스클레로티움 속(Sclerotium sp.)에 속하는 균을 선택적으로 검출 및 분리할 수 있는 선택배지 조성물을 제공하는 것이다.The present invention was developed in response to the above-mentioned needs, and the purpose of the present invention is to provide a selective medium composition capable of selectively detecting and isolating bacteria belonging to the genus Sclerotium sp.
본 발명의 다른 목적은 상기 선택배지 조성물을 이용하여 스클레로티움 속에 속하는 진균을 식물체 및 토양 검체로부터 효과적으로 검출, 분리하고 배양하는 방법을 제공하는 것이다.Another object of the present invention is to provide a method for effectively detecting, isolating, and culturing fungi belonging to the genus Sclerotium from plants and soil samples using the selective medium composition.
상기 목적을 달성하기 위해서, 본 발명에서는 기본배지에 항세균성분, 항진균성분 및 항미생물성 계면활성제를 포함하는 스클레로티움 속(Sclerotium sp.) 진균의 검출 및 분리를 위한 선택배지 조성물을 제공한다.In order to achieve the above object, the present invention provides a selective medium composition for the detection and isolation of Sclerotium sp. fungi containing an antibacterial component, an antifungal component, and an antimicrobial surfactant in a basic medium. do.
또한, 본 발명은 진균 감염이 의심되는 식물체 또는 토양 시료를 상기 선택배지 조성물에 배양하는 단계를 포함하는, 스클레로티움 속 진균의 균핵(sclerotia) 또는 균사(mycelia)를 검출 및 분리하는 방법을 제공한다.In addition, the present invention provides a method for detecting and isolating sclerotia or mycelia of fungi of the genus Sclerotium, comprising culturing a plant or soil sample suspected of fungal infection in the selective medium composition. to provide.
본 발명의 선택배지는 스클레로티움 속(Sclerotium sp.) 진균의 생장을 효과적으로 확인할 수 있으므로, 단시간 내에 검체로부터 스클레로티움 속 진균을 쉽게 검출하고 분리할 수 있다.Since the selective medium of the present invention can effectively confirm the growth of Sclerotium sp. fungi, it can easily detect and isolate Sclerotium sp. fungi from a specimen within a short period of time.
도 1은 기본배지 PDA(potato dextrose agar)에 포함된 항세균성분의 종류에 따른 선택배지의 배양 특성을 확인한 결과이다. cbz: 카벤다짐, sls: 소디움 라우릴 설페이트, PDA: 무처리구, CK: 항세균성분만 미포함된 선택배지, rif: 100 ppm 리팜피신이 포함된 선택배지, amp: 100 ppm 암피실린이 포함된 선택배지.
도 2은 기본배지 PDA에 포함된 항진균성분의 종류에 따른 선택배지의 배양 특성을 확인한 결과이다. rif: 리팜피신, sls: 소디움 라우릴 설페이트, PDA: 무처리구, CK: 항진균성분만 미포함된 선택배지, cbz: 250 ppm 카벤다짐이 포함된 선택배지, thm: 250 ppm 티오파네이트 메틸이 포함된 선택배지, ben: 250 ppm 베노밀이 포함된 선택배지, tbz: 250 ppm 티아벤다졸이 포함된 선택배지, azo: 250 ppm 아족시스트로빈이 포함된 선택배지, teb: 250 ppm 테부코나졸이 포함된 선택배지.
도 3은 기본배지 PDA에 포함된 항미생물성 계면활성제의 종류에 따른 선택배지의 배양 특성을 확인한 결과이다. rif: 리팜피신, cbz: 카벤다짐, PDA: 무처리구, CK: 계면활성제만 미포함된 선택배지, sls: 250 ppm 소디움 라우릴 설페이트가 포함된 선택배지, sdbs: 250 ppm 소디움 도데실벤젠술포네이트가 포함된 선택배지.
도 4는 균핵형성 진균에 대한 선택배지(100 ppm 리팜피신, 250 ppm 티오파네이트 메틸 및 250 ppm 소디움 라우릴 설페이트 포함 PDA 배지)의 균핵형성 억제효과를 확인한 결과이다. S. rolfsii: Sclerotium rolfsii, S. delphinii: Sclerotium delphinii, S. minor: Sclerotinia minor, S. sclerotiorum: Sclerotinia sclerotiorum.
도 5는 선택배지(100 ppm 리팜피신, 250 ppm 티오파네이트 메틸 및 250 ppm 소디움 라우릴 설페이트 포함 PDA 배지)에서 일반적인 진균에 대한 생육 억제효과를 확인한 결과이다. R. solani: Rhizoctonia solani, P. ultimum: Pythium ultimum, C. cladosporiodes: Cladosporium cladosporiodes, T. asperellum: Trichoderma asperellum, A. niger: Aspergillus niger.
도 6은 항세균성분, 항진균성분 및 항미생물성 계면활성제를 포함하는 기본배지 및 무영양배지에서 스클레로티움 롤프시(S. rolfsii) 균주의 배양특성을 확인한 결과이다. PDA: 무처리구, PDArts: PDA 기본배지에 100 ppm 리팜피신, 250 ppm 티오파네이트 메틸 및 250 ppm 소디움 라우릴 설페이트 포함 선택배지, MEArts: MEA(malt extract agar) 기본배지에 100 ppm 리팜피신, 250 ppm 티오파네이트 메틸 및 250 ppm 소디움 라우릴 설페이트 포함 선택배지, NArts: NA(nutrient agar) 기본배지에 100 ppm 리팜피신, 250 ppm 티오파네이트 메틸 및 250 ppm 소디움 라우릴 설페이트 포함 선택배지, TSArts: TSA(tryptic soy agar) 기본배지에 100 ppm 리팜피신, 250 ppm 티오파네이트 메틸 및 250 ppm 소디움 라우릴 설페이트 포함 선택배지, WArts: WA(water agar) 무영양배지에 100 ppm 리팜피신, 250 ppm 티오파네이트 메틸 및 250 ppm 소디움 라우릴 설페이트 포함 선택배지, 상, 중, 하: 균핵의 오염 정도.
도 7은 선택배지에서 병징 또는 표징이 관찰된 식물부위 및 토양검체를 배양하여 스클레로티움 속 진균의 분리를 확인한 결과이다. PDA: 무처리구, 선택배지: 100 ppm 리팜피신, 250 ppm 티오파네이트 메틸 및 250 ppm 소디움 라우릴 설페이트 포함 PDA 선택배지, 토양: 오이 식물체 주변 토양검체.Figure 1 shows the results of confirming the culture characteristics of selective media according to the type of antibacterial ingredient contained in the basic medium PDA (potato dextrose agar). cbz: carbendazim, sls: sodium lauryl sulfate, PDA: untreated, CK: selective medium containing only antibacterial ingredients, rif: selective medium containing 100 ppm rifampicin, amp: selective medium containing 100 ppm ampicillin.
Figure 2 shows the results of confirming the culture characteristics of the selective medium according to the type of antifungal ingredient contained in the basic medium PDA. rif: rifampicin, sls: sodium lauryl sulfate, PDA: untreated, CK: selective medium containing only antifungal ingredients, cbz: selective medium containing 250 ppm carbendazim, thm: selective medium containing 250 ppm thiophanate methyl. , ben: selective medium containing 250 ppm benomyl, tbz: selective medium containing 250 ppm thiabendazole, azo: selective medium containing 250 ppm azoxystrobin, teb: selective medium containing 250 ppm tebuconazole. .
Figure 3 shows the results of confirming the culture characteristics of the selective medium according to the type of antimicrobial surfactant contained in the basic medium PDA. rif: rifampicin, cbz: carbendazim, PDA: untreated, CK: selective medium without surfactant, sls: selective medium containing 250 ppm sodium lauryl sulfate, sdbs: containing 250 ppm sodium dodecylbenzenesulfonate. Selection badge.
Figure 4 shows the results of confirming the sclerotia formation inhibitory effect of a selective medium (PDA medium containing 100 ppm rifampicin, 250 ppm methyl thiophanate, and 250 ppm sodium lauryl sulfate) on sclerotia-forming fungi. S. rolfsii : Sclerotium rolfsii , S. delphinii : Sclerotium delphinii , S. minor : Sclerotinia minor , S. sclerotiorum : Sclerotinia sclerotiorum .
Figure 5 shows the results of confirming the growth inhibition effect on common fungi in a selective medium (PDA medium containing 100 ppm rifampicin, 250 ppm methyl thiophanate, and 250 ppm sodium lauryl sulfate). R. solani : Rhizoctonia solani , P. ultimum : Pythium ultimum , C. cladosporiodes : Cladosporium cladosporiodes , T. asperellum : Trichoderma asperellum , A. niger : Aspergillus niger .
Figure 6 shows the results of confirming the culture characteristics of the S. rolfsii strain in basic medium and nutrient-free medium containing antibacterial ingredients, antifungal ingredients, and antimicrobial surfactants. PDA: untreated, PDArts: selective medium containing 100 ppm rifampicin, 250 ppm methyl thiophanate, and 250 ppm sodium lauryl sulfate on PDA basic medium, MEArts: 100 ppm rifampicin, 250 ppm thiopha on MEA (malt extract agar) basic medium. Selective medium containing methyl methyl and 250 ppm sodium lauryl sulfate, NArts: Selective medium containing 100 ppm rifampicin, 250 ppm methyl thiophanate and 250 ppm sodium lauryl sulfate on NA (nutrient agar) base medium, TSArts: TSA (tryptic soy) agar) basic medium containing 100 ppm rifampicin, 250 ppm thiophanate methyl and 250 ppm sodium lauryl sulfate; WArts: WA (water agar) nutrient-free medium containing 100 ppm rifampicin, 250 ppm thiophanate methyl and 250 ppm Selective medium containing sodium lauryl sulfate, upper, middle, lower: degree of contamination of sclerotia.
Figure 7 shows the results of confirming the isolation of Sclerotium fungi by culturing plant parts and soil samples where symptoms or signs were observed on selective medium. PDA: untreated, selective medium: PDA selective medium containing 100 ppm rifampicin, 250 ppm methyl thiophanate and 250 ppm sodium lauryl sulfate, soil: soil sample around cucumber plants.
본 발명의 목적을 달성하기 위하여, 본 발명은 기본배지에 항세균성분, 항진균성분 및 항미생물성 계면활성제를 포함하는 스클레로티움 속(Sclerotium sp.) 진균의 검출 및 분리를 위한 선택배지 조성물을 제공한다.In order to achieve the purpose of the present invention, the present invention is a selective medium composition for the detection and isolation of Sclerotium sp. fungi containing an antibacterial component, an antifungal component, and an antimicrobial surfactant in a basic medium. provides.
본 발명에 따른 선택배지 조성물에 있어서, 상기 기본배지는 감자한천배지(potato dextrose agar, PDA), 맥아추출물한천배지(malt extract agar, MEA) 또는 물한천배지(water agar, WA)일 수 있으나, 이에 제한되지 않는다.In the selective medium composition according to the present invention, the basic medium may be potato agar (potato dextrose agar (PDA)), malt extract agar (MEA), or water agar (WA). It is not limited to this.
스클레로티움 진균의 생장과 균핵(sclerotia)의 형성에 있어서 탄소원과 질소원 모두 중요한 요인으로 작용하는 것으로 알려져 있다(Fakher. 2020. International Journal of Phytopathology. 9(1):17-27). 따라서 본 발명의 선택배지는 일반적인 진균 배양용 배지를 기본배지로 사용할 수 있다.It is known that both carbon and nitrogen sources act as important factors in the growth of Sclerotium fungi and the formation of sclerotia (Fakher. 2020. International Journal of Phytopathology. 9(1):17-27). Therefore, the selective medium of the present invention can be used as a base medium for general fungal culture.
본 발명의 일 구현 예에 있어서, 상기 기본배지는 PDA, MEA 등을 사용했으나, 이에 제한되는 것은 아니다. 또한 기본배지로써 WA, 1/10 PDA 배지 등 무영양 또는 저영양배지를 사용할 수 있다. 이는 일반적인 세균이나 진균의 분생포자(condia) 크기가 10 ㎛ 이하인 반면, 스클레로티움 균핵은 건조된 이후에도 대부분 100~2,000 ㎛ 수준으로 크기가 크고 자체적으로 보유하고 있는 영양분이 많기 때문에, 무영양 또는 저영양배지에서도 발아 및 생장할 수 있는 특징을 활용하기 위한 것이다.In one embodiment of the present invention, the basic medium used is PDA, MEA, etc., but is not limited thereto. Additionally, non-nutrient or low-nutrient media such as WA and 1/10 PDA media can be used as the basic medium. This is because while the size of condia of common bacteria or fungi is 10 ㎛ or less, Sclerotium sclerotia are mostly large, around 100 to 2,000 ㎛ even after drying, and contain a lot of nutrients themselves, so they are non-nutritious or This is to take advantage of the ability to germinate and grow even in low-nutrient media.
또한, 본 발명에 따른 선택배지 조성물에 있어서, 상기 항세균성분은 리팜피신(rifampicin) 또는 암피실린(ampicillin)일 수 있으나, 이로 제한되는 것은 아니며, 일반적으로 그람양성 및 음성 세균을 저해하는데 사용하는 것이라면, 그 종류에 제한이 없다. 리팜피신은 세균의 DNA-dependent RNA polymerase를 억제함으로써 RNA로 전사(transcription)되는 것과 단백질 합성을 방해한다. 또한, 암피실린은 세균에서 세포벽 생합성에 필수적인 역할을 하는 penicillin-binding proteins(PBPs)를 불활성화한다.In addition, in the selective medium composition according to the present invention, the antibacterial ingredient may be rifampicin or ampicillin, but is not limited thereto, and is generally used to inhibit Gram-positive and Gram-negative bacteria. There is no limit to the type. Rifampicin inhibits bacterial DNA-dependent RNA polymerase, thereby interfering with transcription into RNA and protein synthesis. Additionally, ampicillin inactivates penicillin-binding proteins (PBPs), which play an essential role in cell wall biosynthesis in bacteria.
본 발명의 일 구현 예에 따른 선택배지 조성물에 있어서, 상기 항세균성분은 50~200 ppm의 농도로 포함될 수 있고, 바람직하게는 100~200 ppm의 농도로 포함될 수 있으며, 보다 더 바람직하게는 100~150 ppm의 농도로 포함될 수 있으며, 가장 바람직하게는 100 ppm의 농도로 포함될 수 있으나, 이에 제한되지 않는다.In the selective medium composition according to one embodiment of the present invention, the antibacterial ingredient may be included at a concentration of 50 to 200 ppm, preferably 100 to 200 ppm, and more preferably 100 ppm. It may be included at a concentration of ~150 ppm, most preferably at a concentration of 100 ppm, but is not limited thereto.
또한, 본 발명에 따른 선택배지 조성물에 있어서, 상기 항진균성분은 바람직하게는 벤지미다졸계(Benzimidazole) 항진균성분일 수 있고, 구체적으로, 카벤다짐(carbendazim), 베노밀(benomyl), 티오파네이트 메틸(thiophanate-methyl) 또는 티아벤다졸(thiabendazole)일 수 있으나, 이에 제한되지 않는다. 벤지미다졸계 항진균성분은 세포분열에 관여하는 튜블린(tubulin)과 결합하여 미세소관(microtubule)의 형성을 억제한다. 벤지미다졸계 항진균성분은 담자균류보다 자낭균류(Ascomycota)에 대해 항균 효과가 상대적으로 높다고 알려져 있다(Leadbeater. 2014. Plant health management: fungicides and antibiotics. In Encyclopedia of Agriculture and Food Systems. 4:408-424).In addition, in the selective medium composition according to the present invention, the antifungal component may preferably be a benzimidazole antifungal component, and specifically, carbendazim, benomyl, and thiophanate methyl ( It may be thiophanate-methyl) or thiabendazole, but is not limited thereto. Benzimidazole-based antifungal ingredients bind to tubulin, which is involved in cell division, and inhibit the formation of microtubules. It is known that benzimidazole-based antifungal ingredients have a relatively higher antibacterial effect against Ascomycota than against Basidiomycota (Leadbeater. 2014. Plant health management: fungicides and antibiotics. In Encyclopedia of Agriculture and Food Systems. 4:408-424 ).
본 발명의 일 구현 예에 따른 선택배지 조성물에 있어서, 상기 벤지미다졸계 항진균성분은 200~1,000 ppm의 농도로 포함될 수 있고, 바람직하게는 200~500 ppm의 농도로 포함될수 있으며, 보다 더 바람직하게는 250~500 ppm의 농도로 포함될 수 있으며, 가장 바람직하게는 250 ppm의 농도로 포함될 수 있으나, 이에 제한되지 않는다.In the selective medium composition according to one embodiment of the present invention, the benzimidazole-based antifungal ingredient may be included at a concentration of 200 to 1,000 ppm, preferably at a concentration of 200 to 500 ppm, and even more preferably May be included at a concentration of 250 to 500 ppm, most preferably at a concentration of 250 ppm, but is not limited thereto.
또한, 본 발명에 따른 선택배지 조성물에 있어서, 상기 항미생물성 계면활성제는 소디움 라우릴 설페이트(sodium lauryl sulfate, sls) 또는 소디움 도데실벤젠술포네이트(sodium dodecylbenzenesulfonate, sbds)일 수 있으나, 이로 제한되는 것은 아니며, 항미생물적인 특징을 나타내는 계면활성제라면 그 종류에 제한이 없다. sls는 바이러스 외피를 용해하거나, 게놈을 둘러싸고 있는 캡시드 단백질을 변성시키는 것으로 알려져 있다(Piret et al. 2002. Current Drug Targets. 3(1):17-30). sls는 넓은 범위의 항균활성을 지녔으며, 매우 낮은 농도에서도 활성을 나타내는 계면활성제로 알려져 있다(Fagundes et al. 2020. Materials Science Forum. 1012:500-505).In addition, in the selective medium composition according to the present invention, the antimicrobial surfactant may be sodium lauryl sulfate (sls) or sodium dodecylbenzenesulfonate (sbds), but is limited thereto. There is no limitation to the type of surfactant that exhibits antimicrobial properties. sls is known to dissolve the viral envelope or denature the capsid protein surrounding the genome (Piret et al. 2002. Current Drug Targets. 3(1):17-30). sls has a wide range of antibacterial activity and is known as a surfactant that is active even at very low concentrations (Fagundes et al. 2020. Materials Science Forum. 1012:500-505).
본 발명의 일 구현 예에 따른 선택배지 조성물에 있어서, 상기 항미생물성 계면활성제는 200~1,000 ppm의 농도로 포함될 수 있고, 바람직하게는 200~500 ppm의 농도로 포함될수 있으며, 보다 더 바람직하게는 250~500 ppm의 농도로 포함될 수 있으며, 가장 바람직하게는 250 ppm의 농도로 포함될 수 있으나, 이에 제한되지 않는다.In the selective medium composition according to one embodiment of the present invention, the antimicrobial surfactant may be included at a concentration of 200 to 1,000 ppm, preferably at a concentration of 200 to 500 ppm, and even more preferably May be included at a concentration of 250 to 500 ppm, most preferably at a concentration of 250 ppm, but is not limited thereto.
본 발명에 따른 상기 선택배지는 스클레로티움 속 이외의 균핵형성 진균 및 일반 진균에 대하여 항진균 활성을 나타낸다. 본 발명에 따른 선택배지가 항진균 활성을 나타내는 균핵형성 진균류는 스클레로티니아 마이너(Sclerotinia minor), 스클레로티니아 스클레로티오룸(Sclerotinia sclerotiorum), 일반적인 진균류는 라이족토니아 솔라니(Rhizoctonia solani), 피시움 울티뭄(Pythium ultimum), 클라도스포리움 클라도스포리오드스(Cladosporium cladosporiodes), 트리코데르마 아스퍼렐륨(Trichoderma asperellum) 및 아스파질러스 나이거(Aspergillus niger) 등이 있으나, 이에 제한되는 것은 아니다.The selective medium according to the present invention exhibits antifungal activity against sclerotium-forming fungi other than the Sclerotium genus and general fungi. Sclerotinia minor , Sclerotinia sclerotiorum , and Rhizoctonia solani are common fungi for which the selective medium according to the present invention exhibits antifungal activity. ), Pythium ultimum , Cladosporium cladosporiodes , Trichoderma asperellum , and Aspergillus niger , etc. It is not limited.
본 발명은 또한, 진균 감염이 의심되는 식물체 또는 토양 시료를 스클레로티움 속 진균의 검출 및 분리를 위한 선택배지 조성물에 배양하는 단계를 포함하는, 스클레로티움 속 진균의 균핵 또는 균사(mycelia)를 검출 및 분리하는 방법을 제공한다.The present invention also includes the step of culturing a plant or soil sample suspected of being infected with a fungus on a selective medium composition for detection and isolation of fungi of the genus Sclerotium. ) provides a method for detecting and separating.
본 발명에 따른 방법에 있어서, 상기 선택배지는 기본배지에 항세균성분, 항진균성분 및 항미생물성 계면활성제를 포함하는 것으로, 상세한 설명은 전술한 것과 같다.In the method according to the present invention, the selective medium includes an antibacterial component, an antifungal component, and an antimicrobial surfactant in a basic medium, and the detailed description is the same as the above.
본 발명에 따른 방법에 있어서, 상기 식물체 시료는 식물체의 다양한 조직 예컨대, 뿌리, 줄기, 잎, 꽃, 열매 등을 포함할 수 있으나, 이로 제한되는 것은 아니다.In the method according to the present invention, the plant sample may include various tissues of the plant such as roots, stems, leaves, flowers, fruits, etc., but is not limited thereto.
또한, 시료 접종 및 배양 방법은 당업계의 통상적인 방법에 의해 이루어질 수 있다.Additionally, sample inoculation and culture methods can be performed by conventional methods in the art.
본 발명에 따른 선택배지는 스클레로티움 속 진균 외의 진균류 및 세균류에 대한 성장 억제 효과가 우수하므로, 진균 감염이 의심되는 식물체 또는 토양 시료를 선택배지에 접종하고 배양할 경우 스클레로티움 속의 진균만 생장할 수 있어, 검체로부터 스클레로티움 속 진균을 선택적으로 검출 및 분리할 수 있다.The selective medium according to the present invention has an excellent growth inhibitory effect on fungi and bacteria other than Sclerotium fungi, so when plants or soil samples suspected of being infected with fungi are inoculated into the selective medium and cultured, fungi of the Sclerotium genus Since it can only grow, fungi of the genus Sclerotium can be selectively detected and isolated from specimens.
본 발명의 일 구현 예에 따른 방법에 있어서, 기본배지로서 PDA 또는 MEA 등을 사용한 선택배지에서 시료를 배양하였을 때 스클레로티움 속 진균외 일부 미생물의 오염이 의심되는 경우, 해당 시료를 WA 또는 1/10 PDA 배지 등과 같은 무영양 또는 저영양배지를 기본배지로 한 선택배지에서 배양하면 스클레로티움 속 진균을 특이적으로 검출 및 분리할 수 있다.In the method according to one embodiment of the present invention, when contamination with some microorganisms other than Sclerotium fungi is suspected when culturing a sample in a selective medium using PDA or MEA, etc. as a basic medium, the sample is transferred to WA or By culturing in a selective medium using a non-nutritious or low-nutrient medium such as 1/10 PDA medium as the base medium, fungi of the genus Sclerotium can be specifically detected and isolated.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by examples. However, the following examples only illustrate the present invention, and the content of the present invention is not limited to the following examples.
실시예 1. 선택배지 구성성분 탐색 및 최적농도 확인Example 1. Exploration of selective medium components and confirmation of optimal concentration
스클레로티움 속 선택배지에는 오염세균 및 오염진균의 성장을 억제하는 성분의 포함이 요구된다. 일반적인 진균배양에 사용되는 PDA(potato dextrose agar) 또는 MEA(malt extract agar)를 기본배지로 사용하여, 여기에 항세균성분, 벤지미다졸계 항진균성분 또는 항미생물성 계면활성제를 적절히 첨가하여 선택배지를 제조하였다.Selective media for the Sclerotium genus are required to contain ingredients that inhibit the growth of contaminating bacteria and fungi. PDA (potato dextrose agar) or MEA (malt extract agar), which are used for general fungal culture, is used as a basic medium, and antibacterial ingredients, benzimidazole antifungal ingredients, or antimicrobial surfactants are appropriately added to create a selective medium. Manufactured.
본 발명에 사용한 항세균성분은 리팜피신(rifampicin, rif)과 암피실린(ampicillin, amp)이고, 벤지미다졸계 항진균성분은 카벤다짐(carbendazim, cbz), 티오파네이트 메틸(thiophanate-methyl, thm), 베노밀(benomyl, ben) 및 티아벤다졸(thiabendazole, tbz)을 사용하였고, 다른 계통 항진균성분과의 비교를 위해 스트로빌루린 계열의 아족시스트로빈(azoxystrobin, azo)과 트리아졸 계열의 테부코나졸(tebuconazole, teb)을 사용하였다. 항미생물성 계면활성제는 소디움 라우릴 설페이트(sodium lauryl sulfate, sls)와 소디움 도데실벤젠술포네이트(sodium dodecylbenzenesulfonate, sdbs)를 사용하였다.The antibacterial ingredients used in the present invention are rifampicin (rif) and ampicillin (amp), and the benzimidazole-based antifungal ingredients are carbendazim (cbz), thiophanate-methyl (thm), and benomyl. (benomyl, ben) and thiabendazole (tbz) were used, and for comparison with other antifungal ingredients, azoxystrobin (azo) of the strobilurin series and tebuconazole of the triazole series were used. , teb) was used. Sodium lauryl sulfate (sls) and sodium dodecylbenzenesulfonate (sdbs) were used as antimicrobial surfactants.
또한, 본 실험에 사용한 균주는 스클레로티움 롤프시(Sclerotium rolfsii)이고, 사용한 배지는 PDA(Difco)에 250 ppm 항진균성분과 계면활성제를 각각 넣고 오토클레이브(120℃, 15분)을 진행하였다. 배지온도가 60℃ 이하가 되었을 때 클린벤치에서 100 ppm 항세균성분을 넣고 배지를 제조하였다. 실험은 처리구 당 5개 균핵/배지로 치상하였고 5반복으로 진행되었다. 배양조건은 30℃ 인큐베이터로 7일간 배양한 이후 조사하였다.In addition, the strain used in this experiment was Sclerotium rolfsii , and the medium used was PDA (Difco) with 250 ppm antifungal ingredient and surfactant, respectively, and autoclaved (120°C, 15 minutes). . When the medium temperature was below 60°C, 100 ppm antibacterial ingredient was added and the medium was prepared on a clean bench. The experiment was performed with 5 sclerotia/medium per treatment group and was repeated 5 times. Culture conditions were examined after culturing in an incubator at 30°C for 7 days.
대조구로 PDA에서 균핵발아율 44%, 진균오염율 84%, 세균오염율 25%로 균핵의 상태가 확인되었다. 그에 반해 항세균성분(rif, amp)이 PDA에 첨가되었을 때, 세균오염을 관찰할 수 없었다(도 1). 또한, 벤지미다졸계 항진균성분(cbz, thm, ben, tbz)이 첨가되었을 때, 균핵발아율은 90% 이상을 확인할 수 있었다. 반면에, 스트로빌루린 항진균성분(azo)과 트리아졸 항진균성분(teb)이 첨가되었을 때, 균핵발아율은 각각 12%, 4%로 저조한 것으로 확인할 수 있었다(도 2). 항미생물성 계면활성제(sls, sdbs)가 PDA에 첨가되었을 때, 균핵발아율은 90% 이상으로 확인할 수 있었다(도 3). 이에 스클레로티움 롤프시 선택배지는 항세균성분으로는 리팜피신(rif)과 암피실린(amp)을, 항진균성분은 카벤다짐(cbz)과 티오파네이트 메틸(thm) 등 벤지미다졸계 항진균성분을, 그리고 소디움 라우릴 설페이트(sls)와 소디움 도데실벤젠술포네이트(sdbs) 등 항미생물성 계면활성제가 포함될 수 있음을 알 수 있었다.As a control, the status of sclerotia was confirmed in PDA with a germination rate of 44%, fungal contamination rate of 84%, and bacterial contamination rate of 25%. In contrast, when antibacterial ingredients (rif, amp) were added to PDA, bacterial contamination could not be observed (Figure 1). In addition, when benzimidazole-based antifungal ingredients (cbz, thm, ben, tbz) were added, the sclerotia germination rate was confirmed to be over 90%. On the other hand, when strobilurin antifungal ingredient (azo) and triazole antifungal ingredient (teb) were added, the sclerotia germination rate was confirmed to be low at 12% and 4%, respectively (Figure 2). When antimicrobial surfactants (sls, sdbs) were added to PDA, the sclerotia germination rate was confirmed to be over 90% (Figure 3). Accordingly, the Sclerotium rolfsi selection medium contains rifampicin (rif) and ampicillin (amp) as antibacterial ingredients, and benzimidazole antifungal ingredients such as carbendazim (cbz) and methyl thiophanate (thm) as antifungal ingredients. And it was found that antimicrobial surfactants such as sodium lauryl sulfate (sls) and sodium dodecylbenzenesulfonate (sdbs) may be included.
CK: none
항세균성분: rif(rifampicin), amp(ampicillin)
항진균성분: cbz(carbendazim), thm(thiophanate-methyl), ben(benomyl), tbz(thiabendazole), azo(azoxystrobin), teb(tebuconazole)
계면활성제: sls(sodium lauryl sulfate), sdbs(sodium dodecylbenzenesulfonate)DAI: day after inoculation
CK: none
Antibacterial ingredients: rif(rifampicin), amp(ampicillin)
Antifungal ingredients: cbz (carbendazim), thm (thiophanate-methyl), ben (benomyl), tbz (thiabendazole), azo (azoxystrobin), teb (tebuconazole)
Surfactant: sls(sodium lauryl sulfate), sdbs(sodium dodecylbenzenesulfonate)
실시예 2. 선택배지에 대한 다양한 균 배양확인Example 2. Confirmation of various bacterial cultures on selective media
스클레로티움 선택배지에 대한 균주별 배양특성을 확인하기 위해서 다양한 균주를 테스트하였다. 균주는 크게 균핵형성 균주와 자연상태에서 흔히 볼 수 있는 진균을 사용하였다. 본 발명에 사용된 균주는 국립농업과학원 농업미생물은행(대한민국, 전북)으로부터 분양받아 사용하였다. 균핵형성 진균으로는 스클레로티움 롤프시(Sclerotium rolfsii) 균주 5개, 스클레로티움 델피니(Sclerotium delphinii) 균주 1개, 스클레로티니아 마이너(Sclerotinia minor) 균주 2개, 그리고 스클레로티니아 스클레로티오룸(Sclerotinia sclerotiorum) 균주 2개를 사용하였다. 또한, 일반적인 진균 균주로는 라이족토니아 솔라니(Rhizoctonia solani), 피시움 울티뭄(Pythium ultimum), 클라도스포리움 클라도스포리오드스(Cladosporium cladosporiodes), 트리코데르마 아스퍼렐륨(Trichoderma asperellum), 및 아스파질러스 나이거(Aspergillus niger)로, 토양이나 생활환경에서 일반적으로 관찰되는 진균류를 사용하였다.In order to confirm the culture characteristics of each strain on Sclerotium selective medium, various strains were tested. The strains used were mainly sclerotia-forming strains and fungi commonly found in nature. The strain used in the present invention was purchased from the Agricultural Microbial Bank of the National Institute of Agricultural Sciences (Jeonbuk, Korea). Sclerotinia fungi include 5 strains of Sclerotium rolfsii , 1 strain of Sclerotium delphinii , 2 strains of Sclerotinia minor , and Scleroti. Two Sclerotinia sclerotiorum strains were used. In addition, common fungal strains include Rhizoctonia solani , Pythium ultimum , Cladosporium cladosporiodes, and Trichoderma asperellum . , and Aspergillus niger , fungi commonly observed in soil or living environments were used.
실험재료는 리팜피신(98%, TCI), 톱신엠(70% 티오파네이트 메틸, 경농), 소디움 라우릴 설페이트(94%, 대정)을 각각 사용하였으며, 이 중 리팜피신은 DMSO(dimethyl sulfoxide)를 용매로 하여 저장용 용액(stock solution)을 제조하였다. 제조방법은 멸균 전에 PDA와 증류수를 활용하여 배지를 제조하였고, 이후 원제함량을 기준으로 하여 250 ppm 티오파네이트 메틸이 함유할 수 있게 톱신엠을 첨가하였고, 250 ppm 소디움 라우릴 설페이트를 포함하도록 첨가하였다. 이후 오토클레이브(120℃, 15분)을 진행하였으며, 배지온도가 60℃ 이하가 되었을 때 클린벤치에서 100 ppm 리팜피신을 포함하도록 첨가하여 배지를 제조하였다. 실험방법은 균핵형성 균주는 균핵과 균사가 자란 아가(agar)로 선택배지에 치상하였으며, 균핵과 균사를 구분해서 확인하였다. 균핵을 치상하는 방법은 5개의 균핵/배지로, 균사는 5 mm 코르크 보어로 아가를 절단하여 1개의 아가/배지로 각각 5반복씩 치상하였다. 기타균주는 균사 상태로 선택배지에 치상하여 확인하였다. 마찬가지로 5 mm 코르크 보어로 아가를 절단하여 균주별 5반복으로 치상하였다. 배양조건은 25℃ 인큐베이터에서 배양되었으며, 조사는 7일 후 진행하였다.The experimental materials used were rifampicin (98%, TCI), TopsynM (70% methyl thiophanate, Kyungnong), and sodium lauryl sulfate (94%, Daejeong), of which rifampicin uses DMSO (dimethyl sulfoxide) as a solvent. A stock solution was prepared as follows. The manufacturing method was to prepare a medium using PDA and distilled water before sterilization, and then TopsynM was added to contain 250 ppm methyl thiophanate based on the raw material content, and added to contain 250 ppm sodium lauryl sulfate. did. Afterwards, autoclaving (120°C, 15 minutes) was performed, and when the medium temperature was below 60°C, medium was prepared on a clean bench by adding 100 ppm rifampicin. The experimental method was that sclerotia-forming strains were placed on selective media using agar on which sclerotia and hyphae grew, and sclerotia and hyphae were identified separately. The method of plating sclerotia was with 5 sclerotia/medium, and the hyphae were cut into agar with a 5 mm cork bore and pricked with 1 agar/medium 5 times each. Other strains were confirmed by placing them on selective medium in the mycelial state. Similarly, the agar was cut with a 5 mm cork bore and treated 5 times for each strain. Culture conditions were cultured in an incubator at 25°C, and irradiation was conducted after 7 days.
분석 결과, 균핵형성 균주인 스클레로티움 롤프시(KACC93004, KACC40834, KACC41703, KACC43066, KACC48129)와 스클레로티움 델피니(KACC93031p)는 선택배지에서 균핵과 균사상태 모두 배양이 확인되었다(도 4). 배양 7일차 기준으로 모든 스크레로티움 롤프시와 스클레로티움 델피니의 균핵은 100% 발아율을 확인할 수 있었다. 또한 균사 지름은 각각 8.3, 5.1 cm로 생장하였다. 반면에, 스클레로티니아 마이너(KACC41066, KACC41067)와 스클레로티니아 스클레로티오룸(KACC40922, KACC41065)의 모든 균핵은 발아되지 않은 것을 관찰할 수 있었고, 균사 또한 배양되지 않았다.As a result of the analysis, the sclerotium-forming strains Sclerotium rolfsi (KACC93004, KACC40834, KACC41703, KACC43066, KACC48129) and Sclerotium delphini (KACC93031p) were confirmed to be cultured in both sclerotium and hyphal states on the selective medium (Figure 4 ). As of the 7th day of culture, a 100% germination rate was confirmed for all sclerotia of Sclerotium rolfsi and Sclerotium delphini. Additionally, the mycelium diameters grew to 8.3 and 5.1 cm, respectively. On the other hand, it was observed that all sclerotia of Sclerotinia minor (KACC41066, KACC41067) and Sclerotinia sclerotiorum (KACC40922, KACC41065) did not germinate, and hyphae were also not cultured.
또한, 기타균주인 라이족토니아 솔라니(KACC40132), 피시움 울티뭄(KACC40705), 클라도스포리움 클라도스포리오드스(KACC43532), 트리코데르마 아스퍼렐륨(KACC43821) 및 아스파질러스 나이거(KACC46498)는 선택배지에서 배양되지 않았다(도 5).In addition, other strains such as Rhizoctonia solani (KACC40132), Pycium ultimum (KACC40705), Cladosporium cladosporiods (KACC43532), Trichoderma asperllium (KACC43821), and Aspargillus niger (KACC46498) was not cultured in selective medium (Figure 5).
실시예 3. 선택배지용 기본배지에 대한 평가Example 3. Evaluation of basic medium for selective medium
본 발명에서 사용된 기본배지는 감자한천배지(potato dextrose agar, PDA), 맥아추출물한천배지(malt extract agar, MEA), 보통한천배지(nutrient agar, NA), 대두카제인소화한천배지(tryptic soy agar, TSA), 물한천배지(water agar, WA)이며, 선택배지 조성은 위 실시예 2와 같다. 실험에 사용한 균핵은 스클레로티움 롤프시(S. rolfsii)로 오염정도를 상, 중, 하 그룹으로 구분하여 진행하였다. 상중하 각 균핵의 PDA 배양 7일차 조사에서, 오염정도 상의 그룹은 균핵 발아율 100%, 진균오염율 0%를 보였던 검체이며, 오염정도 중의 그룹은 76%, 진균오염율 36%를 보였고, 오염정도 하의 그룹은 검체균핵을 3시간 동안 밭토양에 혼합한 후 다시 분리하여 사용하였다. 각 그룹의 균핵을 5개의 균핵/배지로 일정하게 치상하고 배양하였다. 균핵의 상중하 그룹을 배지 종류별로 각각 치상하여 30℃에서 배양하였고 배양 7일 후 조사하였다, 각 처리군은 5배지로 총 25개 균으로 구성되었다.The basic media used in the present invention are potato agar (potato dextrose agar, PDA), malt extract agar (MEA), nutrient agar (NA), and soy casein digested agar (tryptic soy agar). , TSA), and water agar (WA), and the composition of the selective medium is the same as Example 2 above. The sclerotia used in the experiment was S. rolfsii , and the degree of contamination was divided into high, medium, and low groups. In the 7th day of PDA culture of upper, middle and lower sclerotia, the group with high contamination level showed 100% sclerotia germination rate and 0% fungal contamination rate, the group with medium contamination level showed 76% and fungal contamination rate 36%, and the group with low contamination level showed 76% and 36% fungal contamination rate. The group mixed the specimen sclerotia with field soil for 3 hours and then separated them again for use. Sclerotia from each group were uniformly plated and cultured with 5 sclerotia/medium. The upper, middle and lower groups of sclerotia were placed on each type of medium and cultured at 30°C and examined after 7 days of culture. Each treatment group consisted of a total of 25 bacteria in 5 media.
조사 결과, 동일한 항세균성분, 항진균성분 및 항미생물성 계면활성제가 포함된 기본배지 중 PDA 배지와 MEA 배지를 사용한 선택배지(PDArts, MEArts)에서 스클레로티움 속 균주의 균핵발아율과 균사생장이 가장 양호하여 선택성이 우수함을 알 수 있었다. 또한, 무영양선택배지(WArts)에서 스클레로티움 속 균주의 균핵발아율과 균사생장을 평가한 결과, 스클레로티움 속 균주의 생장이 느리지만 자라는 것을 확인하였고, 진균오염은 관찰되지 않아 선택적인 생장을 확인할 수 있었다.As a result of the investigation, among the basic media containing the same antibacterial ingredients, antifungal ingredients, and antimicrobial surfactants, the sclerotium germination rate and mycelial growth of Sclerotium strains were increased in selective media (PDArts, MEArts) using PDA medium and MEA medium. It was found to be the best, showing excellent selectivity. In addition, as a result of evaluating the sclerotium germination rate and mycelial growth of the Sclerotium strain on nutrient-free selective medium (WArts), it was confirmed that the Sclerotium strain grew slowly, and fungal contamination was not observed, so it was selected. Positive growth could be confirmed.
기본배지로 평가된 NA, TSA 등(NArts, TSArts) 등은 기본배지로써 적합하지 않았다. NArts와 TSArts 배지에서는 스클레로티움 속 진균의 경우에도 균핵발아율이 낮은 반면 진균오염율은 높게 관찰되었다. NArts 배지에서는 WArts 배지와 유사한 형태로 짧은 균총형성이 관찰되었고 일부 오염진균의 생장을 확인할 수 있었다. TSArts 배지에서는 오염정도 상의 그룹에 속하는 균핵에서만 양호한 균총형성이 관찰되었고, 오염정도 중과 하의 균핵에서는 높은 오염진균의 생장이 관찰되었다.NA, TSA, etc. (NArts, TSArts), which were evaluated as basic badges, were not suitable as basic badges. In NArts and TSArts media, the sclerotium germination rate was low for Sclerotium fungi, while the fungal contamination rate was high. In the NArts medium, short colony formation was observed in a similar manner to the WArts medium, and the growth of some contaminating fungi was confirmed. In TSArts medium, good colony formation was observed only in sclerotia belonging to the high contamination level group, and high growth of contaminated fungi was observed in sclerotia belonging to the medium and low contamination levels.
실시예 4. 검체별 선택배지의 검출 및 분리방법 비교Example 4. Comparison of detection and separation methods of selective media for each sample
본 발명의 선택배지 조성물을 사용하면 스클레로티움 속의 진균이 형성하는 균사와 균핵에 대해서만 선택적으로 생장하는 것을 관찰할 수 있었다. 특히 균핵의 경우에는 토양에 장기간 방치한 후 회수한 균핵에서도 스클레로티움 속의 진균만을 선택적으로 검출하고 분리할 수 있는 효과를 제공하였다. 병징 또는 표징을 나타낸 식물검체와 그 주변의 토양검체를 선택배지에서 배양하여 스클레로티움 속 진균의 검출 및 분리가 가능한지 알아보았다.When using the selective medium composition of the present invention, it was observed that the fungi of the Sclerotium genus selectively grow only on the hyphae and sclerotia formed. In particular, in the case of sclerotia, it provided the effect of selectively detecting and isolating only fungi of the Sclerotium genus even from sclerotia recovered after being left in the soil for a long period of time. Plant samples showing symptoms or signs and soil samples surrounding them were cultured in selective media to determine whether it was possible to detect and isolate Sclerotium fungi.
실험검체는 오이와 토마토를 재배한 포장에서 스클레로티움 롤프시를 처리하고 2개월 후 지표면과 접촉되어 있는 줄기 또는 열매를 채취하였다. 오이는 줄기와 열매를 채취하였고, 토마토는 줄기만 채취하였다. 토양검체는 작물의 주변 토양을 채취하였다. 각 작물 줄기는 균핵균 병징으로 확인되는 부위를 0.5 cm 절단, 그리고 열매는 균사생장이 관찰되는 부분을 1 cm 크기 절편으로 절단하여 선택배지에 치상하였다. 무처리 대조구배지(PDA)와 선택배지(PDArts)의 배양결과를 비교하였다. 한 검체당 5반복으로 배지를 구분하여 치상하였고, 배양은 30℃ 인큐베이터에서 7일간 진행하였다.The experimental samples were treated with Sclerotium rolfsi in fields where cucumbers and tomatoes were grown, and stems or fruits in contact with the ground were collected two months later. For cucumbers, the stems and fruits were collected, and for tomatoes, only the stems were collected. Soil samples were collected from the soil surrounding the crops. Each crop stem was cut into 0.5 cm pieces at the area where sclerotia disease symptoms were identified, and the fruit was cut into 1 cm-sized sections at the area where mycelial growth was observed and placed on selective medium. The culture results of untreated control medium (PDA) and selective medium (PDArts) were compared. Each sample was cultured in five separate mediums, and culture was performed in an incubator at 30°C for 7 days.
무처리 대조구로 사용된 PDA 배지에서는 스클레로티움 롤프시의 생장이 관찰되었지만 오염세균과 오염진균도 동시에 관찰되었다(도 7). 토양검체의 경우 PDA 배지에서 다양한 오염진균의 생장을 관찰할 수 있었고, 병징이 관찰되었던 토마토 줄기에서는 PDA 배지 5개 중 2개에서 스클레로티움 롤프시의 균사 생장을 관찰할 수 있었다. 오이의 줄기 및 열매 검체에서는 스클레로티움 롤프시의 균사생장이 관찰되었지만 오염세균의 생장도 관찰되었다. 반면에 선택배지 PDArts에서는 토마토 줄기 그리고 오이 줄기와 열매에서 스클레로티움 롤프시의 균사가 오염진균의 균사생장 없이 선택적으로 자라는 것을 확인할 수 있었다. 또한, 토양검체에서도 선택적인 균사생장을 관찰할 수 있었다.In the PDA medium used as an untreated control, the growth of Sclerotium rolfsi was observed, but contaminating bacteria and contaminating fungi were also observed at the same time (FIG. 7). In the case of soil samples, the growth of various contaminating fungi was observed on PDA medium, and on tomato stems where disease symptoms were observed, mycelial growth of Sclerotium rolfsi was observed on two out of five PDA medium. Mycelial growth of Sclerotium rolfsi was observed in cucumber stem and fruit samples, but growth of contaminating bacteria was also observed. On the other hand, in the selective medium PDArts, it was confirmed that the mycelium of Sclerotium rolfsi selectively grew on tomato stems and cucumber stems and fruits without mycelial growth of contaminating fungi. Additionally, selective mycelial growth was observed in soil samples.
따라서 본 발명의 선택배지 조성물인 PDArts 배지를 사용하면 식물의 줄기, 열매 또는 토양 검체로부터 스클레로티움 속 진균의 균사 또는 균핵을 선택적으로 배양하고 분리할 수 있었다. 또한 PDArts 선택배지에서 오염진균에 대한 선택적 배양과 분리가 충분하지 않을 경우에는 WAarts 선택배지를 이용하면 스클레로티움 속 진균을 선택적으로 분리하고 배양할 수 있음을 확인할 수 있었다.Therefore, by using PDArts medium, which is the selective medium composition of the present invention, it was possible to selectively culture and isolate mycelia or sclerotia of Sclerotium fungi from plant stems, fruits, or soil samples. In addition, when the PDArts selective medium was not sufficient to selectively culture and isolate contaminating fungi, it was confirmed that fungi of the Sclerotium genus could be selectively isolated and cultured using the WAarts selective medium.
Claims (7)
상기 기본배지는 감자한천배지(potato dextrose agar), 맥아추출물한천배지(malt extract agar) 또는 물한천배지(water agar)이고,
상기 항미생물성 계면활성제는 소디움 라우릴 설페이트(sodium lauryl sulfate) 또는 소디움 도데실벤젠술포네이트(sodium dodecylbenzenesulfonate)인 것을 특징으로 하는 스클레로티움 속 진균의 검출 및 분리를 위한 선택배지 조성물.A selective medium composition for the detection and isolation of Sclerotium sp. fungi containing rifampicin, benzimidazole antifungal ingredients, and an antimicrobial surfactant in a basic medium,
The basic medium is potato dextrose agar, malt extract agar, or water agar,
A selective medium composition for the detection and isolation of fungi of the genus Sclerotium, wherein the antimicrobial surfactant is sodium lauryl sulfate or sodium dodecylbenzenesulfonate.
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Non-Patent Citations (3)
| Title |
|---|
| Canadian Journal of Plant pathology. vol. 16, no. 1, 68-70(1994.03.01.)* * |
| Netherlands Journal of Plant Pathology. vol. 88, 39-45(1982)* * |
| PeerJ. vol. 6, no. e6009(2018) * |
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