Method for detecting fungi in turf grass with a lamp assay having novel primer sets

Abstract

To provide a method for detecting fungal DNA in a turf grass sample.SOLUTION: The present invention provides a method for detecting fungal DNA in a turf grass sample with a loop- mediated isothermal amplification (LAMP) assay which contains primers for fungal DNA of at least one turf pathogenic fungus selected from Sclerotinia homoeocarpa, Rhizoctonia solani spp., Pythium aphanidermatum, Gaeumannomyces graminis spp., Microdochium nivale spp., Magnaporthe poae, Colletotrichum graminicola, Colletotrichum cereale, and Pythium ultimum var. ultimum, comprising: subjecting the turf sample to a LAMP reaction. The LAMP reaction uses a primer set of four or more nucleic acid sequences, with each primer in the set having from 15 to 50 nucleic acids.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for detecting disease in turfgrass caused by fungal pathogens using a loop-mediated isothermal amplification (LAMP) assay of a sample of such turfgrass to detect nucleic acid derived from one or more fungi. [Background technology]

[0002] For example, LAMP, or loop-mediated isothermal amplification, as described in U.S. Patent No. 6,410,278 (Eiken), is a DNA amplification method characterized by the use of at least four different primers specifically designed to recognize six distinct regions on a target gene (see, for example, http: / / loopamp.eiken.co.jp / e / lamp / primer.html). The reaction process proceeds at a constant temperature using strand displacement chemistry. Amplification and detection of a target nucleic acid of interest can be completed in a single step by incubating a mixture of a biological sample or its nucleic acid extract, primers, a DNA polymerase with strand displacement activity, and a substrate at a constant temperature (approximately 65°C). It provides high amplification efficiency, allowing DNA to be amplified multiple times in 15 to 60 minutes. Due to its high specificity, the presence of an amplification product can indicate the presence of the target gene (http: / / loopamp.eiken.co.jp / e / lamp / principle.html).

[0003] There are many challenges that turfgrass managers face in maintaining turfgrass to the quality standards expected by users. While the challenges are many, those related to diseases (including those caused by fungal pathogens) are particularly difficult to manage and control. For example, diseases can infest turfgrass plants on golf courses, causing reduced revenue due to reduced quality, including playability. One common challenge for golf course managers is knowing which diseases are present so that appropriate and timely management measures can be taken. Relevant turf diseases caused by turf pathogenic microorganisms include, for example, anthracnose, take-all patch, summer patch, snow rot, red rot, leaf rot, and dollar spot.

[0004] Agriculturally effective chemicals to control pathogens, such as fungicides, are typically applied on golf courses as needed depending on the level of disease pressure, pathogen populations, weather, etc. However, fungicide application is highly controlled by the course budget, the availability of appropriate equipment, and the availability of qualified personnel to apply the agriculturally effective chemicals.

[0005] In view of these challenges, a rapid and reliable assay for the detection of pathogenic fungi in turfgrass would be extremely useful. Known PCR assays are impractical for use on golf courses or other intensively managed turfgrass or professional landscape environments because PCR requires specialized laboratory skills and equipment. Other molecular biology methods for detecting fungal diseases in turfgrass are known, and are described, for example, in WO 2009147017 for the TRFLP method. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] U.S. Patent No. 6,410,278 [Patent Document 2] International Publication No. 2009147017 Summary of the Invention [Means for solving the problem]

[0007] Thus, the present invention relates to a LAMP assay for detecting the presence of DNA in turf samples associated with selected fungal pathogens that cause related turf diseases including, for example, anthracnose, take-all patch, summer patch, snow rot, red burn, leaf rot, and dollar spot.

[0008] To facilitate timely and efficient detection of turfgrass disease pathogens and improve the cost and effectiveness of turfgrass disease treatments, the LAMP assay of the present invention can be used for early detection of DNA associated with fungal pathogens causing relevant turf diseases. According to the present invention, the LAMP method preferably uses a primer set of at least four, and preferably six or more, nucleic acid sequences derived from the target disease pathogen. More specifically, the method of the present invention provides that each primer used in the selected primer set for the LAMP assay has 15 to 50 nucleic acids, and the primers in the set are selected from specific DNA loci in the target fungus.

[0009] According to the present invention, there is provided a method for detecting fungal DNA in turfgrass samples using a loop-mediated isothermal amplification (LAMP) assay containing primers for the fungal DNA (nucleic acid) of a turf pathogenic fungus selected from the group consisting of Sclerotinia homoeocarpa, Rhizoctonia solani spp., Pythium aphanidermatum, Gaeumannomyces graminis spp., Microdochium nivale spp., Magnaporthe poae, Colletotrichum graminicola, Colletotrichum cereale, and Pythium ultimum var. ultimum (target fungi). The LAMP assay of the present invention uses a primer set of at least four, preferably six or more, nucleic acid sequences, each primer having 15-50 nucleic acids, and the fungal DNA to be detected is obtained from a target fungal pathogen. Primers useful in this LAMP assay method are selected from specific internal transcribed spacer regions or genes of the target fungus to obtain improved assay results.

[0010] In certain embodiments, the target fungus of the species Microdochium nivale is selected from Microdochium nivale var. nivale and Microdochium nivale var. majus. In another embodiment, the target fungus of the species Gaeumannomyces graminis is selected from Gaeumannomyces graminis var. avenae, Gaeumannomyces graminis var. graminis, and Gaeumannomyces graminis var. tritici. In a further embodiment, the target fungus of the species Rhizoctonia solani is selected from Rhizoctonia solani AG2-2IV and Rhizoctonia solani AG2-2IIIB. DETAILED DESCRIPTION OF THE INVENTION

[0011] In the context of the present invention, detection of fungal DNA using the LAMP assay of the present invention in turf samples can indicate the presence of fungal pathogens and can also be useful in assessing the onset or presence of turf disease conditions such as:

[0012] [Table 1]

[0013] In one embodiment, (a) A primer set for dollar spot disease fungus (Sclerotinia homoeocarpa) DNA is selected from the DNA of SEQ ID NO: 1; (b) a primer set for Rhizoctonia solani DNA selected from the DNA of SEQ ID NO: 2 or SEQ ID NO: 9; (c) a primer set for Microdochium nivale species DNA (preferably Microdochium nivale var. nivale) selected from the DNA of SEQ ID NO: 3; (d) a primer set for Pythium aphanidermatum DNA selected from the DNA of SEQ ID NO: 4 or SEQ ID NO: 10; (e) a primer set for Gaeumannomyces graminis species DNA (preferably Gaeumannomyces graminis var. avenae, Gaeumannomyces graminis var. graminis, or Gaeumannomyces graminis var. tritici) selected from the DNA of SEQ ID NO: 5 or SEQ ID NO: 8; (f) a primer set for Microdochium nivale species DNA (preferably Microdochium nivale var. majus) selected from the DNA of SEQ ID NO: 6; (g) a primer set for Magnaporthe poae DNA selected from the DNA of SEQ ID NO: 7; (h) a primer set for Colletotrichum graminicola DNA selected from the DNA of SEQ ID NO: 11; (i) a primer set for Colletotrichum cereale DNA selected from the DNA of SEQ ID NO: 12; (j) A primer set for Pythium ultimum var. Ultimum DNA is selected from the DNA of SEQ ID NO: 13.

[0014] Preferably, a LAMP primer set suitable for use in detecting fungal DNA in turf samples according to the present invention comprises four primers, including a pair of forward (FIP) and reverse (BIP) inner primers and a pair of forward (F3) and reverse (B3) outer primers. More preferably, a LAMP primer set suitable for use in the present invention comprises the addition of a loop-forward (LF) and / or loop-back (LB) primer to accelerate the amplification of nucleic acids present in turf samples and reduce the detection time of any target fungi that may be present in such turf samples. The embodiments of the LAMP primer set listed below relate to the detection of target fungal DNA in turf samples according to the methods of the present invention.

[0015] In the following description of embodiments relating to primers of the present invention having SEQ ID NOs: 14 to 91, it will be understood that primers useful in the present invention each independently and respectively have a sequence that is at least 90%, preferably at least 95%, more preferably at least 96%, and even more preferably at least 97% identical to the primers of SEQ ID NOs: 14 to 91.

[0016] In a particularly preferred embodiment, the primers useful in the present invention each independently and respectively have a sequence that is at least 98%, more preferably at least 99%, identical to the primers of SEQ ID NOs: 14 to 91. Most preferably, the primers useful in the present invention each independently and respectively have a sequence that is identical to the primers of SEQ ID NOs: 14 to 91.

[0017] Thus, the present invention provides a method for the prevention and treatment of dollar spot pathogens (Sclerotinia homoeocarpa), sheath blight pathogens (Rhizoctonia solani) species, Pythium aphanidermatum, damping-off pathogens (Gaeumannomyces graminis) species, pink snow mold pathogens (Microdochium nivale) species, Magnaporthe poae, Colletotrichum graminicola, Colletotrichum cereale and cabbage pythium rot pathogens (Pythium ultimum) The present invention provides a method for detecting fungal DNA in a turfgrass sample using a loop-mediated isothermal amplification (LAMP) assay including primers for the fungal DNA of at least one turf pathogenic fungus selected from the group consisting of Pseudomonas var. ultimum, the method comprising the step of subjecting the turf sample to a LAMP reaction, wherein the LAMP reaction uses a primer set of four or more nucleic acid sequences, each primer in the set having 15 to 50 nucleic acids, and the set of primers includes at least one primer set described below.

[0018] In one embodiment, the primer set for detecting dollar spot disease (Sclerotinia homoeocarpa) DNA comprises or is selected from SEQ ID NOs: 15 and 27.

[0019] In another embodiment, the primer set for detecting Sclerotinia homoeocarpa DNA comprises or is selected from SEQ ID NOs: 14, 15, 16 and 17.

[0020] In a further embodiment, the primer set for detecting Sclerotinia homoeocarpa DNA comprises or is selected from SEQ ID NOs: 14, 15, 16, 17, 18 and 19.

[0021] In a further embodiment, the primer set for detecting Rhizoctonia solani DNA comprises or is selected from SEQ ID NO:23.

[0022] In another embodiment, the primer set for detecting Rhizoctonia solani DNA comprises or is selected from SEQ ID NOs: 63, 64 and 65.

[0023] In another embodiment, the primer set for detecting Rhizoctonia solani DNA comprises or is selected from SEQ ID NOs: 20, 21, 22 and 23.

[0024] In another embodiment, the primer set for detecting Rhizoctonia solani DNA comprises or is selected from SEQ ID NOs: 62, 63, 66 and 67.

[0025] In yet another embodiment, the primer set for detecting Rhizoctonia solani DNA comprises or is selected from SEQ ID NOs: 62, 63, 64, 65, 66 and 67.

[0026] In another embodiment, the primer set for detecting Rhizoctonia solani DNA comprises or is selected from SEQ ID NOs: 20, 21, 22, 23, 24 and 25.

[0027] In another embodiment, the primer set for detecting Microdochium nivale species (preferably Microdochium nivale var. nivale) DNA comprises or is selected from SEQ ID NOs: 27, 28 and 29.

[0028] In another embodiment, the primer set for detecting Microdochium nivale species (preferably Microdochium nivale var. nivale) DNA comprises or is selected from SEQ ID NOs: 26, 27, 28, and 29.

[0029] In a further embodiment, the primer set for detecting Microdochium nivale species (preferably Microdochium nivale var. nivale) DNA comprises or is selected from SEQ ID NOs: 26, 27, 28, 29, 30 and 31.

[0030] In one embodiment, the primer set for detecting Pythium aphanidermatum DNA comprises or is selected from SEQ ID NOs: 33, 36 and 37.

[0031] In another embodiment, the primer set for detecting Pythium aphanidermatum DNA comprises or is selected from SEQ ID NOs: 32, 33, 36, and 37.

[0032] In another embodiment, the primer set for detecting Pythium aphanidermatum DNA comprises or is selected from SEQ ID NOs: 32, 33, 34, 35, 36 and 37.

[0033] In another embodiment, the primer set for detecting Pythium aphanidermatum DNA comprises or is selected from SEQ ID NOs: 69, 70, and 71.

[0034] In another embodiment, the primer set for detecting Pythium aphanidermatum DNA comprises or is selected from SEQ ID NOs: 68, 69, 72, and 73.

[0035] In another embodiment, the primer set for detecting Pythium aphanidermatum DNA comprises or is selected from SEQ ID NOs: 68, 69, 70, 71, 72, and 73.

[0036] In one embodiment, a primer set for detecting Gaeumannomyces graminis species (preferably Gaeumannomyces graminis var. avenae, Gaeumannomyces graminis var. graminis, or Gaeumannomyces graminis var. tritici) DNA comprises or is selected from SEQ ID NO: 60.

[0037] In another embodiment, the primer set for detecting Gaeumannomyces graminis species (preferably Gaeumannomyces graminis var. avenae, Gaeumannomyces graminis var. graminis, or Gaeumannomyces graminis var. tritici) DNA comprises or is selected from SEQ ID NOs: 42 and 43.

[0038] In a further embodiment, the primer set for detecting Gaeumannomyces graminis species (preferably Gaeumannomyces graminis var. avenae, Gaeumannomyces graminis var. graminis, or Gaeumannomyces graminis var. tritici) DNA comprises or is selected from SEQ ID NOs: 38, 39, 42, and 43.

[0039] In a further embodiment, the primer set for detecting Gaeumannomyces graminis species (preferably Gaeumannomyces graminis var. avenae, Gaeumannomyces graminis var. graminis, or Gaeumannomyces graminis var. tritici) DNA comprises or is selected from SEQ ID NOs: 56, 57, 60, and 61.

[0040] In another embodiment, a primer set for detecting Gaeumannomyces graminis species (preferably Gaeumannomyces graminis var. avenae, Gaeumannomyces graminis var. graminis, or Gaeumannomyces graminis var. tritici) DNA comprises or is selected from SEQ ID NOs: 38, 39, 40, 41, 42, and 43.

[0041] In another embodiment, a primer set for detecting Gaeumannomyces graminis species (preferably Gaeumannomyces graminis var. avenae, Gaeumannomyces graminis var. graminis, or Gaeumannomyces graminis var. tritici) DNA comprises or is selected from SEQ ID NOs: 56, 57, 58, 59, 60, and 61.

[0042] In one embodiment, the primer set for detecting Microdochium nivale species (preferably Microdochium nivale var. majus) DNA comprises or is selected from SEQ ID NOs: 48 and 49.

[0043] In a further embodiment, the primer set for detecting Microdochium nivale species (preferably Microdochium nivale var. majus) DNA comprises or is selected from SEQ ID NOs: 44, 45, 48 and 49.

[0044] In yet another embodiment, the primer set for detecting Microdochium nivale species (preferably Microdochium nivale var. majus) DNA comprises or is selected from SEQ ID NOs: 44, 45, 46, 47, 48 and 49.

[0045] In another embodiment, the primer set for detecting Magnaporthe poae DNA comprises or is selected from SEQ ID NOs: 54 and 55.

[0046] In a further embodiment, the primer set for detecting Magnaporthe poae DNA comprises or is selected from SEQ ID NOs: 50, 51, 54 and 55.

[0047] In another embodiment, the primer set for detecting Magnaporthe poae DNA comprises or is selected from SEQ ID NOs: 50, 51, 52, 53, 54, and 55.

[0048] In one embodiment, the primer set for detecting Colletotrichum graminicola DNA comprises or is selected from SEQ ID NOs: 74, 76 and 77.

[0049] In a further embodiment, the primer set for detecting Colletotrichum graminicola DNA comprises or is selected from SEQ ID NOs: 74, 75, 78 and 79.

[0050] In another embodiment, the primer set for detecting Colletotrichum graminicola DNA comprises or is selected from SEQ ID NOs: 74, 75, 76, 77, 78, and 79.

[0051] In another embodiment, the primer set for detecting Colletotrichum cereale DNA comprises or is selected from SEQ ID NOs: 80, 82, and 83.

[0052] In a further embodiment, the primer set for detecting Colletotrichum cereale DNA comprises or is selected from SEQ ID NOs: 80, 81, 84 and 85.

[0053] In another embodiment, the primer set for detecting Colletotrichum cereale DNA comprises or is selected from SEQ ID NOs: 80, 81, 82, 83, 84, and 85.

[0054] In one embodiment, the primer set for detecting Pythium ultimum var. Ultimum ldin-rc DNA comprises or is selected from SEQ ID NOs: 86, 88 and 89.

[0055] In a further embodiment, the primer set for detecting Pythium ultimum var. Ultimum ldin-rc DNA comprises or is selected from SEQ ID NOs: 86, 87, 90 and 91.

[0056] In another embodiment, the primer set for detecting Pythium ultimum var. Ultimum ldin-rc DNA comprises or is selected from SEQ ID NOs: 86, 87, 88, 89, 90, and 91.

[0057] The LAMP assay of the present invention can be used for the detection, including early detection, of DNA from turf fungi selected from the group consisting of Sclerotinia homoeocarpa, Rhizoctonia solani, Pythium aphanidermatum, Gaeumannomyces graminis spp., Microdochium nivale spp., Magnaporthe poae, Colletotrichum graminicola, Colletotrichum cereale, and Pythium ultimum var. Ultimum in turf samples, which is readily available and allows turfgrass management and / or maintenance to be adjusted accordingly.

[0058] According to the invention, "turfgrass" is understood to be an annual or perennial grass of the Gramineae family, preferably belonging to one or more of the genera Agropyron, Agrostis, Axonopus, Bromus, Buchloe, Cynodon, Eremochloa, Festuca, Lolium, Paspulum, Pennisetum, Phleum, Poa, Stenotaphrum or Zoysia. More preferably, the family Gramineae belongs to one or more of the genera Agrostis, Buchloe, Cynodon, Eremochloa, Festuca, Lolium, Paspulum, Pennisetum, Poa, Stenotaphrum or Zoysia.

[0059] In one embodiment, according to the present invention, "turf" is understood to be a group of turfgrass that covers a surface area of ​​the ground and is regularly maintained.

[0060] The present invention can be practiced on all turfgrasses, including cool-season and warm-season turfgrasses.

[0061] Examples of cool-season turfgrasses are strawberry weeds (Poa L.), such as longgrass (Poa pratensis L.), bluegrass (Poa trivialis L.), bluegrass (Poa compressa L.), and annual bluegrass (Poa annua L.); bentgrasses (Agrostis L.), such as creeping bentgrass (Agrostis palustris Huds.), sedge weed (Agrostis tenius Sibth.), and dwarf weed (Agrostis canina L.). L.) and dun grass (Agrostis alba L.); fescue (Festuca L.), e.g., big fescue (Festuca rubra L.), tall fescue (Festuca rubra var. commutata Gaud.), fescue (Festuca ovina L.), Korean fescue (Festuca longifolia), tall fescue (Festuca arundinacea Schreb.) Schreb.), tall fescue (Festuca elatior L.); ryegrass (Lolium L.), e.g., perennial ryegrass (Lolium perenne L.), annual (Italian) ryegrass (Lolium multiflorum Lam.); wheatgrass (Agropyron Gaertn.), e.g., fairway wheatgrass (Agropyron cristatum L. Gaertn.).) and Western wheatgrass (Agropyron smithii Rydb.). Other cool-season turfgrasses include bromegrass (Bromus inermis Leyss.) and timothy grass (Phleum L.).

[0062] Examples of warm-season turfgrasses include Bermudagrass (Cynodon LCRich), Zoysia Willd., St. Augustinegrass (Stenotaphrum secundatum (Walt.) Kuntze), Japanese laurel wattle (Eremochloa ophiuroides (Munro.) Hack.), carpetgrass (Axonopus Beauv.), Pacific bluegrass (Paspalum notatum Flugge.), and kikuyugrass (Pennisetum clandestinum Hochst. ex Chiov.). Hochst. ex Chiov.), buffalo grass (Buchloe dactyloides (Nutt.) Engelm.), and swartzgrass (Paspalum vaginatum swartz).

[0063] The LAMP method invention also contemplates a kit for detecting fungi in turfgrass samples using a LAMP assay. Test strips containing one or more of the primer sets described herein may be used. In one embodiment, multiple primer sets are multiplexed in a test strip for detecting multiple diseases from turfgrass samples collected from a specific location.

[0064] For example, a 1 cm cubic of homogenized grass sample is placed in a ball-and-socket bijou tube containing an appropriate amount of lysis buffer and shaken vigorously for 1 minute. All test strips containing sample wells contain resuspension buffer, and a few drops of this test solution are placed in the sample wells in the test strip, where the wells contain all the components necessary to perform a LAMP reaction (e.g., primer sets and reagents such as an isothermal master mix, catalog number iso-001, available from Optigene). In one embodiment, the test strip is multiplexed. In another embodiment, the test strip contains eight wells (two controls and six for the grass disease of interest). In one embodiment, the test strip is coupled to a diagnostic instrument, such as the Genie® II or III, available from OptiGene.

[0065] Primer design Highly conserved genes were used to design LAMP primers for the detection of DNA from selected turfgrass pathogens (Table 3, column 1). Pure genomic DNA from all fungi of interest was obtained using NucleoSpin Plant II (Macherey-Nagel). PCR techniques were used to amplify the sequences of interest using published primer pairs, followed by Sanger sequencing. The following DNA loci (genes and regions) were sequenced: internal transcribed spacer (ITS), elongation factor 1-α (EF), β-tubulin (Tub), cytochrome c oxidase subunit 1 (Cox), superoxide dismutase (SOD1), and large subunit nuclear ribosomal RNA (LSU). Raw sequences were aligned using the ClustalW alignment method (CLC Main Workbench Software). Gene homologs were identified using BLAST comparison with sequences from GenBank (NCBI). Ideally, good sequences are defined for all target taxa by successful PCR amplification and have no homology with other taxa.

[0066] The best sequences (SEQ ID NOS: 1-13) from the sequenced DNA loci were then used to design LAMP primers for each of the selected turfgrass pathogens using LAMP Designer 1.14 (PREMIER Biosoft). Thus, different parameters were tested to obtain different primer sets for each organism and locus (see Table 3 for a correlation between turf pathogens, selected loci, and the SEQ ID NOS of the best sequences used for primer design). The designed primer sets shown in Table 1 were then tested for their specificity (Table 3) and sensitivity (Table 4).

[0067] [Table 2-1] [Table 2-2]

[0068] specificity To examine the specificity of the reaction (see references below), the assay using the designed primer set was tested using pure genomic DNA extracts from the fungal isolates listed in Table 2. A comprehensive collection of turfgrass pathogens from different geographical origins was collected and grown on various media (potato dextrose / malt / cornmeal / cherry / V8). DNA was extracted from mycelia using 10-day-old fungal cultures (NucleoSpin Plant II-MACHEREY-NAGEL). Genomic DNA was diluted to 5 ng / μl with nuclease-free water, and a 2.5 μl aliquot was used for specificity testing.

[0069] LAMP specificity tests were performed in a LightCycler 480 (Roche) in 96-well plates at 64°C for 55 minutes. Amplicon-specific annealing temperatures were determined during cooling from 98°C to 65°C at a ramp rate of -0.1°C / s. Real-time LAMP assays were performed in 10 μl reaction mixtures containing 5 μl of 1× isothermal master mix (Optigene), 0.4 μM of each external primer, 1.6 μM of each internal primer, and 0.8 μM of each loop primer (synthesized by Microsynth) and 2.5 μl of genomic DNA.

[0070] All reactions were performed in duplicate and on two different days.

[0071] Literature for carrying out specificity: Besuschio, SA, Murcia, ML, Benatar, AF, Monnerat, S., Cruz, I., Picado, A., Schijman, AG (2017). Analytical sensitivity and specificity of a loop-mediated isothermal amplification (LAMP) kit prototype for detection of Trypanosoma cruzi DNA in human blood samples. PLOS Neglected Tropical Diseases,11(7),e0005779.https: / / doi.org / 10.1371 / journal.pntd.0005779 Kitamura, M., Aragane, M., Nakamura, K., Watanabe, K., & Sasaki, Y. (2016). Development of Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of Cannabis sativa. Biological and Pharmaceutical Bulletin, 39(7), 1144-1149. https: / / doi.org / 10.1248 / bpb.b16-00090 Seki, M., Kilgore, P. E., Kim, E. J., Ohnishi, M., Hayakawa, S., & Kim, D. W. (2018). Loop-Mediated Isothermal Amplification Methods for Diagnosis of Bacterial Meningitis. Frontiers in Pediatrics, 6. https: / / doi.org / 10.3389 / fped.2018.00057 Wang, D.-G., Brewster, J. D., Paul, M., & Tomasula, P. M. (2015). Two Methods for Increased Specificity and Sensitivity in Loop-Mediated Isothermal Amplification. Molecules, 20(4), 6048-6059. https: / / doi.org / 10.3390 / molecules20046048

[0072]

Table 3-1

Table 3-2

[0073] Interpretation of Results: The specificity of the LAMP assay was examined against the designed specific targets for the fungal strains listed in Table 2, as summarized in Table 3. As a further confirmation of specificity, consistent melting temperatures of 82.6-89.9°C ± 0.5°C were observed for the different amplification products, also as shown in Table 3.

[0074] [Table 4]

[0075] The sensitivity of the listed primer sets corresponding to the DNA of SEQ ID NOS: 1-13 (Table 1) was determined using serial dilutions (1 ng to 100 fg) of genomic DNA from all fungi of interest, with each reaction performed in duplicate on two different days. Pure genomic DNA from all fungi was obtained using a NucleoSpin Plant II (Macherey-Nagel). LAMP susceptibility tests were performed in a 96-well plate at 64°C for 55 minutes on a LightCycler 480 (Roche). Amplicon-specific annealing temperatures were determined during cooling from 98°C to 65°C at a ramp rate of -0.1°C / s. Real-time LAMP assays were performed in 10 μl reaction mixtures containing 5 μl of 1x isothermal master mix (Optigene), 0.4 μM of each external primer, 1.6 μM of each internal primer, and 0.8 μM of each loop primer (synthesized by Microsynth), and 2.5 μl of genomic DNA.

[0076] [Table 5]

[0077] Methods for detecting fungal pathogens in turfgrass samples Sample collection Turf samples containing grass roots are collected in areas where fungal pathogens are suspected. Turfgrass may also show symptoms. Turf samples are placed in clear 50 ml tubes (Corning) and stored at -20°C until use. DNA is extracted using a Plant Material Lysis Kit (Optigene). 1 cm of the turf sample is then lysed. 3 The cubes are placed in a bijou tube containing 1 ml of lysis buffer (Optigene). The bijou tube is shaken for 1 minute to homogenize the grass sample. A volume of 10 μl of the lysate is transferred to the provided dilution tube (Optigene) and mixed vigorously by shaking. The diluted lysate is then defined as the template.

[0078] LAMP reaction In one embodiment, the LAMP reaction is carried out at about 60° C. to about 70° C., for example, about 64° C. to about 67° C., or about 64° C. to about 66° C. In a particular example, the LAMP reaction is carried out at 64° C.

[0079] In one embodiment, the LAMP reaction is allowed to proceed for about 15 to about 45 minutes, for example, about 20 to about 40 minutes, or about 25 to about 35 minutes.

[0080] In one embodiment, the primer concentrations in the LAMP reaction of the present invention are 1.4 to 1.8 μM, more particularly 1.6 μM, for the forward (FIP) and reverse (BIP) inner primers, and 0.2 to 0.4 μM, more particularly 0.4 μM, and 0.4 to 0.8 μM, more particularly 0.8 μM, for the forward (F3) and reverse (B3) outer primers, loop-forward (LF) and / or loop-back (LB) primers, which are useful for accelerating the amplification of nucleic acids present in turf samples and reducing the detection time of any target fungal DNA that may be present in such turf samples.

[0081] Suitable buffer systems useful for the LAMP assay reaction include: 1X Isothermal Amplification Buffer Pack from New England Biolabs 20 mM Tris-HCl 10mM (NH4)2SO4 50 mM KCl 2mM MgSO4 0.1% Tween® 20 (pH 8.8 (25℃)) 1X Isothermal Amplification Buffer II Pack from New England Biolabs 20 mM Tris-HCl 10mM (NH4)2SO4 150 mM KCl 2mM MgSO4 0.1% Tween® 20 (pH 8.8 (25℃))

[0082] Suitable enzyme systems (such as DNA polymerases) useful in the LAMP assay reaction include the following:

[0083] [Table 6]

[0084] In one embodiment, LAMP reactions are performed in test strips on a Genie instrument (Optigene) using dried reagents (Optigene). In one embodiment, the test strips have eight 150 μl wells (two controls and six for the assay). Real-time LAMP assays are performed in 25 μl reaction mixtures containing 15 μl of 1x isothermal master mix (Optigene), 0.4 μM of each external primer, 1.6 μM of each internal primer, and 0.8 μM of each loop primer (synthesized by Microsynth) (selected from at least one of the primer sets in Table 1). Before adding the template, lyophilized reaction strips are resuspended in 22 μl of resuspension buffer (Optigene). All test strips include negative control and positive plant control primer sets provided by Optigene. For all assays, 3 μl of template is added per reaction and well. The reactions are held at 64°C for 30-55 minutes, followed by an annealing program. The temperature profile of the annealing program is determined during cooling from 98° C. to 65° C. at a ramp rate of −0.1° C. / s.

[0085] The isothermal master mix contains a fluorescent double-stranded DNA binding dye to allow real-time detection of amplicons. The assay is optimized for reaction time, temperature, and volume of DNA added per reaction.

[0086] Fluorescence data acquired during the amplification step at 64°C is reported as amplification time. Fluorescence derivative data acquired during the annealing step is reported as annealing temperature.

[0087] Alternatively, the LAMP assay reaction does not include an annealing program, in which case a pH-sensitive indicator dye can be used to assess the presence of target fungal DNA. In some examples, the pH-sensitive indicator dye is a colored dye detectable in visible light. In particular examples, the colored dye includes cresol red, phenol red, m-cresol purple, bromocresol purple, neutral red, naphtholphthalein, thymol blue, or naphtholphthalein. In other examples, the pH-sensitive indicator dye is a fluorescent indicator dye. In particular examples, the fluorescent dye includes 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein, 5(6)-carboxy-2',7'-dichlorofluorescein, 5(6)-carboxyfluorescein, 3,6-diacetoxyphthalonitrile, 6,8-dihydroxy-1,3-pyrene disulfonic acid, or 5-(and-6)-carboxyl seminaphthorhodafluor.

[0088] Following the procedures described above, detecting the presence of fungal pathogen DNA (Table 3) in turf samples can indicate the presence of turf fungal pathogens that can cause related turf diseases (including, for example, anthracnose, take-all patch, summer patch, snow mold, red burn, leaf rot, and dollar spot). Early and efficient detection allows appropriate turfgrass disease management decisions to be made.

[0089] Sequence Listing <210> 1 <211> 912 <212> DNA <213> Dollar spot fungus (Sclerotinia homoeocarpa) <400> 1 [ka] <210> 2 <211> 629 <212> DNA <213> Rhizoctonia solani AG2-2IIIB <400> 2

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Claims

1. Dollar spot fungus (Sclerotinia homoeocarpa), sheath blight fungus (Rhizoctonia solani) species, Pythium aphanidermatum, damping-off fungus (Gaeumannomyces graminis) species, pink snow mold fungus (Microdochium nivale) species, Magnaporthe poae, Colletotrichum graminicola, Colletotrichum cereale, and cabbage pythium rot fungus (Pythium ultimum).

1. A method for detecting fungal DNA in turfgrass samples using a loop-mediated isothermal amplification (LAMP) assay comprising primers for fungal DNA of at least one turf pathogenic fungus selected from the group consisting of: The method includes subjecting the grass sample to a LAMP reaction, wherein the LAMP reaction uses a primer set of four or more nucleic acid sequences, each primer in the set having 15 to 50 nucleic acids, and the set of primers comprises: (a) a primer set for detecting dollar spot disease fungus (Sclerotinia homoeocarpa) DNA, comprising or selected from primers each having a sequence at least 90% identical to SEQ ID NOs: 14, 15, 16, and 17; (b) a primer set for detecting Rhizoctonia solani DNA, comprising or selected from primers each having a sequence at least 90% identical to SEQ ID NOs: 20, 21, 22, and 23, respectively, or comprising or selected from primers each having a sequence at least 90% identical to SEQ ID NOs: 62, 63, 66, and 67, respectively; (c) a primer set for detecting Microdochium nivale var. nivale DNA, comprising or selected from primers each having a sequence at least 90% identical to SEQ ID NOs: 26, 27, 28, and 29; (d) a primer set for detecting Pythium aphanidermatum DNA, comprising or selected from primers each having a sequence at least 90% identical to SEQ ID NOs: 32, 33, 36, and 37, respectively; (e) a primer set for detecting Gaeumannomyces graminis var. avenae DNA, comprising or selected from primers each having a sequence at least 90% identical to SEQ ID NOs: 38, 39, 42, and 43, respectively; (f) a primer set for detecting Microdochium nivale var. majus DNA, comprising or selected from primers each having a sequence at least 90% identical to SEQ ID NOs: 44, 45, 48, and 49; (g) a primer set for detecting Magnaporthe poae DNA comprising or selected from primers each having a sequence at least 90% identical to SEQ ID NOs: 50, 51, 54, and 55; (h) a primer set for detecting Colletotrichum graminicola DNA, comprising or selected from primers each having a sequence at least 90% identical to SEQ ID NOs: 74, 75, 78, and 79; (i) a primer set for detecting Colletotrichum cereale DNA, comprising or selected from primers each having a sequence at least 90% identical to SEQ ID NOs: 80, 81, 84, and 85; and (j) A primer set for detecting Pythium ultimum var. Ultimum DNA, comprising or selected from primers each having a sequence at least 90% identical to SEQ ID NOs: 86, 87, 90, and 91. The method comprises at least one primer set selected from:

2. 2. The method of claim 1, wherein the primer set for detecting dollar spot disease (Sclerotinia homoeocarpa) DNA comprises or is selected from primers each having a sequence at least 90% identical to SEQ ID NOs: 14, 15, 16, 17, 18, and 19, respectively.

3. 2. The method of claim 1, wherein the primer set for detecting Rhizoctonia solani DNA comprises or is selected from primers each having a sequence at least 90% identical to SEQ ID NOs: 62, 63, 64, 65, 66, and 67, respectively.

4. 2. The method of claim 1, wherein the primer set for detecting Rhizoctonia solani DNA comprises or is selected from primers each having a sequence at least 90% identical to SEQ ID NOs: 20, 21, 22, 23, 24, and 25, respectively.

5. 2. The method of claim 1, wherein the primer set for detecting Microdochium nivale var. nivale DNA comprises or is selected from primers each having a sequence at least 90% identical to SEQ ID NOs: 26, 27, 28, 29, 30, and 31, respectively.

6. 2. The method of claim 1, wherein the primer set for detecting Pythium aphanidermatum DNA comprises or is selected from primers each having a sequence at least 90% identical to SEQ ID NOs: 32, 33, 34, 35, 36, and 37, respectively.

7. 2. The method of claim 1, wherein the primer set for detecting Pythium aphanidermatum DNA comprises or is selected from primers each having a sequence at least 90% identical to SEQ ID NOs: 68, 69, 70, 71, 72, and 73, respectively.

8. 2. The method of claim 1, wherein the primer set for detecting Gaeumannomyces graminis var. avenae DNA comprises or is selected from primers each having a sequence at least 90% identical to SEQ ID NOs: 38, 39, 40, 41, 42, and 43, respectively.

9. 2. The method of claim 1, wherein the primer set for detecting Gaeumannomyces graminis var. avenae DNA comprises or is selected from primers each having a sequence at least 90% identical to SEQ ID NOs: 56, 57, 58, 59, 60, and 61, respectively.

10. 2. The method of claim 1, wherein the primer set for detecting Microdochium nivale var. majus DNA comprises or is selected from primers each having a sequence at least 90% identical to SEQ ID NOs: 44, 45, 46, 47, 48, and 49, respectively.

11. 2. The method of claim 1, wherein the primer set for detecting Magnaporthe poae DNA comprises or is selected from primers each having a sequence that is at least 90% identical to SEQ ID NOs: 50, 51, 52, 53, 54, and 55, respectively.

12. 2. The method of claim 1, wherein the primer set for detecting Colletotrichum graminicola DNA comprises or is selected from primers each having a sequence at least 90% identical to SEQ ID NOs: 74, 75, 76, 77, 78, and 79, respectively.

13. 2. The method of claim 1, wherein the primer set for detecting Colletotrichum cereale DNA comprises or is selected from primers each having a sequence that is at least 90% identical to SEQ ID NOs: 80, 81, 82, 83, 84, and 85, respectively.

14. 2. The method of claim 1, wherein the primer set for detecting Pythium ultimum var. Ultimum DNA comprises or is selected from primers each having a sequence at least 90% identical to SEQ ID NOs: 86, 87, 88, 89, 90, and 91, respectively.

15. A kit for the detection of fungal DNA in turfgrass samples using a LAMP assay, comprising one or more of the primer sets identified in claims 1-14.

16. The kit of claim 15, further comprising a buffer solution and a DNA polymerase.

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