KR-20260064858-A - Primer set for detecting soil latent pathogen and uses thereof

Abstract

The present invention relates to a primer set for detecting soil latent pathogens and the use thereof. Since the primer set of the present invention can specifically detect eight types of soil latent pathogens ( Alternaria tenuissima , Botryosphaeria dothidea , Fusarium oxysporum , Glomerella cingulata , Phytophthora cactorum , Rosellinia necatrix , Sclerotium rolfsii , Sclerotinia sclerotiorum ), it can be effectively utilized to detect pathogens latent in the soil at an early stage and control them in advance.

Inventors

• 김국형
• 권구담

Assignees

• 서울대학교산학협력단

Dates

Publication Date

20260508

Application Date

20241029

Claims (7)

1. A primer set composition for detecting soil latent pathogens, comprising one or more oligonucleotide primer sets selected from the group consisting of the oligonucleotide primer set of SEQ ID NOs 1 and 2, the oligonucleotide primer set of SEQ ID NOs 3 and 4, the oligonucleotide primer set of SEQ ID NOs 5 and 6, the oligonucleotide primer set of SEQ ID NOs 7 and 8, the oligonucleotide primer set of SEQ ID NOs 9 and 10, the oligonucleotide primer set of SEQ ID NOs 11 and 12, the oligonucleotide primer set of SEQ ID NOs 13 and 14, and the oligonucleotide primer set of SEQ ID NOs 15 and 16.
2. A primer set composition for detecting soil latent pathogens according to claim 1, characterized in that the soil latent pathogen is one or more selected from the group consisting of Alternaria tenuissima , Botryosphaeria dothidea , Fusarium oxysporum , Glomerella cingulata , Phytophthora cactorum , Rosellinia necatrix , Sclerotium rolfsii , and Sclerotinia sclerotiorum .
3. A primer set composition for detecting soil latent pathogens according to claim 1, wherein the oligonucleotide primer set of SEQ ID NOs 1 and 2 is for detecting Alternaria tenuisima, the oligonucleotide primer set of SEQ ID NOs 3 and 4 is for detecting Botriosperia dotidia, the oligonucleotide primer set of SEQ ID NOs 5 and 6 is for detecting Fusarium oxysporum, the oligonucleotide primer set of SEQ ID NOs 7 and 8 is for detecting Glomerella shingurata, the oligonucleotide primer set of SEQ ID NOs 9 and 10 is for detecting Phytophthora cactorum, the oligonucleotide primer set of SEQ ID NOs 11 and 12 is for detecting Roselinia neckatrix, the oligonucleotide primer set of SEQ ID NOs 13 and 14 is for detecting Sclerotium rolfsi, and the oligonucleotide primer set of SEQ ID NOs 15 and 16 is for detecting Sclerotinia sclerothiorum.
4. A kit for detecting latent soil pathogens comprising: an oligonucleotide primer set composition of claim 1; and a reagent for performing an amplification reaction.
5. In claim 4, the kit comprises a reagent for performing the amplification reaction, the reagent comprising DNA polymerase, dNTPs, and a buffer.
6. Step of isolating genomic DNA from a sample suspected of being infected with a soil latent pathogen; A step of amplifying a target sequence by performing an amplification reaction using the isolated genomic DNA as a template and the oligonucleotide primer set composition of claim 1; and A method for detecting soil latent pathogens, comprising a step of detecting the product of the amplification step.
7. A method according to claim 6, wherein the detection of the amplified product is performed via gel electrophoresis, radiometric measurement, fluorescence measurement, phosphorescence measurement, or DNA chip.

Description

Primer set for detecting soil latent pathogen and uses thereof The present invention relates to a primer set for detecting soil latent pathogens and the use thereof. Recently, with the opening of agricultural imports and the liberalization of trade, crop movement has become free and cultivated crops have become more diverse. Additionally, as year-round cultivation in facilities such as vinyl greenhouses has become prevalent, new diseases are being discovered every year. Furthermore, due to climate change caused by factors such as global warming resulting from rising overall temperatures and increasingly severe pollution, the types of diseases occurring are showing different patterns compared to the past. Due to the diversification of cultivation methods and the prevalence of year-round cultivation leading to continuous cropping, the occurrence of soil-borne diseases has become severe, and the resulting damage from continuous cropping has emerged as an urgent issue that needs to be resolved. First, the most problematic diseases in Solanaceae crops are wilt caused by Fusarium , late blight caused by Phytophthora , and bacterial wilt caused by bacteria; these occur annually in both greenhouse and hydroponic cultivation, causing significant damage. Regarding above-ground diseases, powdery mildew and gray mold are major problems, similar to cucumbers, while in the case of peppers, bacterial spot also occurs severely depending on the year. For strawberries, wilt, anthracnose, and powdery mildew are major diseases causing significant damage, while for lettuce, sclerotinia rot, soft rot, and downy mildew commonly occur, causing damage every year. Apple anthracnose was the most important disease in the 1970s, but with the change in varieties, it passed its position to apple blight and is now a disease that occurs only slightly from year to year. Apple blight became a major target for control after the 1980s following the introduction of the Fuji variety, and apple leaf spot is also a disease that occurs more frequently in recent years than in 1970. The occurrence of these plant diseases is closely related to the soil environment. Although soil fumigation or chemical spraying can be used to control soilborne diseases, these methods are difficult to implement in reality due to the destruction of soil microbial communities and their significant impact on the environment. Furthermore, securing disease-free seedlings requires the cultivation of healthy seedlings through tissue culture, which presents the problem of requiring a significant amount of time and effort. While research on the biological control of plant diseases has a long history, practical control methods are either ineffective or difficult to implement. Accordingly, the inventors have developed a primer set capable of rapidly and accurately detecting soil latent pathogens that cause massive damage to major economic crops, including horticultural crops. Meanwhile, Korean Registered Patent No. 1913742 discloses a "primer set for specifically detecting the strawberry wilt disease pathogen ( Fusarium oxysporum ) and the use thereof," and Korean Published Patent No. 2012-0070931 discloses a "primer pair for detecting deoxynivalenol-producing red mold ( Fusarium graminearum ) and a kit using the same," but the primer set for detecting soil latent pathogens and the use thereof according to the present invention are not described. Figure 1 is the result of qPCR ( quantitativereal-time PCR) analysis of the specificityfor 8 types of soil latent pathogens using the primer set of the present invention (Table 2) which is specific to each of the 8 types of soil latent pathogens ( Alternaria tenuissima, At; Botryosphaeria dothidea , Bd; Fusarium oxysporum , Fo; Glomerella cingulata , Gc; Phytophthora cactorum , Pc; Rosellinia necatrix, Rn; Sclerotium rolfsii, Sr; Sclerotinia sclerotiorum , Ss). Figure 2 shows the qPCR results analyzing the sensitivity of the primer set of the present invention (Table 2) specific to each of the eight types of soil latent pathogens ( Alternaria tenuissima , At; Botryosphaeria dothidea , Bd; Fusarium oxysporum , Fo; Glomerella cingulata , Gc; Phytophthora cactorum , Pc; Rosellinia necatrix, Rn; Sclerotium rolfsii , Sr; Sclerotinia sclerotiorum , Ss). To achieve the objective of the present invention, the present invention provides a primer set composition for detecting latent soil pathogens, comprising one or more oligonucleotide primer sets selected from the group consisting of the oligonucleotide primer set of SEQ ID NOs 1 and 2, the oligonucleotide primer set of SEQ ID NOs 3 and 4, the oligonucleotide primer set of SEQ ID NOs 5 and 6, the oligonucleotide primer set of SEQ ID NOs 7 and 8, the oligonucleotide primer set of SEQ ID NOs 9 and 10, the oligonucleotide primer set of SEQ ID NOs 11 and 12, the oligonucleotide primer set of SEQ ID NOs 13 and 14, and the oligonucleotide primer set of SEQ ID NOs 15 and 16. The primer set composition o