KR20220062725A - Primer sets for diagnosing five fungi infecting peach and diagnostic methods using thereof - Google Patents
Primer sets for diagnosing five fungi infecting peach and diagnostic methods using thereof Download PDFInfo
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Abstract
Description
서열번호 1 및 서열번호 2로 표시되는 프라이머 쌍, 서열번호 3 및 서열번호 4로 표시되는 프라이머 쌍, 서열번호 5 및 서열번호 6으로 표시되는 프라이머 쌍, 서열번호 7 및 서열번호 8로 표시되는 프라이머 쌍, 및 서열번호 9 및 서열번호 10으로 표시되는 프라이머 쌍으로 이루어진 군으로부터 선택되는 하나 이상의 프라이머 쌍을 포함하는, 시토스포라 레우코스토마(Cytospora leucostoma), 마크로포미나 파세올리나(Macrophomina phaseolina), 디아포르테 헬리안티(Diaporthe helianthi), 모닐리니아 프룩티콜라(Monilinia fructicola), 파이토프소라 캑토룸(Phytophthora cactorum)으로 이루어진 군으로부터 선택되는 하나 이상의 복숭아 곰팡이 진단용 프라이머 세트; 상기 프라이머 쌍을 포함하는 복숭아 곰팡이 진단용 조성물; 상기 조성물을 포함하는 복숭아 곰팡이 진단용 키트; 및 복숭아 곰팡이 진단 방법에 관한 것이다.Primer pair represented by SEQ ID NO: 1 and SEQ ID NO: 2, primer pair represented by SEQ ID NO: 3 and SEQ ID NO: 4, primer pair represented by SEQ ID NO: 5 and SEQ ID NO: 6, primers represented by SEQ ID NO: 7 and SEQ ID NO: 8 Cytospora leucostoma ( Cytospora leucostoma ) , Macrophomina phaseolina ), Diapor the helianti ( Diaporthe helianthi ), Monilinia fructicola ( Monilinia fructicola ), Phytophthora cactorum ( Phytophthora cactorum ) One or more sets of primers for diagnosis of peach mold selected from the group consisting of; A composition for diagnosis of peach fungus comprising the primer pair; A kit for diagnosing peach fungus comprising the composition; and to a method for diagnosing peach fungus.
복숭아 (Prunus persica (L.) Batch)는 장미과(The family Rosaceae), 벚나무속(The genus Prunus)에 속하는 과실나무이다. 전세계적으로 복숭아는 살구, 매실, 자두, 체리와 더불어 인기가 많은 핵과류(Stone fruits) 중의 하나이며, 복숭아 과실은 주로 신선한 과일 또는 캔으로 가공되어 소비된다. 현재 국내에는 다양한 종류의 수입 과일들이 대량으로 수입되어 소비되고 있으며, 기존 국내에서 생산되고 있는 많은 과일들을 대체하고 있다. 하지만 복숭아의 경우 저장의 어려움으로 인해 수입이 불가하여, 국내에서 재배되고 있는 복숭아 과실들은 수입과일과 비교하여 여름철에 좋은 경쟁력을 가지고 있다. 국내에서 재배되고 있는 복숭아 품종은 매우 다양하며, 주로 7월부터 10월까지 생산되고 있다. 그러나, 다양한 병원성 균들과 해충들이 복숭아 나무 재배에 많은 경제적 피해를 주고 있다. 그 중에서도 곰팡이병은 복숭아의 수확량뿐만 아니라 복숭아 품질에 악영향을 주고 있어, 곰팡이병을 예방하거나 조절할 수 있는 방법들에 대한 개발이 필요하다. Peach ( Prunus persica (L.) Batch) is a fruit tree belonging to the family Rosaceae , The genus Prunus . Worldwide, peaches are one of the most popular stone fruits along with apricots, plums, plums, and cherries, and peach fruits are mainly consumed as fresh fruits or canned fruits. Currently, various kinds of imported fruits are imported and consumed in large quantities in Korea, replacing many fruits produced in Korea. However, in the case of peaches, it is impossible to import them due to storage difficulties, so peach fruits grown in Korea have good competitiveness in summer compared to imported fruits. Peach varieties cultivated in Korea are very diverse, and mainly produced from July to October. However, various pathogenic bacteria and pests cause a lot of economic damage to peach tree cultivation. Among them, fungal disease adversely affects not only peach yield but also peach quality, so it is necessary to develop methods for preventing or controlling fungal disease.
한편, 대부분의 복숭아 품종들은 영양번식(Clonal propagation) 방법인 접목(Grafting)을 통해 번식 및 재배되고 있다. 하지만 복숭아 나무들은 영양번식 작물들의 특징 중의 하나인, 바이러스, 세균, 곰팡이들을 비롯한 다양한 병원성 미생물에 의해 심각하게 감염되어있다. 이러한 문제점을 극복하기 위하여 최근에는 농촌진흥청을 비롯한 국가연구기관들을 중심으로 주요 병원성 미생물에 대한 진단 방법들이 개발되었고, 알려진 병원성 미생물에 대한 진단법을 토대로 무병묘 복숭아 묘목들이 개발되고 있다. 최근까지 복숭아 나무에 병을 일으키는 다양한 종류의 곰팡이균들이 동정되었다. 대표적인 복숭아 감염 곰팡이로는 잿빛무늬병(brown rot)을 일으키는 Monilinia fructicola와 M. laxa가 있으며, gray mold (Botrytis cinerea), Alternaria spp., Aspergillus spp., Cladosporium spp.와 blue mold (Penicillium spp.)들이 복숭아 과실에 곰팡이병을 자주 일으키는 것으로 알려져있다. 또한 복숭아 과수원의 토양에는 주로 붉은 곰팡이 종(Fusarium species)들이 많이 있는 것으로 알려져있다. 다양한 복숭아 곰팡이 병 중에서도 잿빛무늬병을 일으키는 M. fructicola의 경우 복숭아 과실 생산에 많은 피해를 주고 있으며, 특히 겨울철에 미라화된(mummified) 복숭아 과실에 살아남아 꽃과 어린 슈트(shoot)를 감염시킬 수 있는 포자들을 생산한다. On the other hand, most peach varieties are propagated and cultivated through grafting, which is a clonal propagation method. However, peach trees are seriously infected with various pathogenic microorganisms, including viruses, bacteria, and fungi, which are characteristic of vegetatively propagated crops. In order to overcome this problem, recently, diagnostic methods for major pathogenic microorganisms have been developed centered on national research institutes including the Rural Development Administration, and disease-free peach seedlings are being developed based on the diagnostic methods for known pathogenic microorganisms. Until recently, various types of fungi that cause disease on peach trees have been identified. Representative peach infecting molds include Monilinia fructicola and M. laxa , which cause brown rot, gray mold ( Botrytis cinerea ), Alternaria spp., Aspergillus spp., Cladosporium spp. and blue mold ( Penicillium spp.) are known to frequently cause fungal diseases on peach fruits. In addition, it is known that there are mainly red mold species ( Fusarium species) in the soil of the peach orchard. Among various peach fungal diseases, M. fructicola , which causes gray spots, causes a lot of damage to peach fruit production. produce spores
최근 차세대염기서열분석(Next-generation sequencing; NGS) 기술의 빠른 발달로 인해 이를 이용하여 곰팡이를 비롯한 다양한 미생물의 유전체, 전사체 정보의 확보 및 미생물 동정 등을 손쉽게 할 수 있게 되었다. 특히 NGS와 생물정보학 분석을 통해 새롭게 분리된 곰팡이균의 정확한 동정이 가능하며, 관련 유전자 정보등을 손쉽게 확보할 수 있다. Due to the recent rapid development of next-generation sequencing (NGS) technology, it has become possible to easily obtain information about the genome and transcriptome of various microorganisms, including fungi, and to identify microorganisms. In particular, accurate identification of newly isolated fungi is possible through NGS and bioinformatics analysis, and related genetic information can be easily obtained.
정확한 곰팡이 동정은 곰팡이병을 예방하는데 매우 중요하다. 곰팡이병을 진단하는 가장 일반적인 방법으로는 곰팡이균 배양을 통한 방법이 있다. 이 방법의 경우 특이적인 배지에 자라는 곰팡이로부터 DNA를 추출한 후, 곰팡이 특이적인 internal-transcribed-space (ITS) 프라이머를 이용하여 PCR 증폭한다. 증폭된 PCR 산물은 클로닝(Cloning)과 Sanger-sequencing을 통해 염기서열을 결정하고, 새롭게 결정된 ITS 염기서열은 BLAST를 이용해 기존 곰팡이 ITS 데이타베이스에 유전자 서열을 비교한 후 새롭게 동정된 곰팡이 균의 종을 결정한다. 그러나, ITS 프라이머를 이용한 방법의 경우 곰팡이 종을 동정하는데 많은 시간이 걸리며, 때때로 식물기주에서도 ITS 유전자 부분이 증폭되기 때문에 곰팡이 진단에 많은 어려움이 있다. 따라서, 곰팡이를 효과적으로 진단하기 위해서는 곰팡이 종 특이적 유전자들을 이용한 진단방법 개발이 필수적이다. Accurate mold identification is very important in preventing fungal diseases. The most common method for diagnosing fungal diseases is through fungal culture. In this method, DNA is extracted from a mold growing in a specific medium, and then PCR amplified using a mold-specific internal-transcribed-space (ITS) primer. The amplified PCR product is sequenced through cloning and Sanger-sequencing, and the newly determined ITS sequence is compared to the existing mold ITS database using BLAST to determine the newly identified fungal species. decide However, in the case of the method using the ITS primer, it takes a lot of time to identify the fungal species, and since the ITS gene part is sometimes amplified even in the plant host, there are many difficulties in diagnosing the fungus. Therefore, in order to effectively diagnose mold, it is essential to develop a diagnostic method using fungal species-specific genes.
이에, 본 발명의 발명자들은 복숭아 감염 곰팡이의 진단 및 모니터링, 나아가 무병묘 복숭아 묘목 개발 및 복숭아 나무 및 과실의 피해 예방에 활용하고자, 다양한 지역에서 채취한 복숭아 나무로부터 5종의 곰팡이를 분리 동정하여, 5종의 복숭아 곰팡이 각각에 선택적, 특이적으로 결합 가능한 프라이머 쌍을 설계함으로써 본 발명을 완성하였다.Accordingly, the inventors of the present invention areolate and identify 5 types of fungi from peach trees harvested from various regions in order to diagnose and monitor peach infection fungus, further develop disease-free peach seedlings, and prevent damage to peach trees and fruits, The present invention was completed by designing a primer pair capable of selectively and specifically binding to each of the five types of peach mold.
본 발명의 하나의 목적은 시토스포라 레우코스토마(Cytospora leucostoma), 마크로포미나 파세올리나(Macrophomina phaseolina), 디아포르테 헬리안티(Diaporthe helianthi), 모닐리니아 프룩티콜라(Monilinia fructicola), 파이토프소라 캑토룸(Phytophthora cactorum)으로 이루어진 군으로부터 선택되는 하나 이상의 복숭아 곰팡이 진단용 프라이머 세트를 제공하는 것이다.One object of the present invention is Cytospora leucostoma ( Cytospora leucostoma ), Macrophomina phaseolina ( Macrophomina phaseolina ), Diaporte helianti ( Diaporthe helianthi ), Monilinia fructicola ( Monilinia fructicola ), phytope It is to provide a primer set for diagnosing one or more peach molds selected from the group consisting of Phytophthora cactorum .
본 발명의 다른 하나의 목적은 상기 프라이머 쌍을 포함하는 복숭아 곰팡이 진단용 조성물을 제공하는 것이다.Another object of the present invention is to provide a composition for diagnosis of peach fungus comprising the primer pair.
본 발명의 또 다른 하나의 목적은 상기 조성물을 포함하는 복숭아 곰팡이 진단용 키트를 제공하는 것이다.Another object of the present invention is to provide a kit for diagnosing peach fungus comprising the composition.
본 발명의 또 다른 하나의 목적은 시토스포라 레우코스토마(Cytospora leucostoma), 마크로포미나 파세올리나(Macrophomina phaseolina), 디아포르테 헬리안티(Diaporthe helianthi), 모닐리니아 프룩티콜라(Monilinia fructicola), 파이토프소라 캑토룸(Phytophthora cactorum)으로 이루어진 군으로부터 선택되는 하나 이상의 복숭아 곰팡이 진단 방법을 제공하는 것이다.Another object of the present invention is Cytospora leucostoma ( Cytospora leucostoma ), Macrophomina phaseolina ( Macrophomina phaseolina ), Diaporte helianthi ( Diaporthe helianthi ), Monilinia fructicola ( Monilinia fructicola ), To provide a method for diagnosing at least one peach mold selected from the group consisting of Phytophthora cactorum .
이를 구체적으로 설명하면 다음과 같다. 한편, 본 출원에서 개시된 각각의 설명 및 실시형태는 각각의 다른 설명 및 실시 형태에도 적용될 수 있다. 즉, 본 출원에서 개시된 다양한 요소들의 모든 조합이 본 출원의 범주에 속한다. 또한, 하기 기술된 구체적인 서술에 의하여 본 출원의 범주가 제한된다고 볼 수 없다.This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present application may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present application fall within the scope of the present application. In addition, it cannot be seen that the scope of the present application is limited by the detailed description described below.
상기 목적을 달성하기 위한 본 발명의 하나의 양태는 서열번호 1 및 서열번호 2로 표시되는 프라이머 쌍, 서열번호 3 및 서열번호 4로 표시되는 프라이머 쌍, 서열번호 5 및 서열번호 6으로 표시되는 프라이머 쌍, 서열번호 7 및 서열번호 8로 표시되는 프라이머 쌍, 및 서열번호 9 및 서열번호 10으로 표시되는 프라이머 쌍으로 이루어진 군으로부터 선택되는 하나 이상의 프라이머 쌍을 포함하는, 시토스포라 레우코스토마(Cytospora leucostoma), 마크로포미나 파세올리나(Macrophomina phaseolina), 디아포르테 헬리안티(Diaporthe helianthi), 모닐리니아 프룩티콜라(Monilinia fructicola), 파이토프소라 캑토룸(Phytophthora cactorum)으로 이루어진 군으로부터 선택되는 하나 이상의 복숭아 곰팡이 진단용 프라이머 세트를 제공한다.One aspect of the present invention for achieving the above object is a primer pair represented by SEQ ID NO: 1 and SEQ ID NO: 2, a primer pair represented by SEQ ID NO: 3 and SEQ ID NO: 4, primers represented by SEQ ID NO: 5 and SEQ ID NO: 6 Cytospora leucostoma ( Cytospora ) comprising at least one primer pair selected from the group consisting of a pair, a primer pair represented by SEQ ID NO: 7 and SEQ ID NO: 8, and a primer pair represented by SEQ ID NO: 9 and SEQ ID NO: 10 leucostoma ), Macrophomina phaseolina, Diaporthe helianthi , Monilinia fructicola , Phytophthora cactorum at least one selected from the group consisting of A primer set for diagnosing peach fungus is provided.
본 발명에서 용어 "프라이머"는 카피하려는 핵산 가닥에 상보적인 단일 가닥 올리고뉴클레오티드 서열을 말하며, 프라이머 연장 산물의 합성을 위한 개시점으로서 작용할 수 있다. 상기 프라이머의 길이 및 서열은 연장 산물의 합성을 시작하도록 허용해야 한다. 프라이머의 구체적인 길이 및 서열은 요구되는 DNA 또는 RNA 표적의 복합도(complexity)뿐만 아니라 온도 및 이온 강도와 같은 프라이머 이용 조건에 의존할 것이다.As used herein, the term "primer" refers to a single-stranded oligonucleotide sequence complementary to a nucleic acid strand to be copied, and can serve as a starting point for synthesis of a primer and an extension product. The length and sequence of the primers should allow the synthesis of extension products to begin. The specific length and sequence of the primer will depend on the conditions of use of the primer, such as temperature and ionic strength, as well as the complexity of the DNA or RNA target required.
본 발명에 있어서, 프라이머로서 이용된 올리고뉴클레오티드는 또한 뉴클레오티드 유사체(analogue), 예를들면, 포스포로티오에이트(phosphorothioate), 알킬포스포로티오에이트 또는 펩티드 핵산 (peptide nucleic acid)을 포함할 수 있거나 또는 삽입 물질(intercalating agent)을 포함할 수 있다. 또한, 프라이머는 DNA 합성의 개시점으로 작용하는 프라이머의 기본 성질을 변화시키지 않는 추가의 특징을 혼입할 수 있다. 본 발명의 프라이머 서열은 필요한 경우, 분광학적, 광화학적, 생화학적, 면역화학적 또는 화학적 수단에 의해 직접적으로 또는 간접적으로 검출 가능한 표지를 포함할 수 있다. 표지의 예로는, 효소(예를 들어, HRP (horse radish peroxidase), 알칼리 포스파타아제), 방사성 동위원소(예를 들어, 32P), 형광성 분자, 화학그룹(예를 들어, 비오틴) 등이 있다.In the present invention, the oligonucleotide used as a primer may also contain a nucleotide analogue, for example, a phosphorothioate, an alkylphosphorothioate or a peptide nucleic acid or An intercalating agent may be included. In addition, the primer may incorporate additional features that do not change the basic properties of the primer to serve as the starting point of DNA synthesis. If necessary, the primer sequence of the present invention may include a label detectable directly or indirectly by spectroscopic, photochemical, biochemical, immunochemical or chemical means. Examples of labels include enzymes (eg, horse radish peroxidase (HRP), alkaline phosphatase), radioactive isotopes (eg, 32 P), fluorescent molecules, chemical groups (eg, biotin), and the like. there is.
본 발명에서 용어 "복숭아 곰팡이"는 복숭아에 나타나는 질병의 원인이 되는 곰팡이를 총칭하는 것으로, 예컨대 시토스포라 레우코스토마(Cytospora leucostoma), 마크로포미나 파세올리나(Macrophomina phaseolina), 디아포르테 헬리안티(Diaporthe helianthi), 모닐리니아 프룩티콜라(Monilinia fructicola), 및 파이토프소라 캑토룸(Phytophthora cactorum) 등을 모두 포함하는 것일 수 있으며, 이에 특별히 제한되는 것은 아니다. In the present invention, the term "peach mold" is a generic term for molds that cause diseases appearing in peaches, for example, Cytospora leucostoma, Macrophomina phaseolina, Diaporte Helianti ( Diaporthe helianthi ), Monilinia fructicola ( Monilinia fructicola ), and Phytophthora cactorum ) It may include all of them, and is not particularly limited thereto.
구체적으로, 본 발명에서는 다양한 지역에서 채취한 복숭아 나무로부터 5종의 복숭아 곰팡이인 시토스포라 레우코스토마(Cytospora leucostoma), 마크로포미나 파세올리나(Macrophomina phaseolina), 디아포르테 헬리안티(Diaporthe helianthi), 모닐리니아 프룩티콜라(Monilinia fructicola), 및 파이토프소라 캑토룸(Phytophthora cactorum)을 동정하였다. 구체적으로, 동정된 곰팡이의 베타-튜불린(beta-tubulin) 유전자를 확보하여 이를 바탕으로 복숭아 곰팡이 진단용 프라이머 세트를 제작하였다. Specifically, in the present invention, five types of peach fungus, Cytospora leucostoma , Macrophomina phaseolina, Diaporthe helianthi , Monilinia fructicola , and Phytophthora cactorum were identified. Specifically, the identified fungal beta-tubulin (beta-tubulin) gene was obtained and based on this, a primer set for diagnosing peach fungus was prepared.
본 발명에서 복숭아 곰팡이 진단용은 복숭아 곰팡이 검출용을 의미한다.In the present invention, for peach mold diagnosis means for peach mold detection.
본 발명에서 상기 복숭아 곰팡이 진단용 프라이머는 PCR용 프라이머 쌍을 의미한다. 본 발명의 프라이머 쌍은 정방향 프라이머와 역방향 프라이머를 포함하며, 구체적으로는 시토스포라 레우코스토마(Cytospora leucostoma)를 진단할 수 있는 서열번호 1 및 서열번호 2로 표시되는 프라이머 쌍, 마크로포미나 파세올리나(Macrophomina phaseolina)를 진단할 수 있는 서열번호 3 및 서열번호 4로 표시되는 프라이머 쌍, 디아포르테 헬리안티(Diaporthe helianthi)를 진단할 수 있는 서열번호 5 및 서열번호 6으로 표시되는 프라이머 쌍, 모닐리니아 프룩티콜라(Monilinia fructicola)를 진단할 수 있는 서열번호 7 및 서열번호 8로 표시되는 프라이머 쌍, 및 파이토프소라 캑토룸(Phytophthora cactorum)을 진단할 수 있는 서열번호 9 및 서열번호 10을 진단할 수 있는 프라이머 쌍을 의미할 수 있다. 본 발명의 일 실시예에서는, 복숭아 나무의 줄기, 잎, 과실로부터 동정한 5종의 복숭아 곰팡이 각각에서 베타-튜불린(beta-tubulin) 유전자 정보를 확보하여, 이를 바탕으로 PCR 방법을 이용해 각 곰팡이를 특이적으로 정확하게 진단할 수 있는 프라이머 세트를 디자인하였다.In the present invention, the primer for diagnosis of peach fungus means a primer pair for PCR. The primer pair of the present invention includes a forward primer and a reverse primer, and specifically, a pair of primers represented by SEQ ID NO: 1 and SEQ ID NO: 2 capable of diagnosing Cytospora leucostoma , macroformina pase Olina ( Macrophomina phaseolina ) A pair of primers represented by SEQ ID NO: 3 and SEQ ID NO: 4 that can diagnose, Diaporthe helianthi ( Diaporthe helianthi ) A pair of primers represented by SEQ ID NO: 5 and SEQ ID NO: 6 that can diagnose Nilinia fructicola ( Monilinia fructicola ) A pair of primers represented by SEQ ID NO: 7 and SEQ ID NO: 8 capable of diagnosing, and Phytophthora cactorum ) capable of diagnosing SEQ ID NO: 9 and SEQ ID NO: 10 It may mean a pair of primers that can be diagnosed. In one embodiment of the present invention, beta-tubulin gene information is obtained from each of five types of peach molds identified from the stems, leaves, and fruits of the peach tree, and based on this, each mold is obtained using a PCR method. A primer set capable of diagnosing specifically and accurately was designed.
구체적으로, 서열번호 1 및 서열번호 2로 표시되는 프라이머 쌍은 시토스포라 레우코스토마(Cytospora leucostoma)의 베타-튜불린 유전자에 특이적으로 결합할 수 있는 프라이머 쌍이고, 서열번호 3 및 서열번호 4로 표시되는 프라이머 쌍은 마크로포미나 파세올리나(Macrophomina phaseolina)의 베타-튜불린 유전자에 특이적으로 결합할 수 있는 프라이머 쌍이고, 서열번호 5 및 서열번호 6으로 표시되는 프라이머 쌍은 디아포르테 헬리안티(Diaporthe helianthi)의 베타-튜불린 유전자에 특이적으로 결합할 수 있는 프라이머 쌍이고, 서열번호 7 및 서열번호 8로 표시되는 프라이머 쌍은 모닐리니아 프룩티콜라(Monilinia fructicola)의 베타-튜불린 유전자에 특이적으로 결합할 수 있는 프라이머 쌍이며, 서열번호 9 및 서열번호 10으로 표시되는 프라이머 쌍은 파이토프소라 캑토룸(Phytophthora cactorum)의 베타-튜불린 유전자에 특이적으로 결합할 수 있는 프라이머 쌍이다.Specifically, the primer pair represented by SEQ ID NO: 1 and SEQ ID NO: 2 is a primer pair capable of specifically binding to the beta-tubulin gene of Cytospora leucostoma , SEQ ID NO: 3 and SEQ ID NO: The primer pair represented by 4 is a primer pair capable of specifically binding to the beta-tubulin gene of Macrophomina phaseolina , and the primer pair represented by SEQ ID NO: 5 and SEQ ID NO: 6 is Diaporte heli Anti ( Diaporthe helianthi ) Beta- A primer pair capable of binding specifically to the tubulin gene, and the primer pair represented by SEQ ID NO: 7 and SEQ ID NO: 8 is Monilinia fructicola ( Monilinia fructicola ) Beta-tu It is a primer pair capable of specifically binding to a bulin gene, and the primer pair represented by SEQ ID NO: 9 and SEQ ID NO: 10 is a beta-tubulin gene of Phytophthora cactorum that can specifically bind a pair of primers.
상기 목적을 달성하기 위한 본 발명의 다른 하나의 양태는 서열번호 1 및 서열번호 2로 표시되는 프라이머 쌍, 서열번호 3 및 서열번호 4로 표시되는 프라이머 쌍, 서열번호 5 및 서열번호 6으로 표시되는 프라이머 쌍, 서열번호 7 및 서열번호 8로 표시되는 프라이머 쌍, 및 서열번호 9 및 서열번호 10으로 표시되는 프라이머 쌍으로 이루어진 군으로부터 선택되는 하나 이상의 프라이머 쌍을 포함하는, 시토스포라 레우코스토마(Cytospora leucostoma), 마크로포미나 파세올리나(Macrophomina phaseolina), 디아포르테 헬리안티(Diaporthe helianthi), 모닐리니아 프룩티콜라(Monilinia fructicola), 및 파이토프소라 캑토룸(Phytophthora cactorum)으로 이루어진 군으로부터 선택되는 하나 이상의 복숭아 곰팡이 진단용 조성물 및 이를 포함하는 복숭아 곰팡이 진단용 키트를 제공한다. Another aspect of the present invention for achieving the above object is a primer pair represented by SEQ ID NO: 1 and SEQ ID NO: 2, a primer pair represented by SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 Cytospora leucostoma ( Cytospora leucostoma ) , Macrophomina phaseolina , Diaporthe helianthi , Monilinia fructicola , and Phytophthora cactorum selected from the group consisting of Provided are a composition for diagnosing at least one peach mold and a kit for diagnosing peach mold comprising the same.
상기 키트는 역전사-중합효소연쇄반응(reverse transcription PCR) 키트, 중합효소 연쇄반응(PCR) 키트, 실시간 중합효소연쇄반응(real-time PCR) 키트, 실시간 중합효소연쇄반응 정량검사(RQ-PCR) 키트, 역 중합효소연쇄반응(inverse PCR) 키트 및 다중 중합효소연쇄반응(multiplex PCR) 키트로 이루어진 군으로부터 선택되는 하나 이상일 수 있으며, 구체적으로는 다중 중합효소연쇄반응(multiplex PCR) 키트일 수 있으나, 이에 특별히 제한되는 것은 아니다. 상기 다중 중합효소연쇄반응(multiplex PCR) 키트를 사용할 경우, 1회 PCR 반응 조건에서 실시하여도 검체 내에 존재하는 5종의 복숭아 곰팡이를 한번에 진단할 수 있다.The kit includes a reverse transcription-polymerase chain reaction (PCR) kit, a polymerase chain reaction (PCR) kit, a real-time PCR kit, a real-time polymerase chain reaction quantitative test (RQ-PCR) It may be at least one selected from the group consisting of a kit, an inverse PCR kit, and a multiplex PCR kit, and specifically, it may be a multiplex PCR kit. , but is not particularly limited thereto. In the case of using the multiplex PCR kit, it is possible to diagnose five types of peach mold present in a sample at once even if it is performed under one PCR reaction condition.
상기 조성물 및 키트는 서열번호 1 및 서열번호 2로 표시되는 프라이머 쌍, 서열번호 3 및 서열번호 4로 표시되는 프라이머 쌍, 서열번호 5 및 서열번호 6으로 표시되는 프라이머 쌍, 서열번호 7 및 서열번호 8로 표시되는 프라이머 쌍, 및 서열번호 9 및 서열번호 10으로 표시되는 프라이머 쌍으로 이루어진 군에서 선택되는 하나 이상의 프라이머 쌍과, 추가로 증폭 반응을 수행하기 위한 시약을 포함할 수 있다. 본 발명의 목적 상 상기 키트에서 증폭 반응을 수행하기 위한 시약으로 DNA 폴리머라제(polymerase), dNTPs, Taq 중합효소 및 PCR 완충액 등을 포함할 수 있으며, 또한 PCR 산물의 증폭 여부를 확인하기 위해 전기영동(electrophoresis)의 수행에 필요한 구성성분들이 본 발명의 키트에 추가로 포함될 수 있다. 나아가, 필요한 경우 RNase 억제제, DEPC-water 및 멸균수 등이 포함될 수 있다.The composition and kit are a primer pair represented by SEQ ID NO: 1 and SEQ ID NO: 2, a primer pair represented by SEQ ID NO: 3 and SEQ ID NO: 4, a primer pair represented by SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: It may include one or more primer pairs selected from the group consisting of a primer pair represented by 8, and a primer pair represented by SEQ ID NO: 9 and SEQ ID NO: 10, and a reagent for further performing an amplification reaction. For the purpose of the present invention, DNA polymerase, dNTPs, Taq polymerase and PCR buffer may be included as reagents for performing the amplification reaction in the kit, and electrophoresis to confirm whether the PCR product is amplified Components necessary for performing (electrophoresis) may be additionally included in the kit of the present invention. Furthermore, if necessary, RNase inhibitors, DEPC-water and sterile water may be included.
상기 목적을 달성하기 위한 본 발명의 또 다른 하나의 양태는 (a) 시료로부터 복숭아 곰팡이 핵산을 분리하는 단계; (b) 상기 (a) 단계에서 분리된 핵산을 주형으로 제1항의 프라이머 쌍을 이용하여 표적 핵산을 증폭하는 단계; 및 (c) 상기 (b) 단계에서 증폭된 표적 핵산을 검출하는 단계를 포함하는 것을 특징으로 하는, 시토스포라 레우코스토마(Cytospora leucostoma), 마크로포미나 파세올리나(Macrophomina phaseolina), 디아포르테 헬리안티(Diaporthe helianthi), 모닐리니아 프룩티콜라(Monilinia fructicola), 파이토프소라 캑토룸(Phytophthora cactorum)으로 이루어진 군으로부터 선택되는 하나 이상의 복숭아 곰팡이 진단 방법을 제공한다.Another aspect of the present invention for achieving the above object is (a) isolating the peach fungal nucleic acid from the sample; (b) amplifying a target nucleic acid using the primer pair of
상기 (a) 단계의 시료는 본 발명의 복숭아 곰팡이 존재 여부를 확인하기 위해 필요한 핵산, 구체적으로는 RNA를 추출하기 위한 미지의 물질을 의미하는 것으로, 예컨대 상기 바이러스가 감염된 복숭아 나무의 잎, 줄기, 과실, 뿌리 등을 포함할 수 있으며, 그밖에도 식품, 천연물(예컨대, 물) 또는 생물학적 시료 등을 포함할 수 있으나, 이에 제한되는 것은 아니다. The sample of step (a) refers to an unknown material for extracting nucleic acids, specifically RNA, necessary to confirm the presence of the peach mold of the present invention, for example, leaves, stems, It may include fruits, roots, and the like, and may include, but is not limited to, food, natural products (eg, water) or biological samples.
상기 (b) 표적 핵산을 증폭하는 단계는 역전사-중합효소연쇄반응(reverse transcription PCR) 키트, 중합효소 연쇄반응(PCR) 키트, 실시간 중합효소연쇄반응(real-time PCR) 키트, 실시간 중합효소연쇄반응 정량검사(RQ-PCR) 키트, 역 중합효소연쇄반응(inverse PCR) 키트 및 다중 중합효소연쇄반응(multiplex PCR)로 이루어진 군으로부터 선택된 하나 이상을 이용하여 수행할 수 있으며, 구체적으로 다중 중합효소연쇄반응(multiplex PCR)을 이용하여 수행할 수 있으나, 이에 제한되는 것은 아니다. 본 발명의 일 실시예에서는, PCR 조건으로 94℃에서 3분, 이어서 94℃에서 20초, 60℃에서 40 초 (어닐링 온도는 모든 프라이머에서 동일함), 및 72℃에서 40초 동안 35회 사이클이었으며, 72℃에서 5분 동안 최종 신장(final extension)하였다.The step of (b) amplifying the target nucleic acid is reverse transcription-polymerase chain reaction (reverse transcription PCR) kit, polymerase chain reaction (PCR) kit, real-time polymerase chain reaction (real-time PCR) kit, real-time polymerase chain It can be carried out using at least one selected from the group consisting of a reaction quantitative test (RQ-PCR) kit, an inverse PCR kit, and a multiplex PCR, specifically, a multiplex polymerase It may be performed using a multiplex PCR, but is not limited thereto. In one embodiment of the present invention, 35 cycles of PCR conditions at 94°C for 3 minutes, then at 94°C for 20 seconds, at 60°C for 40 seconds (annealing temperature is the same for all primers), and at 72°C for 40 seconds and final extension at 72° C. for 5 minutes.
상기 (c) 단계에서 증폭된 표적 핵산의 검출은 모세관 전기영동(capillary electrophoresis), DNA 칩, 겔 전기영동(gel electrophoresis), 방사성 측정, 형광 측정 및 인광 측정으로 이루어진 군에서 선택된 하나 이상의 방법을 이용하여 수행될 수 있으며, 이에 특별히 제한되는 것은 아니다. 본 발명의 일 실시예에서는, 증폭된 PCR 산물을 겔 전기 영동에 이어 EtBr 염색으로 확인하였으며, 나아가 pGEM-T-Easy Vector(Promega, Wisconsin, US)에서 증폭된 PCR 산물을 클로닝 한 후 Sanger 시퀀싱하여 증폭된 PCR 산물의 서열을 확인하였다.The detection of the target nucleic acid amplified in step (c) uses one or more methods selected from the group consisting of capillary electrophoresis, DNA chip, gel electrophoresis, radiometric measurement, fluorescence measurement, and phosphorescence measurement. may be carried out, and is not particularly limited thereto. In one embodiment of the present invention, the amplified PCR product was confirmed by gel electrophoresis followed by EtBr staining, and further, the PCR product amplified in pGEM-T-Easy Vector (Promega, Wisconsin, US) was cloned and then Sanger sequencing was performed. The sequence of the amplified PCR product was confirmed.
본 발명은 다양한 지역에서 채취한 복숭아 나무로부터 5종의 곰팡이를 분리 동정하였으며, 직접 확보한 5종의 복숭아 곰팡이 각각에 선택적, 특이적으로 결합 가능한 프라이머 쌍을 설계하여 진단의 신속성 및 정확성을 높일 수 있는바, 복숭아 곰팡이 진단, 무병묘 복숭아 묘목 개발 및 복숭아 나무 및 과실의 피해 예방에 유용하게 활용될 수 있다.The present invention isolates and identifies 5 types of fungi from peach trees harvested from various regions, and can increase the speed and accuracy of diagnosis by designing primer pairs that can selectively and specifically bind to each of the 5 types of directly secured peach fungi. It can be usefully used to diagnose peach fungus, develop disease-free peach seedlings, and prevent damage to peach trees and fruits.
도 1은 5 종의 복숭아 곰팡이로부터 추출한 DNA, template 및 디자인한 곰팡이 특이적 프라이머를 사용하여 얻어진 증폭된 PCR 산물을 1% 아가로스젤 전기영동을 통해 확인한 결과를 나타낸 것이다. M은 100 bp DNA 마커를 나타내고, 1번부터 5번 레인은 각각 시토스포라 레우코스토마(Cytospora leucostoma), 마크로포미나 파세올리나(Macrophomina phaseolina), 디아포르테 헬리안티(Diaporthe helianthi), 모닐리니아 프룩티콜라(Monilinia fructicola), 및 파이토프소라 캑토룸(Phytophthora cactorum)을 나타내며, 화살표는 곰팡이 특이적 프라이머로부터 증폭된 PCR 산물을 표시한다.1 shows the results of confirming the amplified PCR product obtained by using the DNA, template, and designed mold-specific primers extracted from 5 kinds of peach mold through 1% agarose gel electrophoresis. M represents a 100 bp DNA marker, and
이하, 본 발명을 하기 실시예를 통하여 보다 상세하게 설명한다. 그러나 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예만으로 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.
실시예 1: 복숭아 곰팡이 연구를 위한 샘플 수집 및 곰팡이 분리 동정Example 1 Sample Collection and Fungal Isolation Identification for Peach Fungus Study
복숭아 감염 곰팡이를 식별하고자, 5개 지역(경북 상주, 전북 전주, 전남 장성, 전북 완주 및 경기도 장호원)에서 줄기마름병, 줄기썩음병, 잿빛무늬병 또는 역병 증상을 보이는 복숭아 줄기들을 수집하였다. 수집된 샘플에서 총 5종의 곰팡이 시토스포라 레우코스토마(Cytospora leucostoma), 마크로포미나 파세올리나(Macrophomina phaseolina), 디아포르테 헬리안티(Diaporthe helianthi), 모닐리니아 프룩티콜라(Monilinia fructicola), 및 파이토프소라 캑토룸(Phytophthora cactorum)을 분리 동정하였고(표 1), 이렇게 분리 동정된 곰팡이들은 total RNA 추출에 사용하였다. To identify the peach-infecting fungus, peach stems showing symptoms of stem blight, stem rot, gray blotch or late blight were collected from 5 regions (Gyeongbuk Sangju, Jeonbuk Jeonju, Jeonnam Jangseong, Jeonbuk Wanju and Gyeonggi Province Janghowon). In the collected samples, a total of five types of fungi Cytospora leucostoma , Macrophomina phaseolina, Diaporthe helianthi , Monilinia fructicola , and Phytophthora cactorum were isolated and identified (Table 1), and the isolated and identified fungi were used for total RNA extraction.
실시예 2: Total RNA 추출 및 라이브러리 제작Example 2: Total RNA extraction and library construction
분리 동정된 5종의 각 곰팡이들을 potato dextrose agar (PDA)배지에서 28°C에서 키운 후 곰팡이 균사체(mycelia)를 수확하여 RNeasy Plant Mini Kit (Qiagen, Hilden, Germany)을 이용하여 총 RNA를 추출하였다. 추출한 총 RNA를 기초로 Illumina사의 TruSeq RNA Library Prep Kit v2를 이용하여 라이브러리를 제작하였다. 제작된 라이브러리는 Illumina사의 NovaSeq6000 시스템을 이용하여 paired-end (100bp X 2) sequencing을 수행하였다.Each of the five isolated and identified fungi was grown at 28 °C in potato dextrose agar (PDA) medium, and the fungal mycelia were harvested and total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). . Based on the extracted total RNA, a library was prepared using Illumina's TruSeq RNA Library Prep Kit v2. The prepared library was subjected to paired-end (100bp X 2) sequencing using Illumina's NovaSeq6000 system.
실시예 3: 전사체 조립 및 곰팡이 식별Example 3: Transcript assembly and fungal identification
각 곰팡이로부터 얻어진 raw sequence data는 Trinity 프로그램에서 전사체 조립을 위해 사용되었다. 각 곰팡이별로 조립된 콘티그(contigs)를 확인하기 위해, NCBI의 non-redundant (NR) 단백질 데이터베이스에서 DIAMOND 프로그램을 이용해 BLAST를 수행하였다. MEGAN6 프로그램을 이용해 BLAST결과를 분석하여 5종 곰팡이의 정확한 종을 확인하였다.Raw sequence data from each fungus was used for transcript assembly in the Trinity program. BLAST was performed using the DIAMOND program in NCBI's non-redundant (NR) protein database to identify the assembled contigs for each fungus. BLAST results were analyzed using the MEGAN6 program to confirm the exact species of 5 types of mold.
실시예 4: 복숭아 곰팡이의 베타-튜불린(beta-tubulin) 유전자 동정Example 4: Beta-tubulin gene identification of peach fungus
BLAST 결과를 이용하여 각 곰팡이별로 베타-튜불린 유전자를 동정하였다. 그 결과, 시토스포라 레우코스토마(Cytospora leucostoma), 마크로포미나 파세올리나(Macrophomina phaseolina), 디아포르테 헬리안티(Diaporthe helianthi), 모닐리니아 프룩티콜라(Monilinia fructicola), 및 파이토프소라 캑토룸(Phytophthora cactorum)의 베타-튜불린 유전자는 각각 서열번호 11 내지 15인 것으로 확인되었다. 각 곰팡이 전사체로부터 동정된 beta-tubulin partial 유전자의 크기는 1,672 bp에서 2,427 bp 이었으며, 각 beta-tubulin 유전자 서열을 가지고, NCBI nucleotide 데이터베이스에 BLASTN를 수행하였다(표 2).Using BLAST results, beta-tubulin genes were identified for each fungus. As a result, Cytospora leucostoma ( Cytospora leucostoma ) , Macrophomina phaseolina ( Macrophomina phaseolina ), Diaporte helianti ( Diaporthe helianthi ), Monilinia fructicola ( Monilinia fructicola ), and Phytope Sora cactorum ( Phytophthora cactorum ) Beta-tubulin gene was confirmed to be SEQ ID NOs: 11 to 15, respectively. The size of the beta-tubulin partial gene identified from each fungal transcript was 1,672 bp to 2,427 bp, and BLASTN was performed on the NCBI nucleotide database with each beta-tubulin gene sequence (Table 2).
수행결과 Cytospora leucostoma의 경우 40%의 낮은 coverage로 기존 Cytospora sp.과 유사성을 보여주었다. Macrophomina phaseolina는 Paracoccidioides brasiliensis와 73% coverage와 77.29% 염기서열 identity를 보여주었다. Diaporthe helianthi 의 경우 Colletotrichum scovillei 와 65%의 낮은 coverage를 보여주었다. Monillinia fructicola와 Phytophthora cactorum는 각각 Botrytis cinerea와 Phytophthora parasitica에 99%의 높은 염기서열 coverage를 보여주었지만, 염기서열의 identity는 93.72%와 90.37%로 밝혀졌다. 이 결과들을 보았을 때 본 연구에서 동정된 곰팡이는 기존 곰팡이와 매우 다른 것을 알 수 있었으며, 특히 5종 곰팡이로 동정된 beta-tublin 유전자 정보가 현재 NCBI GenBank 데이터베이스에 전혀 없다는 것을 확인할 수 있었다.As a result, Cytospora leucostoma showed similarity to the existing Cytospora sp. with a low coverage of 40%. Macrophomina phaseolina showed 73% coverage and 77.29% sequence identity with Paracoccidioides brasiliensis . Diaporthe helianthi showed a lower coverage of 65% than Colletotrichum scovillei . Monillinia fructicola and Phytophthora cactorum showed high sequence coverage of 99% to Botrytis cinerea and Phytophthora parasitica , respectively, but sequence identity was found to be 93.72% and 90.37%. Looking at these results, it was found that the fungus identified in this study was very different from the existing mold, and in particular, it could be confirmed that the beta-tublin gene information identified as 5 types of fungi was not present in the NCBI GenBank database at all.
실시예 5: 5종 복숭아 곰팡이의 특이적 진단을 위한 PCR 프라이머 개발Example 5: Development of PCR primers for specific diagnosis of 5 types of peach fungus
5종 복숭아 곰팡이 전사체 분석을 통해 각 곰팡이별로 베타-튜불린(Beta-tubulin) 유전자들을 동정하여 유전자서열을 확보하였다. 확보된 베타-튜불린 유전자 서열을 바탕으로 각 곰팡이를 특이적으로 진단할 수 있는 PCR 프라이머를 디자인하였다. 5종의 곰팡이들을 하나의 PCR 반응을 통해 진단할 수 있는 Multiplex PCR을 위해 증폭되는 PCR 산물의 길이를 서로 다르게 하여 다양한 프라이머들을 디자인하였다.Beta-tubulin genes were identified for each fungus by analyzing the transcriptome of 5 types of peach mold, and the gene sequence was obtained. Based on the obtained beta-tubulin gene sequence, PCR primers capable of specifically diagnosing each fungus were designed. For Multiplex PCR, which can diagnose 5 types of fungi through a single PCR reaction, various primers were designed by varying the length of the PCR product to be amplified.
곰팡이로부터 추출한 DNA를 template과 상기 디자인된 프라이머들을 이용하여 PCR을 수행하였다. PCR 결과를 바탕으로 각 곰팡이 특이적으로 증폭되는 프라이머를 최종적으로 선발하였다. PCR was performed on the DNA extracted from the fungus using the template and the designed primers. Based on the PCR results, primers to be amplified specifically for each fungus were finally selected.
PCR 반응을 수행하기 위해, 50 ml PCR 튜브에 DNA template 1 μL, Forward primer (10 pmol/ul) 1 μL, Reverse primer (10 pmol/ul) 1 μL, 10 X Buffer 3 μL, dNTP (2.5 mM) 4 μL, EX-Taq 0.5 μL를 넣고 최종적으로 증류수로 최종 볼륨을 30 μL로 맞췄다. Genomic DNA의 경우 DNeasy Plant Mini Kit (Qiagen, Hilden, Germany)를 사용하였다. PCR은 Solg 2X Multiplex PCR Series (SolGent, Daejeon, Korea)를 사용하였다. 곰팡이 PCR 조건은 94℃ 에서 3분, 이어서 94℃에서 20초, 60℃에서 40초 (어닐링 온도는 모든 프라이머에서 동일함), 및 72℃에서 40초 동안 35 사이클이었으며, 72℃에서 5분 동안 최종 신장(final extension)하였다. To perform the PCR reaction, in a 50 ml PCR tube, 1 µL of DNA template, 1 µL of Forward primer (10 pmol/ul), 1 µL of Reverse primer (10 pmol/ul), 3 µL of 10 X Buffer, 3 µL of dNTP (2.5 mM) Add 4 μL, 0.5 μL of EX-Taq, and finally adjust the final volume to 30 μL with distilled water. For genomic DNA, DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) was used. For PCR, Solg 2X Multiplex PCR Series (SolGent, Daejeon, Korea) was used. Fungal PCR conditions were 35 cycles of 94 °C for 3 min, followed by 94 °C for 20 s, 60 °C for 40 s (annealing temperature was the same for all primers), and 72 °C for 40 s, followed by 35 cycles at 72 °C for 5 min. It was final extension (final extension).
증폭된 PCR 결과물들은 전기 영동 후 EtBr 염색을 통해 확인하였다(도 1). PCR 수행 결과 각 곰팡이 특이적 프라이머들은 타겟 곰팡이에서만 PCR 증폭산물(amplicon)을 나타내었다. 또한 증폭된 PCR 산물 사이즈는 시토스포라 레우코스토마(Cytospora leucostoma) 100 bp, 마크로포미나 파세올리나(Macrophomina phaseolina) 549 bp, 디아포르테 헬리안티(Diaporthe helianthi) 200 bp, 모닐리니아 프룩티콜라(Monilinia fructicola) 300 bp, 파이토프소라 캑토룸(Phytophthora cactorum) 400 bp로 나타났다. 추가적으로 증폭된 모든 PCR 산물들을 pGEM-T-Easy Vector (Promega, Wisconsin, US)에서 클로닝 한 후 Sanger 시퀀싱하여 증폭된 PCR 산물의 서열을 확인하였다. 곰팡이 특이적 프라이머로 증폭된 베타-튜불린 유전자의 partial 염기서열은 서열번호 16 내지 20에 나타내었다.The amplified PCR products were confirmed by EtBr staining after electrophoresis (FIG. 1). As a result of PCR, each mold-specific primer showed PCR amplification only in the target mold. In addition, the amplified PCR product size is
최종적으로, PCR 결과를 바탕으로 선발된 복숭아 곰팡이 특이적 프라이머를 아래 표 3에 나타내었다.Finally, the peach mold-specific primers selected based on the PCR results are shown in Table 3 below.
이상의 설명으로부터, 본 발명이 속하는 기술분야의 당업자는 본 발명이 그 기술적 사상이나 필수적 특징을 변경하지 않고서 다른 구체적인 형태로 실시될 수 있다는 것을 이해할 수 있을 것이다. 이와 관련하여, 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적인 것이 아닌 것으로 이해해야만 한다. 본 발명의 범위는 상기 상세한 설명보다는 후술하는 특허 청구범위의 의미 및 범위 그리고 그 등가 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims to be described later and their equivalents.
<110> Seoul National University R&DB Foundation <120> Primer sets for diagnosing five fungi infecting peach and diagnostic methods using thereof <130> KPA01554 <160> 20 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 18-484-tub-F2 <400> 1 ccaagggtct caagatgtcc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 18-484-tub-R2 <400> 2 cgacggaaca tagcactgaa 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 19-016-tub-F1 <400> 3 atcgcatgat ggctacgttt 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 19-016-tub-R1 <400> 4 ttcgacctcc tttgtggaaa 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 19-168-tubb-F2 <400> 5 tcttttctcc cagacccaga 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 19-168-tubb-R2 <400> 6 aggttggacg acgctattgt 20 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 19-382-tub-F3 <400> 7 ttgagtggat ccctaacaac g 21 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 19-382-tub-R3 <400> 8 gggcctcctc ttcatactcc 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 19-435-tub-F3 <400> 9 cacgctagcc ttgatgttgt 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 19-435-tub-R3 <400> 10 cacctacggt gacctgaacc 20 <210> 11 <211> 2427 <212> DNA <213> Unknown <220> <223> Cytospora leucostoma_18-484_beta-tubulin <400> 11 ccacctccag accggccaat gcggtaacca aatcggtgct gctttctggt gcgtaccagc 60 cttcgaccac ggcagcaaac gatacgcgac agtaatcatg tcgaaggatt gaaactgacc 120 ccgattaaac aggcaaacca tctctggcga gcacggtctc gacagcaatg gagtttacaa 180 cggcacttct gagctccagc tggagcgcat gaacgtctac ttcaacgagg cttccggcaa 240 caagtatgtt cctcgcgctg tcctcatcga tctcgagccc ggtaccatgg acgccgtccg 300 tgccggtcct ttcggccagc tgttccgtcc cgacaacttc gttttcggcc agtccggtgc 360 tggaaacaac tgggccaagg gtcactacac tgagggtgcg gagctggtcg accaggtcct 420 ggatgtcgtc cgtcgcgagg ctgagggttg cgactgcctg cagggcttcc agatcaccca 480 ctccctgggt ggtggtaccg gtgccggtat gggtactctg ctcatctcca agattcgcga 540 ggagttcccc gaccgcatga tggctacctt ctccgtcatg ccctctccca aggtctccga 600 caccgtcgtt gagccctaca acgccaccct ctccgtccac cagctggtcg agaactcgga 660 cgagactttc tgtatcgaca acgaggccct ctacgacatt tgcatgcgga cgctgaagct 720 gtccaacccc tcttacggtg accttaacta cctggtgtcg gctgtcatgt ccggcgtcac 780 cgtctcgctg cgtttccccg gtcagctgaa ctctgacctc cgcaagctgg ctgtgaacat 840 ggtgccgttc cctcgtctgc acttcttcat ggttggcttc gctcccctga ccagccgtgg 900 cgcccactcc ttccgcgctg tcaccgtccc tgagctcacc cagcagatgt tcgaccccaa 960 gaacatgatg gctgcgtctg acttccgtaa cggtcgctac ctgacgtgct ctgccatctt 1020 gtgagtcctc ccgcccccaa gtgtctctat ccgtgcttgt ggtgctaatt atgtttccag 1080 ccgtggcaag gtctccatga aggaggttga ggaccagatg cgcaacgtcc agagcaagaa 1140 ctcgtcctac ttcgtcgagt ggatccccaa caacgtccag accgccctct gctctattcc 1200 ccccaagggt ctcaagatgt cctcgacctt cgttggtaac tcgactgcta tccaggagct 1260 gttcaagcgt gtcggcgagc agttcagtgc tatgttccgt cgcaaggctt tcttgcattg 1320 gtacactggt gagggtatgg acgagatgga gttcaccgag gctgagtcca acatgaacga 1380 tcttgtctcg gaataccagc agtaccagga tgccagcatc gacgaggagg aggaggagta 1440 cgaggaggag gctccccttg aggaggagtg aactatggtg tgattaaaaa gtgtttcccg 1500 gcggtcttga tggccaagga cctatgtcca taatttgtca ccttgggtac ccacggcact 1560 tcccgcatgt attctgcttg tgtgggttgt gtgtcacgtt tgcgtgggta gctatgcgat 1620 ccggagatgg gttctggtaa tcgattggcc gtatgaacaa agggacacaa cattgatttg 1680 aaatatacct cttgtagttg accctttacc acgaatacaa aatcggacaa agccgtccac 1740 atgatagccc caagcgtgtg tgatacaata tcagtagtaa cgatttgcca aacagaggcc 1800 aagattgtct taatccactt gcttgagtcc ataccacagg ggaccttgca ccgcctcttc 1860 tacgtaggta ttgcaggtaa tcctagggca gtgcagttga ctttgatagt cgcttgaggc 1920 tggtagtatg ttccactatc gtgatctagc ccatggcacc cacaactggt caaggagagg 1980 ctgaaaggca tcgccgactt tcagcggcag gcggccgacc agcggcgtaa actggaaatg 2040 tagattgtgc gcgaggtcac ccgagatcaa aatctcatgc tgatcagcgt atcaggcgcc 2100 tattaagcgc tatagtgact gactgactgc accgatatgt agtaggtagg taggcccagc 2160 ccaccttagg gacagattac aggcaccgaa ccggatacct actaagtcgt caggtatgta 2220 atgtgtgaat ctgtgtgggc ttcacatagg ctaagttagg tagtgtagat aagcggcaag 2280 gaggatgcgg cattgccatg cgttgatcaa taaatcctcc tagtcactca tctttccccc 2340 ttctgttata gatctgatgt aagcgctgaa ggaggtgaga ggcagcctgc gtgtcagttt 2400 tcttacggta tagtgtatgc gtgtgac 2427 <210> 12 <211> 1705 <212> DNA <213> Unknown <220> <223> Macrophomina phaseolina_19-016_beta-tubulin <400> 12 cgtctgcaac caatttgcgc tgtccacctc acagaaccca caacagacgt gatcaattcg 60 tcacccgctg ccaatatgcg cgagattatc catgttcagg ctggccaatg tggtaaccaa 120 atcggatctg ctttctggca gacgatctgc aaagagcatg gtgtcgatag ctctggaaac 180 tacaacggca catcggatct ccaactcgag cgcatgaatg tctatttcaa tgagccttcg 240 agcaagaaat tcgtaccccg cgccgtgctc gttgatcttg aacccggcac catggatgcc 300 gttcgtgccg gcccagctgg acggctcttc cgccccgaca attttgtttt cgggcagtct 360 ggagccggaa acaactgggc aaaaggccat tacaccgaag gcgctgagct ggtggatcag 420 gttcttgatg ttgtgcgccg ggagtccgaa ggttgcgatt ccctgcaagg gtttcaaatc 480 actcattcgt tgggcggcgg cactggctcc ggcatgggca ccttgctcac ggctaagatt 540 cgggaagagt ttccggatcg catgatggct acgttttcgg tcatgccttc gccgaaggtg 600 tccgaggtcg ttgttgagcc ctacaatgct actctgtcga ttcatgagtt ggttgaaaat 660 gccgatgaga cgttctgcat cgacaacgag gcgctgtatg atatctgtag gcgcacgctc 720 aaactcgacc atccatcgta cggagatctc aactcgctcg tgtccaccgt catgtccggc 780 gtcaccaccg gcttccgctt tcctggccaa ctcaattcag atctccggaa actggccgtg 840 aacatggtcc cgtttcctcg tcttcacttc tttacggttg gctttgcgcc tctcacggcc 900 cgcggtggca gctcattccg caattcatcc gtgtcagact tgacgcagca actgttcgac 960 ccaaggaaca tgatggccgc cgccgacttc cggaatggca ggtacttgac gtgctcgact 1020 ttcttccgtg gcaacgtttc cacaaaggag gtcgaagatc aaatgatcaa gttgcaagac 1080 aagaactctt cctatttcgt cgaatggatt ccgaacaatt tccaaaccgc cgtgtgctcc 1140 gtccccccgc caggcttgaa aatgtctgcc acctttgttg ggaataccac ttctattcag 1200 gagctcttca agcgtgtcgg agatcagttc actgccatgt tccgccgcaa agccttcttg 1260 cattggtata ccgccgaagg aatggacgag atggaattca cggaggcaga gtccaatatg 1320 aatgatctcg tggcggagta tcagcagtac caggaggcct cgatcagcga caccgaagac 1380 tacgatgagg aagctccatt ggaagaggag gaggagtgag gaagtgtacg acaaacgtta 1440 ctctgattgt ctgtcgtcat ttgttgcatc tttgagaaga caagaaccat atagcgtacc 1500 actagcgaat catcattgaa ttgcgccaac gtatcacctc attcatctcg ttgaccatga 1560 tccgtcctat gtacctattc aaacttcaaa ttgggcattt ttcgtagtag atcttgcacc 1620 tccaaggcgc gcagaattgc tccttagtat aggaagacct tagcgttact taaaaaaggc 1680 aacgcgcgat gatgctcgcc gtgcc 1705 <210> 13 <211> 2050 <212> DNA <213> Unknown <220> <223> Diaporthe helianthi_19-168_beta-tubulin <400> 13 ctcttccgat ctttcacccc tggcacctgg cacctggcac tggcagcagc agcagctccg 60 gcacaacaga gtgaaagagg gaaagcgaag ggcctccact ccactctttt ctcccagacc 120 cagactggcc ctgccagaca atccatccca atcccaacac caccaccgtc gccgccgata 180 cccaaacctc accttctcaa tctcgctccg agctaccggc ccttcgtctt ggacctcgct 240 gctctcgttt gtctaccgcc attcttctgt ctcgacccga ctcgacaata gcgtcgtcca 300 accttcaaaa tgcgtgagat tgttcacctt cagaccggcc aatgcggtaa ccaaatcggt 360 gctgctttct ggcaaaccat ctctggcgag cacggcctcg acagcaatgg cgtttacaac 420 ggcacttctg agctccagct cgagcgcatg aacgtctact tcaacgaggc ctccggcaac 480 aagtatgttc ctcgcgccgt cctcgtcgat ctcgagcccg gtaccatgga cgccgtccgt 540 gccggtccct ttggccagct cttccgcccc gacaacttcg tcttcggcca gtccggtgct 600 ggaaacaact gggccaaggg tcactacact gagggtgctg agctggtcga ccaggtcctc 660 gatgtcgtcc gtcgcgaggc tgagggctgc gactgcctcc agggcttcca gatcacccac 720 tccctgggtg gtggtactgg tgctggtatg ggtacgctgc ttatctccaa gatccgcgag 780 gagttccccg accgcatgat ggccaccttc tccgtcgtgc cctcccccaa ggtctccgac 840 accgtcgtcg agccctacaa cgccaccctg tccgtccacc agctggtcga gaactcggac 900 gagaccttct gcatcgacaa cgaggccctg tacgacatct gcatgcgtac gctcaagctg 960 tccaacccct cctacggcga cctgaactac ctggtgtcgg ctgtcatgtc cggcgtcacc 1020 gtctccctgc gtttccccgg tcagctgaac tctgacctgc gcaagctggc cgtgaacatg 1080 gtgcccttcc ctcgtctgca cttcttcatg gtcggctttg ctcccctgac cagccgtggc 1140 gcccactcct tccgcgccgt taccgttcct gagctcaccc agcagatgtt cgaccccaag 1200 aacatgatgg ctgcctctga cttccgtaac ggtcgctacc tgacgtgctc tgctatcttc 1260 cgtggaaagg tctccatgaa ggaggtcgag gaccagatgc gcaacgtcca gagcaagaac 1320 tcatcctact tcgtcgagtg gatccccaac aacgtccaga ccgccctctg ctcgatccct 1380 cccaagggcc tcaagatgtc ctctaccttc gttggtaact cgactgctat ccaggagctg 1440 ttcaagcgtg tgggtgagca gttcactgcc atgttccggc gcaaggcttt cttgcattgg 1500 tacactggtg agggtatgga cgagatggag ttcaccgagg ctgagtccaa catgaacgat 1560 ctggtctccg aataccagca gtaccaggac gcctccattg atgaggagga ggaggagtac 1620 gaggaggagg ttccccttga gggcgaggag tgaatagtgg tctgatgaga tctgctgtct 1680 ggattggttc aatgatctag gactctgcta ccgtccttaa tccgtcatca ttgtacccct 1740 gacacttctc actcatttct tgtgtgcgtg tgcgcgcgtg tgcgaatgtg tgggacgctt 1800 gaagctggtg tgaatagcta ttcgattcat gattcgggag gagcgggggt gttctggtaa 1860 ttattgaatg acagacccgg ccgatcgtcc gaagaagaga gcagagcaaa aaggggaaac 1920 attgatttta aaaatatacc tctttgtagt tgaaccctgt gcccacatcc ggcctgccag 1980 tttcgcaccc tttcatgcat acatgcaacc acgtttccat ttggaatgaa attcacagga 2040 tgattgcttc 2050 <210> 14 <211> 1767 <212> DNA <213> Unknown <220> <223> Monillinia fructicola_19-382_beta-tubulin <400> 14 ctccactatc cacaaggaca tctcagcaat ccttaacctc ttactttctc tatctacaac 60 ctcgacttct caatatccaa actaccatac tttaacacct caacacaaga tcctaaatct 120 accttcaaaa tgcgtgagat tgttcatctt caaaccggtc aatgtggtaa ccaaattggt 180 gctgctttct ggcaaactat ctctggcgag cacggtcttg atggctctgg tgtctacaat 240 ggtacctccg acctccaact tgagcgtatg aacgtctact tcaacgaggc ttccggcaac 300 aagtatgttc cccgtgccgt tctcgtcgat ttggagccag gtaccatgga cgctgtccgt 360 gccggtcctt tcggtcaact cttccgcccc gataacttcg tcttcggtca atccggtgct 420 ggtaacaact gggctaaggg tcattacact gagggtgctg agcttgttga ccaagttctt 480 gatgttgttc gtcgtgaagc tgaaggctgt gactgccttc aaggtttcca aatcacccac 540 tctctcggtg gtggaactgg tgccggtatg ggtacgcttt tgatctccaa gatccgtgag 600 gagttccccg atcgtatgat ggctaccttt tctgtcgttc catcgccaaa ggtttccgat 660 accgttgtcg agccatataa cgctaccctc tccgttcatc aattggtaga aaactctgat 720 gaaaccttct gtatcgataa cgaggctctt tacgacatct gcatgagaac cttgaagctc 780 agcaacccat cctacggaga tcttaaccac ttggtttctg ctgtcatgtc cggtgttacc 840 acctgtctcc gtttccctgg tcaacttaac tcagatctcc gaaagttggc tgttaacatg 900 gttccattcc cacgtctcca tttcttcatg gttggatttg ctcctttgac cagccgtggt 960 gcacactctt tccgtgctgt taccgttcca gagttgactc aacaaatgta cgaccctaag 1020 aacatgatgg ccgcttccga tttccgtaac ggtcgttact tgacctgctc tgctatcttc 1080 cgtggtaagg tttccatgaa agaggtcgag gaccaaatgc gcaatgtcca aaacaagaac 1140 tcttcttact tcgttgagtg gatccctaac aacgtccaaa ccgccctttg ctcgattcct 1200 ccacgtggtc ttaagatgtc ttccaccttc gtcggtaact cgacctctat ccaagaactc 1260 ttcaagcgtg ttggtgatca attcactgct atgttccgaa gaaaggcttt cttgcattgg 1320 tacactggtg agggtatgga cgagatggag ttcactgagg ctgagtccaa catgaacgat 1380 ttggtttccg agtaccaaca ataccaggat gcctccatct ccgagggaga ggaggagtat 1440 gaagaggagg ccccaatcga gggcgaggaa tagatgtttg attctgttcc tacttcgctt 1500 ctttgacaaa acgacgggct ggttactgat attgcattag tggcaacacg atgtcgaatc 1560 atatttatta gtagcaacgt tgataatttc tcgggtgtgg tttgttttag tctttttgtt 1620 aattaggagc gagtggtctt aagggcgtag agcgactaga catcgtcgat accctatgaa 1680 gtagttcatc ttgatggatt aagaaatcaa gacaatatta caaaactggt gtttactgca 1740 aatatacggt gatgtcaaat gtggtgg 1767 <210> 15 <211> 1672 <212> DNA <213> Unknown <220> <223> Phytophthora cactorum_19-435_beta-tubulin <400> 15 ttttttttga aaacggatga aaaaggcagg tttgcattga agttatgtgt agcaaaaagc 60 agcgggttaa gcaaggtaat atgatctctg ttctgtagat gctaagccac taacctctcg 120 ggctaaacta ctcacgaggt gcacgaaggt gatcagcaag gaggtagtaa cgactacgac 180 gggtctacat catctcgtcc atctcctcgt cctcgtcgaa ctcgccctcc tcctctgcgg 240 tggcgtcctg gtactgctgg tactcagaca ccagatcgtt catgttggac tcggcctcag 300 tgaactccat ctcatccata ccctcaccag tgtaccagtg caagaaagcc ttacgacgga 360 acattgccgt aaactgttcg gacacacgct tgaacatctc ctggatggcg gtcgagttac 420 caatgaacgt ggtgctcatc ttcagaccct tgggaggaat atcacacacg ctagccttga 480 tgttgttggg gatccactca acgaagtacg atgagttctt gttctgcacg tttagcatct 540 gttcgtcaac ctccttcgtg ctcatccgtc cgcggaacat acacgcggca gttaaatagc 600 gaccgtggcg agggtcagcg gcacacatca tgttcttagc atcgaactgt tgctgggtca 660 gctcagggac cgtgagggca cggtactgct gagatccacg cgaggtcaac ggggcgaaac 720 caatcataaa gaagtggaga cgcgggaacg ggatcaggtt cacagccaac ttacgcaggt 780 ccgagttcaa ctgaccgggg aaacgcaggc acgtcgtgat accggacatg gcagcacaca 840 ccaagtggtt caggtcaccg taggtgggcg tggtgagctt cagcgtacgg aagcaaatat 900 cgtacagggc ctcgttatcc aggcacatga cctcatcagc gttctcgaca agctggtgta 960 ccgacaacgt ggcgttgtag ggctccacga cagtgtccga caccttaggc gacgggcaca 1020 ccgagtacgt gcacatgata cggtcggggt actcctcacg aatcttcgag ataagaagcg 1080 tacccatacc ggaaccggta ccgccaccaa gcgagtgggt gatctggaaa ccctgcaggc 1140 agtcacagct ctcagcctcc ttgcggacga catcaagcac cgagtcgatc agctcagcac 1200 cctctgtata gtgtcccttg gcccagttgt taccagcgcc tgtttggccg aacacgaagt 1260 tgtccggacg gaagagctga ccgtaagggc cagcgcggac cgagtccatt gtgccaggct 1320 ccagatccat aaggatggcg cggggcacgt aacggccgcc cgtagcctcg ttgtagtaca 1380 cattgatgcg ctccagctgc aggtccgagt cgccgtggta cgagcccgtc gggtccacgc 1440 catgttcgtc agagatgacc tcccagaact tggcaccgat ctggttaccg cactgaccac 1500 cctggatgtg aacaagctct ctcatgttat ggtctgttgc tttacgtgcg cgaagacgtc 1560 gggggttcct tcctgcagcg gctcttggct ggctgtgagg agaattcgga cttttggcga 1620 atgaaggagt cgcgttgcag gcggccgcgt gggacgcgcg ggaaaaatta cc 1672 <210> 16 <211> 100 <212> DNA <213> Artificial Sequence <220> <223> 18-484-tub-100 <400> 16 ccaagggtct caagatgtcc tcgaccttcg ttggtaactc gactgctatc caggagctgt 60 tcaagcgtgt cggcgagcag ttcagtgcta tgttccgtcg 100 <210> 17 <211> 549 <212> DNA <213> Artificial Sequence <220> <223> 19-016-tub-549 <400> 17 atcgcatgat ggctacgttt tcggtcatgc cttcgccgaa ggtgtccgag gtcgttgttg 60 agccctacaa tgctactctg tcgattcatg agttggttga aaatgccgat gagacgttct 120 gcatcgacaa cgaggtgaga aaccccccaa ttgatgccaa atcaggcgct aattgactgc 180 caggcgctgt atgatatctg taggcgcacg ctcaaactcg accatccatc gtacggagat 240 ctcaactcgc tcgtgtccac cgtcatgtcc ggcgtcacca ccggcttccg ctttcctggc 300 caactcaatt cagatctccg gaaactggcc gtgaacatgg tcccgtttcc tcgtcttcac 360 ttctttacgg ttggctttgc gcctctcacg gcccgcggtg gcagctcatt ccgcaattca 420 tccgtgtcag acttgacgca gcaactgttc gacccaagga acatgatggc cgccgccgac 480 ttccggaatg gcaggtactt gacgtgctcg actttcttcc gtggcaacgt ttccacaaag 540 gaggtcgaa 549 <210> 18 <211> 200 <212> DNA <213> Artificial Sequence <220> <223> 19-168-tub-200 <400> 18 tcttttctcc cagacccaga ctggccctgc cagacaatcc atctcaatcc caacaccacc 60 accgtcgccg ccgataccca aacctcacct tctcaatctc gctccgagct accggccctc 120 cgtcttggac ctcgctgctc tcgtttgtct accgccattc ttctgtctcg acccgactcg 180 acaatagcgt cgtccaacct 200 <210> 19 <211> 300 <212> DNA <213> Artificial Sequence <220> <223> 19-382-tub-300 <400> 19 ttgagtggat ccctaacaac gtccaaaccg ccctttgctc gattcctcca cgtggtctta 60 agatgtcttc caccttcgtc ggtaactcga cctctatcca agaactcttc aagcgtgttg 120 gtgatcaatt cactgctatg ttccgaagaa aggctttctt gcattggtac actggtgagg 180 gtatggacga gatggagttc actgaggctg agtccaacat gaacgatttg gtttccgagt 240 accaacaata ccaggatgcc tccatctccg agggagagga ggagtatgaa gaggaggccc 300 300 <210> 20 <211> 400 <212> DNA <213> Artificial Sequence <220> <223> 19-453-tub-400 <400> 20 cacgctagcc ttgatgttgt tggggatcca ctcaacgaag tacgatgagt tcttgttctg 60 cacgtttagc atctgttcgt caacctcctt cgtgctcatc cgtccgcgga acatacacgc 120 ggcagttaaa tagcgaccgt ggcgagggtc agcggcacac atcatgttct tagcatcgaa 180 ctgttgctgg gtcagctcag ggaccgtgag ggcacggtac tgctgagatc cacgcgaagt 240 caacggggcg aaaccaatca taaagaagtg gagacgcggg aacgggatca ggttcacagc 300 caacttacgc aggtccgagt tcaactgacc ggggaaacgc aggcacgtcg tgataccgga 360 catggcagca cacaccaagt ggttcaggtc accgtaggtg 400 <110> Seoul National University R&DB Foundation <120> Primer sets for diagnosing five fungi infecting peach and diagnostic methods using thereof <130> KPA01554 <160> 20 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA < 213> Artificial Sequence <220> <223> 18-484-tub-F2 <400> 1 ccaagggtct caagatgtcc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 18-484 -tub-R2 <400> 2 cgacggaaca tagcactgaa 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 19-016-tub-F1 <400> 3 atcgcatgat ggctacgttt 20 <210 > 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 19-016-tub-R1 <400> 4 ttcgacctcc tttgtggaaa 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 19-168-tub-F2 <400> 5 tcttttctcc cagacccaga 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 19-168-tub -R2 <400> 6 aggttggacg acgctattgt 20 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 19-382-tub-F3 <400> 7 ttgagtggat ccctaacaac g 21 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 19-382-tub-R3 <400> 8 gggcctcctc ttcatactcc 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 19 -435-tub-F3 <400> 9 cacgctagcc ttgatgttgt 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 19-435-tub-R3 <400> 10 cacctacggt gacctgaacc 20 <210> 11 <211> 2427 <212> DNA <213> Unknown <220> <223> Cytospora leucostoma_18-484_beta-tubulin <400> 11 ccacctccag accggccaat gcggtaacca aatcggtgct gctttctggt gcgtaccagc 60 cttcgaccac ggcagcaaac gatacgcgac agtaatcatg tcgaaggatt gaaactgacc 120 ccgattaaac aggcaaacca tctctggcga gcacggtctc gacagcaatg gagtttacaa 180 cggcacttct gagctccagc tggagcgcat gaacgtctac ttcaacgagg cttccggcaa 240 caagtatgtt cctcgcgctg tcctcatcga tctcgagccc ggtaccatgg acgccgtccg 300 tgccggtcct ttcggccagc tgttccgtcc cgacaacttc gttttcggcc agtccggtgc 360 tggaaacaac tgggccaagg gtcactacac tgagggtgcg gagctggtcg accaggtcct 420 ggatgtcgtc cgtcgcgagg ctgagggttg cgactgcctg cagggcttcc agatcaccca 480 ctccctgggt ggtggtac cg gtgccggtat gggtactctg ctcatctcca agattcgcga 540 ggagttcccc gaccgcatga tggctacctt ctccgtcatg ccctctccca aggtctccga 600 caccgtcgtt gagccctaca acgccaccct ctccgtccac cagctggtcg agaactcgga 660 cgagactttc tgtatcgaca acgaggccct ctacgacatt tgcatgcgga cgctgaagct 720 gtccaacccc tcttacggtg accttaacta cctggtgtcg gctgtcatgt ccggcgtcac 780 cgtctcgctg cgtttccccg gtcagctgaa ctctgacctc cgcaagctgg ctgtgaacat 840 ggtgccgttc cctcgtctgc acttcttcat ggttggcttc gctcccctga ccagccgtgg 900 cgcccactcc ttccgcgctg tcaccgtccc tgagctcacc cagcagatgt tcgaccccaa 960 gaacatgatg gctgcgtctg acttccgtaa cggtcgctac ctgacgtgct ctgccatctt 1020 gtgagtcctc ccgcccccaa gtgtctctat ccgtgcttgt ggtgctaatt atgtttccag 1080 ccgtggcaag gtctccatga aggaggttga ggaccagatg cgcaacgtcc agagcaagaa 1140 ctcgtcctac ttcgtcgagt ggatccccaa caacgtccag accgccctct gctctattcc 1200 ccccaagggt ctcaagatgt cctcgacctt cgttggtaac tcgactgcta tccaggagct 1260 gttcaagcgt gtcggcgagc agttcagtgc tatgttccgt cgcaaggctt tcttgcattg 1320 gtacactggt gagggtatgg acgagatggagttcaccgag gctgagtcca acatgaacga 1380 tcttgtctcg gaataccagc agtaccagga tgccagcatc gacgaggagg aggaggagta 1440 cgaggaggag gctccccttg aggaggagtg aactatggtg tgattaaaaa gtgtttcccg 1500 gcggtcttga tggccaagga cctatgtcca taatttgtca ccttgggtac ccacggcact 1560 tcccgcatgt attctgcttg tgtgggttgt gtgtcacgtt tgcgtgggta gctatgcgat 1620 ccggagatgg gttctggtaa tcgattggcc gtatgaacaa agggacacaa cattgatttg 1680 aaatatacct cttgtagttg accctttacc acgaatacaa aatcggacaa agccgtccac 1740 atgatagccc caagcgtgtg tgatacaata tcagtagtaa cgatttgcca aacagaggcc 1800 aagattgtct taatccactt gcttgagtcc ataccacagg ggaccttgca ccgcctcttc 1860 tacgtaggta ttgcaggtaa tcctagggca gtgcagttga ctttgatagt cgcttgaggc 1920 tggtagtatg ttccactatc gtgatctagc ccatggcacc cacaactggt caaggagagg 1980 ctgaaaggca tcgccgactt tcagcggcag gcggccgacc agcggcgtaa actggaaatg 2040 tagattgtgc gcgaggtcac ccgagatcaa aatctcatgc tgatcagcgt atcaggcgcc 2100 tattaagcgc tatagtgact gactgactgc accgatatgt agtaggtagg taggcccagc 2160 ccaccttagg gacagattac aggcaccgaa ccggat acct actaagtcgt caggtatgta 2220 atgtgtgaat ctgtgtgggc ttcacatagg ctaagttagg tagtgtagat aagcggcaag 2280 gaggatgcgg cattgccatg cgttgatcaa taaatcctcc tagtcactca tctttccccc 2340 ttctgttata gatctgatgt aagcgctgaa ggaggtgaga ggcagcctgc gtgtcagttt 2400 tcttacggta tagtgtatgc gtgtgac 2427 <210> 12 <211> 1705 <212> DNA <213> Unknown <220> <223 > Macrophomina phaseolina_19-016_beta-tubulin <400> 12 cgtctgcaac caatttgcgc tgtccacctc acagaaccca caacagacgt gatcaattcg 60 tcacccgctg ccaatatgcg cgagattatc catgttcagg ctggccaatg tggtaaccaa 120 atcggatctg ctttctggca gacgatctgc aaagagcatg gtgtcgatag ctctggaaac 180 tacaacggca catcggatct ccaactcgag cgcatgaatg tctatttcaa tgagccttcg 240 agcaagaaat tcgtaccccg cgccgtgctc gttgatcttg aacccggcac catggatgcc 300 gttcgtgccg gcccagctgg acggctcttc cgccccgaca attttgtttt cgggcagtct 360 ggagccggaa acaactgggc aaaaggccat tacaccgaag gcgctgagct ggtggatcag 420 gttcttgatg ttgtgcgccg ggagt ccgaa ggttgcgatt c ggtt caaggt ccgaa ggttgcgatt c ggtt caaggt cc ggt ac ggctaagatt 540 cgggaagagt ttccggatcg catgatggct acgttttcgg tcatgccttc gccgaaggtg 600 tccgaggtcg ttgttgagcc ctacaatgct actctgtcga ttcatgagtt ggttgaaaat 660 gccgatgaga cgttctgcat cgacaacgag gcgctgtatg atatctgtag gcgcacgctc 720 aaactcgacc atccatcgta cggagatctc aactcgctcg tgtccaccgt catgtccggc 780 gtcaccaccg gcttccgctt tcctggccaa ctcaattcag atctccggaa actggccgtg 840 aacatggtcc cgtttcctcg tcttcacttc tttacggttg gctttgcgcc tctcacggcc 900 cgcggtggca gctcattccg caattcatcc gtgtcagact tgacgcagca actgttcgac 960 ccaaggaaca tgatggccgc cgccgacttc cggaatggca ggtacttgac gtgctcgact 1020 ttcttccgtg gcaacgtttc cacaaaggag gtcgaagatc aaatgatcaa gttgcaagac 1080 aagaactctt cctatttcgt cgaatggatt ccgaacaatt tccaaaccgc cgtgtgctcc 1140 gtccccccgc caggcttgaa aatgtctgcc acctttgttg ggaataccac ttctattcag 1200 gagctcttca agcgtgtcgg agatcagttc actgccatgt tccgccgcaa agccttcttg 1260 cattggtata ccgccgaagg aatggacgag atggaattca cggaggcaga gtccaatatg 1320 aatgatctcg tggcggagta tcagcagtac caggaggcct cgatcagcga caccgaagac1380 tacgatgagg aagctccatt ggaagaggag gaggagtgag gaagtgtacg acaaacgtta 1440 ctctgattgt ctgtcgtcat ttgttgcatc tttgagaaga caagaaccat atagcgtacc 1500 actagcgaat catcattgaa ttgcgccaac gtatcacctc attcatctcg ttgaccatga 1560 tccgtcctat gtacctattc aaacttcaaa ttgggcattt ttcgtagtag atcttgcacc 1620 tccaaggcgc gcagaattgc tccttagtat aggaagacct tagcgttact taaaaaaggc 1680 aacgcgcgat gatgctcgcc gtgcc 1705 <210> 13 <211> 2050 <212 > DNA <213> Unknown <220> <223> Diaporthe helianthi_19-168_beta-tubulin <400> 13 ctcttccgat ctttcacccc tggcacctgg cacctggcac tggcagcagc agcagctccg 60 gcacaacaga gtgaaagagg gaaagcgaag ggcctccact ccactctttt ctcccagacc 120 cagactggcc ctgccagaca atccatccca atcccaacac caccaccgtc gccgccgata 180 cccaaacctc accttctcaa tctcgctccg agctaccggc ccttcgtctt ggacctcgct 240 gctctcgttt gtctaccgcc attcttctgt ctcgacccga ctcgacaata gcgtcgtcca 300 accttcaaaa tgcgtgagat tgttcacctt cagaccggcc aatgcggtaa ccaaacccggt 360 gctgcttttct gt gtcaacca ctccagct cgagcgcatg aacgtctact tcaacgaggc ctccggcaac 480 aagtatgttc ctcgcgccgt cctcgtcgat ctcgagcccg gtaccatgga cgccgtccgt 540 gccggtccct ttggccagct cttccgcccc gacaacttcg tcttcggcca gtccggtgct 600 ggaaacaact gggccaaggg tcactacact gagggtgctg agctggtcga ccaggtcctc 660 gatgtcgtcc gtcgcgaggc tgagggctgc gactgcctcc agggcttcca gatcacccac 720 tccctgggtg gtggtactgg tgctggtatg ggtacgctgc ttatctccaa gatccgcgag 780 gagttccccg accgcatgat ggccaccttc tccgtcgtgc cctcccccaa ggtctccgac 840 accgtcgtcg agccctacaa cgccaccctg tccgtccacc agctggtcga gaactcggac 900 gagaccttct gcatcgacaa cgaggccctg tacgacatct gcatgcgtac gctcaagctg 960 tccaacccct cctacggcga cctgaactac ctggtgtcgg ctgtcatgtc cggcgtcacc 1020 gtctccctgc gtttccccgg tcagctgaac tctgacctgc gcaagctggc cgtgaacatg 1080 gtgcccttcc ctcgtctgca cttcttcatg gtcggctttg ctcccctgac cagccgtggc 1140 gcccactcct tccgcgccgt taccgttcct gagctcaccc agcagatgtt cgaccccaag 1200 aacatgatgg ctgcctctga cttccgtaac ggtcgctacc tgacgtgctc tgctatcttc 1260 cgtggaaagg tctccatgaa ggaggt cgag gaccagatgc gcaacgtcca gagcaagaac 1320 tcatcctact tcgtcgagtg gatccccaac aacgtccaga ccgccctctg ctcgatccct 1380 cccaagggcc tcaagatgtc ctctaccttc gttggtaact cgactgctat ccaggagctg 1440 ttcaagcgtg tgggtgagca gttcactgcc atgttccggc gcaaggcttt cttgcattgg 1500 tacactggtg agggtatgga cgagatggag ttcaccgagg ctgagtccaa catgaacgat 1560 ctggtctccg aataccagca gtaccaggac gcctccattg atgaggagga ggaggagtac 1620 gaggaggagg ttccccttga gggcgaggag tgaatagtgg tctgatgaga tctgctgtct 1680 ggattggttc aatgatctag gactctgcta ccgtccttaa tccgtcatca ttgtacccct 1740 gacacttctc actcatttct tgtgtgcgtg tgcgcgcgtg tgcgaatgtg tgggacgctt 1800 gaagctggtg tgaatagcta ttcgattcat gattcgggag gagcgggggt gttctggtaa 1860 ttattgaatg acagacccgg ccgatcgtcc gaagaagaga gcagagcaaa aaggggaaac 1920 attgatttta aaaatatacc tctttgtagt tgaaccctgt gcccacatcc ggcctgccag 1980 tttcgcaccc tttcatgcat acatgcaacc acgtttccat ttggaatgaa attcacagga 2040 tgattgcttc 2050 <210> 14 <211> 1767 < 212> DNA <213> Unknown <220> <223> Monillinia fructicola_19-382_beta -tubulin <400> 14 ctccactatc cacaaggaca tctcagcaat ccttaacctc ttactttctc tatctacaac 60 ctcgacttct caatatccaa actaccatac tttaacacct caacacaaga tcctaaatct 120 accttcaaaa tgcgtgagat tgttcatctt caaaccggtc aatgtggtaa ccaaattggt 180 gctgctttct ggcaaactat ctctggcgag cacggtcttg atggctctgg tgtctacaat 240 ggtacctccg acctccaact tgagcgtatg aacgtctact tcaacgaggc ttccggcaac 300 aagtatgttc cccgtgccgt tctcgtcgat ttggagccag gtaccatgga cgctgtccgt 360 gccggtcctt tcggtcaact cttccgcccc gataacttcg tcttcggtca atccggtgct 420 ggtaacaact gggctaaggg tcattacact gagggtgctg agcttgttga ccaagttctt 480 gatgttgttc gtcgtgaagc tgaaggctgt gactgccttc aaggtttcca aatcacccac 540 tctctcggtg gtggaactgg tgccggtatg ggtacgcttt tgatctccaa gatccgtgag 600 gagttccccg atcgtatgat ggctaccttt tctgtcgttc catcgccaaa ggtttccgat 660 accgttgtcg agccatataa cgctaccctc tccgttcatc aattggtaga aaactctgat 720 gaaaccttct gtatcgataa cgaggctctt tacgacatct gcatgagaac cttgaagctc 780 agcaacccat cctacggaga tcttaaccac ttggtttctg ctgtcatgtc cggtgttacc 840 acc tgtctcc gtttccctgg tcaacttaac tcagatctcc gaaagttggc tgttaacatg 900 gttccattcc cacgtctcca tttcttcatg gttggatttg ctcctttgac cagccgtggt 960 gcacactctt tccgtgctgt taccgttcca gagttgactc aacaaatgta cgaccctaag 1020 aacatgatgg ccgcttccga tttccgtaac ggtcgttact tgacctgctc tgctatcttc 1080 cgtggtaagg tttccatgaa agaggtcgag gaccaaatgc gcaatgtcca aaacaagaac 1140 tcttcttact tcgttgagtg gatccctaac aacgtccaaa ccgccctttg ctcgattcct 1200 ccacgtggtc ttaagatgtc ttccaccttc gtcggtaact cgacctctat ccaagaactc 1260 ttcaagcgtg ttggtgatca attcactgct atgttccgaa gaaaggcttt cttgcattgg 1320 tacactggtg agggtatgga cgagatggag ttcactgagg ctgagtccaa catgaacgat 1380 ttggtttccg agtaccaaca ataccaggat gcctccatct ccgagggaga ggaggagtat 1440 gaagaggagg ccccaatcga gggcgaggaa tagatgtttg attctgttcc tacttcgctt 1500 ctttgacaaa acgacgggct ggttactgat attgcattag tggcaacacg atgtcgaatc 1560 atatttatta gtagcaacgt tgataatttc tcgggtgtgg tttgttttag tctttttgtt 1620 aattaggagc gagtggtctt aagggcgtag agcgactaga catcgtcgat accctatgaa 1680 gtagttcatc ttgatggatt aagaaatcaa gacaatatta caaaactggt gtttactgca 1740 aatatacggt gatgtcaaat gtggtgg 1767 <210> 15 <211> 1672 <212> DNA <213> Unknown <220> <223> Phytophthora cactorum_19-435_beta-tubulin <400> 15 ttttttttga aaacggatga aaaaggcagg tttgcattga agttatgtgt agcaaaaagc 60 agcgggttaa gcaaggtaat atgatctctg ttctgtagat gctaagccac taacctctcg 120 ggctaaacta ctcacgaggt gcacgaaggt gatcagcaag gaggtagtaa cgactacgac 180 gggtctacat catctcgtcc atctcctcgt cctcgtcgaa ctcgccctcc tcctctgcgg 240 tggcgtcctg gtactgctgg tactcagaca ccagatcgtt catgttggac tcggcctcag 300 tgaactccat ctcatccata ccctcaccag tgtaccagtg caagaaagcc ttacgacgga 360 acattgccgt aaactgttcg gacacacgct tgaacatctc ctggatggcg gtcgagttac 420 caatgaacgt ggtgctcatc ttcagaccct tgggaggaat atcacacacg ctagccttga 480 tgttgttggg gatccactca acgaagtacg atgagttctt gttctgcacg tttagcatct 540 gttcgtcaac ctccttcgtg ctcatccgtc cgcggaacat acacgcggca gttaaatagc 600 gaccgtggcg agggtcagcg gcacagtcatca tcgaactggt caggt cggtcagcg gcacagtcatca tcgttctgt cgg gct gagatccacg cgaggtcaac ggggcgaaac 720 caatcataaa gaagtggaga cgcgggaacg ggatcaggtt cacagccaac ttacgcaggt 780 ccgagttcaa ctgaccgggg aaacgcaggc acgtcgtgat accggacatg gcagcacaca 840 ccaagtggtt caggtcaccg taggtgggcg tggtgagctt cagcgtacgg aagcaaatat 900 cgtacagggc ctcgttatcc aggcacatga cctcatcagc gttctcgaca agctggtgta 960 ccgacaacgt ggcgttgtag ggctccacga cagtgtccga caccttaggc gacgggcaca 1020 ccgagtacgt gcacatgata cggtcggggt actcctcacg aatcttcgag ataagaagcg 1080 tacccatacc ggaaccggta ccgccaccaa gcgagtgggt gatctggaaa ccctgcaggc 1140 agtcacagct ctcagcctcc ttgcggacga catcaagcac cgagtcgatc agctcagcac 1200 cctctgtata gtgtcccttg gcccagttgt taccagcgcc tgtttggccg aacacgaagt 1260 tgtccggacg gaagagctga ccgtaagggc cagcgcggac cgagtccatt gtgccaggct 1320 ccagatccat aaggatggcg cggggcacgt aacggccgcc cgtagcctcg ttgtagtaca 1380 cattgatgcg ctccagctgc aggtccgagt cgccgtggta cgagcccgtc gggtccacgc 1440 catgttcgtc agagatgacc tcccagaact tggcaccgat ctggttaccg cactgaccac 1500 cctggatgtg aacaagctct ctcatgttat ggtctgt tgc tttacgtgcg cgaagacgtc 1560 gggggttcct tcctgcagcg gctcttggct ggctgtgagg agaattcgga cttttggcga 1620 atgaaggagt cgcgttgcag gcggggcgt 100 gggacgc > 16 ccaagggtct caagatgtcc tcgaccttcg ttggtaactc gactgctatc caggagctgt 60 tcaagcgtgt cggcgagcag ttcagtgcta tgttccgtcg 100 <210> 17 <211> 549 <212> DNA <213> ggc 19ac-016-tubgat <223> Artificial Sequence <220> 49 tcggtcatgc cttcgccgaa ggtgtccgag gtcgttgttg 60 agccctacaa tgctactctg tcgattcatg agttggttga aaatgccgat gagacgttct 120 gcatcgacaa cgaggtgaga aaccccccaa ttgatgccaa atcaggcgct aattgactgc 180 caggcgctgt atgatatctg taggcgcacg ctcaaactcg accatccatc gtacggagat 240 ctcaactcgc tcgtgtccac cgtcatgtcc ggcgtcacca ccggcttccg ctttcctggc 300 caactcaatt cagatctccg gaaactggcc gtgaacatgg tcccgtttcc tcgtcttcac 360 ttctttacgg ttggctttgc gcctctcacg gcccgcggtg gcagctcatt ccgcaattca 420 tccgtgtcag acttgacgca gcaactgttc gacccaagga aca tgatggc cgccgccgac 480 ttccggaatg gcaggtactt gacgtgctcg actttcttcc gtggcaacgt ttccacaaag 540 gaggtcgaa 549 <210> 18 <211> 200 <212> DNA <213> Artificial Sequence <aga220> <223> ctcc 19-cctctttga cctt atctcaatcc caacaccacc 60 accgtcgccg ccgataccca aacctcacct tctcaatctc gctccgagct accggccctc 120 cgtcttggac ctcgctgctc tcgttttgtct accgccattc ttctgtctcg accctubc-223 <> 213cc Sequenceacc ttctgtgtctcg accctubc-212> <19gt> aca <400> 19 ttgagtggat ccctaacaac gtccaaaccg ccctttgctc gattcctcca cgtggtctta 60 agatgtcttc caccttcgtc ggtaactcga cctctatcca agaactcttc aagcgtgttg 120 gtgatcaatt cactgctatg ttccgaagaa aggctttctt gcattggtac actggtgagg 180 gtatggacga gatggagttc actgaggctg agtccaacat gaacgatttg gtttccgagt 240 accaacaata ccaggatgcc tccatctccg agggagagga ggagtatgaa gaggaggccc 300 300 <210> 20 <211> 400 <212 > DNA <213> Artificial Sequence <220> <223> 19-453-tub-400 <400> 20 cacgctagcc ttgatgttgt tggggatcca ctcaacgaag tacgatgagt tcttgttctg 60 cacgtttagc atctgttcgt caacctcctt cgtgctcatc cgtccgcgga acatacacgc 120 ggcagttaaa tagcgaccgt ggcgagggtc agcggcacac atcatgttct tagcatcgaa 180 ctgttgctgg gtcagctcag ggaccgtgag ggcacggtac tgctgagatc cacgcgaagt 240 caacggggcg aaaccaatca taaagaagtg gagacgcggg aacgggatca ggttcacagc 300 caacttacgc aggtccgagt tcaactgacc ggggaaacgc aggcacgtcg tgataccgga 360catggcagca cacaccaagt ggttcaggtc accgtaggtg 400
Claims (6)
시토스포라 레우코스토마(Cytospora leucostoma), 마크로포미나 파세올리나(Macrophomina phaseolina), 디아포르테 헬리안티(Diaporthe helianthi), 모닐리니아 프룩티콜라(Monilinia fructicola), 및 파이토프소라 캑토룸(Phytophthora cactorum)으로 이루어진 군으로부터 선택되는 하나 이상의 복숭아 곰팡이 진단용 프라이머 세트.
Primer pair represented by SEQ ID NO: 1 and SEQ ID NO: 2, primer pair represented by SEQ ID NO: 3 and SEQ ID NO: 4, primer pair represented by SEQ ID NO: 5 and SEQ ID NO: 6, primers represented by SEQ ID NO: 7 and SEQ ID NO: 8 a pair, and one or more primer pairs selected from the group consisting of a pair of primers represented by SEQ ID NO: 9 and SEQ ID NO: 10;
Cytospora leucostoma , Macrophomina phaseolina, Diaporthe helianthi , Monilinia fructicola , and Phytophthora cactorum ) one or more peach fungus diagnostic primer sets selected from the group consisting of.
Claim 1 comprising the pair of primers, Cytospora leucostoma ( Cytospora leucostoma ), Macrophomina phaseolina ( Macrophomina phaseolina ), Diaporthe helianthi ( Diaporthe helianthi ), Monilinia fructicola ( Monilinia fructicola ), Phytophthora cactorum ( Phytophthora cactorum ) At least one composition for diagnosis of peach fungus selected from the group consisting of.
A kit for diagnosing peach fungus, comprising the composition of claim 2.
According to claim 3, wherein the kit is a reverse transcription-polymerase chain reaction (reverse transcription PCR) kit, polymerase chain reaction (PCR) kit, real-time polymerase chain reaction (real-time PCR) kit, real-time polymerase chain reaction quantitative Test (RQ-PCR) kit, reverse polymerase chain reaction (inverse PCR) kit, and multiplex PCR kit, characterized in that at least one selected from the group consisting of, Peach mold diagnosis kit.
(b) 상기 (a) 단계에서 분리된 핵산을 주형으로 제1항의 프라이머 쌍을 이용하여 표적 핵산을 증폭하는 단계; 및
(c) 상기 (b) 단계에서 증폭된 표적 핵산을 검출하는 단계를 포함하는 것을 특징으로 하는,
시토스포라 레우코스토마(Cytospora leucostoma), 마크로포미나 파세올리나(Macrophomina phaseolina), 디아포르테 헬리안티(Diaporthe helianthi), 모닐리니아 프룩티콜라(Monilinia fructicola), 파이토프소라 캑토룸(Phytophthora cactorum)으로 이루어진 군으로부터 선택되는 하나 이상의 복숭아 곰팡이 진단 방법.
(a) isolating the peach fungal nucleic acid from the sample;
(b) amplifying a target nucleic acid using the primer pair of claim 1 as a template using the nucleic acid isolated in step (a); and
(c) characterized in that it comprises the step of detecting the target nucleic acid amplified in step (b),
Cytospora leucostoma , Macrophomina phaseolina, Diaporthe helianthi , Monilinia fructicola , Phytophthora cactorum At least one method for diagnosing peach fungus selected from the group consisting of.
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