Method for detecting fungi in turfgrass using LAMP assays with novel primer sets

Abstract

The present invention provides a method for detecting fungal DNA in a turfgrass sample using a loop-mediated isothermal amplification (LAMP) assay containing primers for fungal DNA of at least one turfgrass pathogenic fungus, the present invention relates to a method for LAMP detection of lawn pathogenic fungi selected from the group consisting of: Sclerotinia colletotrichum, Rhizoctonia solani species, Pythium aphanidermatum, Cyclotheca graminearum species, Gerlachia nilaparis species, Pseudomonas aeruginosa, Colletotrichum graminearum, Colletotrichum gloeosporioides, and Pythium ultimate variants, the method comprising: subjecting a lawn sample to an LAMP reaction, wherein the LAMP reaction uses a primer set of four or more nucleic acid sequences, each primer in the set having from 15 to 50 nucleic acids. The primers useful in the method are selected from specifically selected internal transcriptional spacers or genes of the target fungi to provide improved assay results.

Description

[0001] This application is a divisional application. The corresponding parent application has the application number 201980011873.3, the application date is February 5, 2019, and the name of the invention is “A method for detecting fungi in lawn grass using a LAMP assay with a novel primer set”. Technical Field

[0002] The present invention relates to methods for detecting disease in turfgrass caused by fungal pathogens by assaying such turfgrass samples using loop-mediated isothermal amplification (LAMP) to detect nucleic acids from one or more fungi. Background Art

[0003] As described in, for example, US 6410278 (Eiken), LAMP or loop-mediated isothermal amplification is a DNA amplification method characterized by the use of at least 4 or more different primers (see, for example, http: / / loopamp.eiken.co.jp / e / lamp / primer.html ), these primers are specifically designed to recognize 6 different regions on the target gene, and the reaction process is carried out by strand displacement reaction at a constant temperature. 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 (about 65°C), the amplification and detection of the target nucleic acid of interest can be completed in a single step. It provides high amplification efficiency, in which DNA is amplified multiple times within 15-60 minutes. Due to its high specificity, the presence of the amplified product can indicate the presence of the target gene (http: / / loopamp.eiken.co.jp / e / lamp / principle.html).

[0004] Turfgrass managers face many problems in keeping turfgrass at the quality standards expected by users. Although there are many problems, those related to diseases (including diseases caused by fungal pathogens) are particularly challenging to manage and control. For example, diseases can affect turfgrass plants on golf courses, causing loss of revenue due to reduced quality (including playability). For golf course managers, an example of a common problem is knowing which diseases are present so that appropriate and timely management techniques can be adopted. Turfgrass diseases caused by turf pathogenic microorganisms include, for example, anthracnose, take-all, summer spot, snow rot, pythium blight, brown spot, dollar spot, etc.

[0005] Agriculturally active chemicals, such as fungicides, used to control pathogens are often applied to golf courses as needed based on the level of disease pressure, pathogen populations, weather, etc. However, the application of fungicides is highly controlled by the course budget, the availability of appropriate equipment, and the availability of qualified personnel to apply the agriculturally active chemicals.

[0006] In view of these problems, it would be very useful to establish a rapid and reliable method for detecting pathogenic fungi in lawns. Known PCR assays are not practical for golf courses or other intensively managed lawn grasses or professional landscape environments because PCR requires specialized laboratory skills and instrumentation. For example, certain other molecular biological methods for detecting fungal diseases in lawn grasses are known and described in WO 2009147017, which involves the TRFLP method. Summary of the invention

[0007] Thus, the present invention relates to LAMP assays for detecting the presence of DNA in turfgrass samples that is associated with selected fungal pathogens that cause relevant turfgrass diseases, including, for example, anthracnose, take-all, summer blight, snow rot, pythium wilt, brown spot, and dollar spot.

[0008] In order to facilitate timely and effective detection of turfgrass disease pathogens and to improve the cost and effectiveness of turfgrass disease treatment, the LAMP assay according to the present invention can be used for early detection of DNA associated with fungal pathogens that cause relevant turfgrass diseases. According to the present invention, the LAMP method is suitable for using a primer set derived from at least four and preferably six or more nucleic acid sequences of the target disease pathogen. More particularly, the method of the present invention provides that each primer used in the primer set selected for the LAMP assay has 15 to 50 nucleic acids, and wherein the primers in the set are selected from specific DNA loci within the target fungus.

[0009] According to the present invention, a method is provided for detecting fungal DNA in a turfgrass sample using a loop-mediated isothermal amplification (LAMP) assay containing primers for fungal DNA (nucleic acid) of turf pathogenic fungi 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 and preferably six or more nucleic acid sequences, wherein each primer in the set has 15 to 50 nucleic acids, and wherein the fungal DNA to be detected is obtained from a target fungal pathogen. Primers useful in the present LAMP assay method are selected from specific internal transcribed spacer regions or genes of the target fungus to provide improved assay results.

[0010] In a particular embodiment, 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 other embodiments, the target fungus of the species Rhizoctonia solani is selected from Rhizoctonia solani AG2-2IV and Rhizoctonia solani AG2-2IIIB.

[0011] In the context of the present invention, detection of fungal DNA in turfgrass samples using the LAMP assay of the present invention can indicate the presence of fungal pathogens and can also assist in assessing the occurrence or presence of turfgrass disease symptoms such as:

[0012]

[0013] In one embodiment,

[0014] (a) the primer set for the DNA of Sclerotinia sclerotiorum is selected from the DNA of SEQ ID NO: 1;

[0015] (b) the primer set for Rhizoctonia solani DNA is selected from the DNA of SEQ ID NO: 2 or SEQ ID NO: 9;

[0016] (c) a primer set for a DNA of a species of Pseudomonas nivalis (preferably Pseudomonas nivalis var. nivalis) is selected from the DNA of SEQ ID NO: 3;

[0017] (d) the primer set for Pythium aphanidermatum DNA is selected from the DNA of SEQ ID NO: 4 or SEQ ID NO: 10;

[0018] (e) a primer set for a G. graminis species DNA (preferably G. graminis var. avenae, G. graminis var. oryzae or G. graminis var. tritici) selected from the DNA of SEQ ID NO: 5 or SEQ ID NO: 8;

[0019] (f) a primer set for a DNA of a species of Pseudomonas nivalis (preferably Pseudomonas nivalis var. macrosporum) is selected from the DNA of SEQ ID NO: 6;

[0020] (g) the primer set for the DNA of summer leaf spot pathogen is selected from the DNA of SEQ ID NO: 7;

[0021] (h) a primer set for Colletotrichum graminearum DNA is selected from the DNA of SEQ ID NO: 11;

[0022] (i) a primer set for corn anthracnose DNA is selected from the DNA of SEQ ID NO: 12; and

[0023] (j) The primer set for the Pythium ultimum var. ultimum DNA is selected from the DNA of SEQ ID NO: 13.

[0024] Preferably, the LAMP primer set suitable for use in detecting fungal DNA in lawn 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, the LAMP primer set suitable for use in the present invention includes the addition of a forward loop primer (LF) and / or a reverse loop primer (LB) to accelerate the amplification of nucleic acids present in lawn samples and reduce the detection time of any target fungi that may be present in such lawn samples. The LAMP primer set embodiments listed below relate to detecting target fungal DNA in lawn samples according to the method of the present invention.

[0025] In the following description of the embodiments associated with primers of SEQ ID No 14-91 according to the present invention, it will be understood that the primers useful in the present invention each independently and separately 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 No 14-91.

[0026] In a particularly preferred embodiment, the primers useful in the present invention each independently and separately have a sequence that is at least 98%, more preferably at least 99% identical to the primers of SEQ IDs 14 to 91. Most preferably, the primers useful in the present invention each independently and separately have a sequence that is identical to the SEQ IDs 14 to 91.

[0027] Therefore, the present invention provides a method for detecting fungal DNA in a turf grass sample using a loop-mediated isothermal amplification (LAMP) assay, the assay containing primers for the fungal DNA of at least one turf pathogenic fungus, which are selected from: Sclerotinia sclerotiorum, Rhizoctonia solani species, Pythium aphanidermatum, Gramineus species, Nyctolagus nivalis species, Pythium ultimum blight, Colletotrichum graminearum, Colletotrichum ulminum and Pythium ultimum var. ultimum, the method comprising: subjecting the turf 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 wherein the set of primers comprises at least one primer set as described below.

[0028] In one embodiment, the primer set for detecting Sclerotinia sclerotiorum DNA comprises or is selected from SEQ ID NOs: 15 and 27.

[0029] In another embodiment, the primer set for detecting Sclerotinia sclerotiorum DNA comprises or is selected from SEQ ID NOs: 14, 15, 16 and 17.

[0030] In other embodiments, the primer set for detecting Sclerotinia sclerotiorum DNA comprises or is selected from SEQ ID NOs: 14, 15, 16, 17, 18 and 19.

[0031] In other embodiments, the primer set for detecting Rhizoctonia solani DNA comprises or is selected from SEQ ID NO:23.

[0032] In another embodiment, the primer set for detecting Rhizoctonia solani DNA comprises or is selected from SEQ ID NOs: 63, 64 and 65.

[0033] In another embodiment, the primer set for detecting Rhizoctonia solani DNA comprises or is selected from SEQ ID NOs: 20, 21, 22 and 23.

[0034] In another embodiment, the primer set for detecting Rhizoctonia solani DNA comprises or is selected from SEQ ID NO:62, 63, 66 and 67.

[0035] 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.

[0036] In another embodiment, the primer set for detecting Rhizoctonia solani DNA comprises or is selected from SEQ ID NO: 20, 21, 22, 23, 24 and 25.

[0037] In another embodiment, the primer set for detecting Pseudomonas aeruginosa species (preferably Pseudomonas aeruginosa var. nivalis) DNA comprises or is selected from SEQ ID NOs: 27, 28 and 29.

[0038] In another embodiment, the primer set for detecting Pseudomonas aeruginosa species (preferably Pseudomonas aeruginosa var. nivalis) DNA comprises or is selected from SEQ ID NOs: 26, 27, 28 and 29.

[0039] In other embodiments, the primer set for detecting the DNA of the species of Pseudomonas nivalis (preferably Pseudomonas nivalis var. nivalis) comprises or is selected from SEQ ID Nos: 26, 27, 28, 29, 30 and 31.

[0040] In one embodiment, the primer set for detecting Pythium aphanidermatum DNA comprises or is selected from SEQ ID NOs: 33, 36 and 37.

[0041] In another embodiment, the primer set for detecting Pythium aphanidermatum DNA comprises or is selected from SEQ ID NOs: 32, 33, 36 and 37.

[0042] In another embodiment, the primer set for detecting Pythium aphanidermatum DNA comprises or is selected from SEQ ID NO:32, 33, 34, 35, 36 and 37.

[0043] In another embodiment, the primer set for detecting Pythium aphanidermatum DNA comprises or is selected from SEQ ID NOs: 69, 70 and 71.

[0044] In another embodiment, the primer set for detecting Pythium aphanidermatum DNA comprises or is selected from SEQ ID NOs: 68, 69, 72 and 73.

[0045] In another embodiment, the primer set for detecting Pythium aphanidermatum DNA comprises or is selected from SEQ ID NO:68, 69, 70, 71, 72 and 73.

[0046] In one embodiment, the primer set for detecting the DNA of a species of G. graminis (preferably G. graminis var. avenae, G. graminis var. oryzae or G. graminis var. tritici) comprises or is selected from SEQ ID NO:60.

[0047] In another embodiment, the primer set for detecting the DNA of a species of G. graminis (preferably G. graminis var. avenae, G. graminis var. oryzae or G. graminis var. tritici) comprises or is selected from SEQ ID NOs: 42 and 43.

[0048] In other embodiments, the primer set for detecting the DNA of a species of G. graminis (preferably G. graminis var. avenae, G. graminis var. oryzae or G. graminis var. tritici) comprises or is selected from SEQ ID NOs: 38, 39, 42 and 43.

[0049] In other embodiments, the primer set for detecting the DNA of a species of G. graminis (preferably G. graminis var. avenae, G. graminis var. oryzae or G. graminis var. tritici) comprises or is selected from SEQ ID NOs: 56, 57, 60 and 61.

[0050] In another embodiment, the primer set for detecting the DNA of a species of G. graminis (preferably G. graminis var. avenae, G. graminis var. oryzae or G. graminis var. tritici) comprises or is selected from SEQ ID NOs: 38, 39, 40, 41, 42 and 43.

[0051] In another embodiment, the primer set for detecting the DNA of a species of G. graminis (preferably G. graminis var. avenae, G. graminis var. oryzae or G. graminis var. tritici) comprises or is selected from SEQ ID NOs: 56, 57, 58, 59, 60 and 61.

[0052] In one embodiment, the primer set for detecting Pseudomonas nivalis species (preferably Pseudomonas nivalis var. macrosporum) DNA comprises or is selected from SEQ ID NOs: 48 and 49.

[0053] In other embodiments, the primer set for detecting the DNA of a species of Pseudomonas nivalis (preferably Pseudomonas nivalis var. macrosporum) comprises or is selected from SEQ ID NOs: 44, 45, 48 and 49.

[0054] In yet another embodiment, the primer set for detecting the DNA of a species of Pseudomonas nivalis (preferably Pseudomonas nivalis var. macrosporum) comprises or is selected from SEQ ID Nos: 44, 45, 46, 47, 48 and 49.

[0055] In another embodiment, the primer set for detecting the DNA of Pseudomonas aeruginosa comprises or is selected from SEQ ID NOs: 54 and 55.

[0056] In other embodiments, the primer set for detecting the DNA of Pseudomonas aeruginosa comprises or is selected from SEQ ID NOs: 50, 51, 54 and 55.

[0057] In another embodiment, the primer set for detecting the DNA of Pseudomonas aeruginosa comprises or is selected from SEQ ID NOs: 50, 51, 52, 53, 54 and 55.

[0058] In one embodiment, the primer set for detecting Colletotrichum graminearum DNA comprises or is selected from SEQ ID NOs: 74, 76 and 77.

[0059] In other embodiments, the primer set for detecting Colletotrichum graminearum DNA comprises or is selected from SEQ ID NO:7475, 78 and 79.

[0060] In another embodiment, the primer set for detecting Colletotrichum graminearum DNA comprises or is selected from SEQ ID NO:74, 75, 76, 77, 78 and 79.

[0061] In another embodiment, the primer set for detecting corn anthracnose DNA comprises or is selected from SEQ ID No 80, 82 and 83.

[0062] In other embodiments, the primer set for detecting corn anthracnose DNA comprises or is selected from SEQ ID NO: 80, 81, 84 and 85.

[0063] In another embodiment, the primer set for detecting corn anthracnose DNA comprises or is selected from SEQ ID Nos: 80, 81, 82, 83, 84 and 85.

[0064] 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.

[0065] In other embodiments, 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.

[0066] 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.

[0067] The LAMP assay of the present invention can be used to detect (including early detection) DNA from turfgrass fungi, which are easily accessible and allow the management and / or maintenance of turfgrass to be adjusted accordingly. These turfgrass fungi are selected from the group consisting of: Sclerotinia sclerotiorum, Rhizoctonia solani, Pythium aphanidermatum, Aspergillus graminis species, Pythium nivale species, Pythium summer leaf spot, Colletotrichum graminearum, Colletotrichum graminearum and Pythium ultimum var. ultimum in turfgrass samples.

[0068] According to the present invention, "lawn grass" is understood to be an annual or perennial grass. The grass preferably belongs to one or more of the following genera: Agropyron, Bentgrass, Carpetgrass, Bromus, Buffalo, Cynodon, Centipedegrass, Fescue, Lolium, Paspalum, Pennisetum, Timothy, Poa, Agrimony or Zoysia. More preferably, the grass belongs to one or more of the following genera: Bentgrass, Buffalo, Cynodon, Centipedegrass, Fescue, Lolium, Paspalum, Pennisetum, Poa, Agrimony or Zoysia.

[0069] In one embodiment, a "lawn" according to the present invention is understood to be a group of lawn grasses which covers a surface area of ​​the ground and is subject to regular maintenance.

[0070] The present invention can be practiced with all turf grasses, including cool-season turf grasses as well as warm-season turf grasses.

[0071] Examples of cool-season turf grasses are: bluegrass (Poa L.), such as meadow bluegrass (Poa pratensis L.), rough bluegrass (Poa trivialis L.), Canada bluegrass (Poa compressa L.) and annual bluegrass (Poa annua L.); bentgrass (Agrostis L.), such as creeping bentgrass (Agrostis palustris Huds.), thin bentgrass (Agrostis tenius Sibth.), velvety bentgrass (Agrostis canina L.) and small chaff grass (Agrostis alba L.); Festuca L., such as creeping red fescue (Festuca rubra L.), fine fescue (Festuca carubra var. commutata Gaud.), fescue (Festuca ovina L.), hard fescue (Festuca longifolia, tall fescue (Festuca arundinacea Schreb.), meadow fescue (Festuca elatior L.); ryegrass (Lolium L.), such as perennial ryegrass (Lolium perenne L.), multiflorum ryegrass (Lolium multiflorum Lam.); wheatgrass (Agropyron Gaertn.), such as flat spike wheatgrass (Agropyron cristatum (L.) Gaertn.), bluestem wheatgrass (Western wheatgrass, Agropyron smithii Rydb.). Other cool-season turfgrasses include Bromus inermis Leyss.; and timothy (Phleum L.).

[0072] Examples of warm-season turf grasses are Bermuda grass (Cynodon LCRich), Zoysia Willd., St. Augustine grass (Stenotaphrum secundatum (Walt.) Kuntze), Centipede grass (Eremochloaophiuroides (Munro.) Hack.), Carpet grass (Axonopus Beauv.), Bahia grass (Paspalum notatumFlugge.), Pennisetum clandestinum Hochst. ex Chiov., Buffalo grass (Buchloedactyloides (Nutt.) Engelm.), and Seashore paspalum (Paspalum vaginatum swartz).

[0073] The LAMP method invention also contemplates a kit for detecting fungi in a lawn grass sample using a LAMP assay. As described herein, a test strip containing one or more than one primer set can be used. In one embodiment, multiple primer sets are multiplexed on a test strip for detecting multiple diseases from a lawn grass sample collected from a specific locus.

[0074] For example, a bijou tube with a ball bearing and an appropriate amount of lysis buffer is provided, in which 1 cubic centimeter of homogenized lawn sample is placed and shaken vigorously for 1 minute. The sample wells of the test strip contain all the resuspension buffer, and a drop of this test solution is placed in the sample wells of the test strip, wherein these wells have all the components required for the LAMP reaction (e.g., primer sets and reagents, such as isothermal mastermix (catalog number iso-001) obtained from Optigene). In one embodiment, the test strip is multiplexed. In another embodiment, the test strip includes 8 wells (two controls and 6 for the desired lawn disease). In one embodiment, the test strip is combined with a diagnostic instrument (e.g., obtained from OptiGene). II or III). DETAILED DESCRIPTION

[0075] Primer design

[0076] Highly conservative genes are used to design LAMP primers to detect the DNA (Table 3 the 1st column) from the lawn grass pathogens selected. Use NucleoSpin Plant II (MACHEREY-NAGEL) to obtain pure genomic DNA from all purpose fungi. In conjunction with PCR technology, use disclosed primers to and subsequently use Sanger sequencing to amplify the target sequence. The following DNA loci (gene and region) are sequenced: internal transcribed spacer (ITS), elongation factor 1-α (EF), beta-tubulin (Tub), cytochrome c oxidase subunit 1 (Cox), superoxide dismutase (SOD1) and large subunit nuclear ribosomal RNA (LSU). ClustalW alignment method (CLC Main Workbench software) is used to compare the original sequence. Use BLAST to compare with the sequence from GenBank (NCBI) to identify gene homologues. Ideally, a good sequence is defined as a successful PCR amplification for all target taxa and does not have homology with other taxa.

[0077] The best sequences (SEQ ID No 1-13) from the sequenced DNA loci were then used to design LAMP primers for each selected turfgrass pathogen using LAMP Designer 1.14 (PREMIER Biosoft). Thus, different parameters were tested to obtain different primer sets for each organism and each locus (see Table 3 for correlation of SEQ IDs for turfgrass pathogens, selected loci, and best sequences for primer design). The designed primer sets shown in Table 1 were then tested for specificity (Table 3) and sensitivity (Table 4).

[0078] Table 1DNA SEQ ID

[0079]

[0080]

[0081]

[0082] Specificity

[0083] To check the specificity of the reaction (see literature below), the assay (using the designed primer set) was tested with pure genomic DNA extracts from fungal isolates as described in Table 2. A comprehensive collection of different turfgrass pathogens from different geographical origins was collected and grown on different media (potato dextrose / malt / corn meal / cherry / V8). DNA was extracted from mycelia (NucleoSpin Plant II-MACHEREY-NAGEL) using 10-day-old fungal cultures. Genomic DNA was diluted to 5 ng / μl with nuclease-free water and a portion of 2.5 μl was used for specificity testing.

[0084] At 64 ℃, in LightCycler 480 (Roche (Roche)), carry out LAMP specificity test in 96 well plates and continue 55min.During cooling from 98 ℃ to 65 ℃, the amplicon specific annealing temperature is determined with the rate of change of-0.1 ℃ / second.Real-time LAMP determination is carried out in the reaction mixture of 10 μ l, and these reaction mixtures contain the isothermal premix (master mix, Optigene company) of 1x concentration of 5 μ l, 0.4 μ M every kind of outer primer, 1.6 μ M every kind of inner primer and 0.8 μ M every kind of loop primer (synthesized by Microsynth company) and 2.5 μ l of genomic DNA.

[0085] All reactions were performed in duplicate on two different days.

[0086] Specific literature:

[0087] 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

[0088] Kitamura, M., Aragane, M., Nakamura, K., Watanabe, K., and 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

[0089] Seki, M., Kilgore, P.E., Kim, E.J., Ohnishi, M., Hayakawa, S., and 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

[0090] Wang, D.-G., Brewster, JD, Paul, M., and Tomasula, PM (2015). Two Methods for Increased Specificity and Sensitivity in Loop-Mediated Isothermal Amplification. Molecules, 20(4), 6048-6059. https: / / doi.org / 10.3390 / molecules20046048

[0091] Table 2:

[0092]

[0093]

[0094]

[0095] Stein and SYN strains: Syngenta, CH-4332Stein, Switzerland CBS strain: Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands

[0096] ZHAW strain: Zurich University of Applied Sciences, PO Box 8820, Wädenswil, Switzerland

[0097] Result interpretation:

[0098] The specificity of the LAMP assay was examined for the specific targets designed for the fungal strains listed in Table 2 as summarized in Table 3. As an additional confirmation of specificity, matching melting temperatures of 82.6°C-89.9°C±0.5°C were also observed for different amplification products as shown in Table 3.

[0099]

[0100] The sensitivity of the described primer sets corresponding to the DNA of Seq ID No. 1-13 (Table 1) was determined using serial dilutions (1 ng to 100 fg) of the genomic DNA of all the fungi of interest, wherein each reaction was performed in duplicate on two different days. Pure genomic DNA from all fungi was obtained using NucleoSpin Plant II (MACHEREY-NAGEL). LAMP sensitivity testing was performed in 96-well plates on a LightCycler 480 (Roche) at 64°C for 55 min. During cooling from 98°C to 65°C, the amplicon-specific annealing temperature was determined at a rate of change of -0.1°C / second. 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 outer primer, 1.6 μM of each inner primer, and 0.8 μM of each loop primer (synthesized by Microsynth), and 2.5 μl of genomic DNA.

[0101] Table 4

[0102]

[0103] Methods for Detection of Fungal Pathogens in Turfgrass Samples

[0104] Sample collection

[0105] Collect a turfgrass sample including the roots at a location where the fungal pathogen is expected. Turfgrass may also show symptoms. Place the turfgrass sample in a clean 50 ml tube (Corning) and store at -20°C until use. DNA is extracted using the Plant Material Lysis Kit (Optigene). Place 1 cm 3 The lawn sample was placed in a Bijou tube containing 1 ml of lysis buffer (Optigene). The lawn sample was homogenized by shaking the Bijou tube for 1 min. A volume of 10 μl of the lysate was transferred to the provided dilution tube (Optigene) and mixed vigorously by shaking. The diluted lysate was then defined as the template.

[0106] LAMP reaction

[0107] In some embodiments, the LAMP reaction is performed at about 60° C. to about 70° C. (eg, about 64° C. to about 67° C., or about 64° C. to about 66° C.). In a specific example, the LAMP reaction is performed at 64° C.

[0108] In some embodiments, the LAMP reaction is allowed to proceed for about 15 to about 45 minutes, such as about 20 to about 40 minutes, or about 25 to about 35 minutes.

[0109] In some embodiments, the primer concentrations in the LAMP reaction according to the present invention are; 1.4-1.8 μM for the forward inner primer (FIP) and the reverse inner primer (BIP), more particularly 1.6 μM; 0.2-0.4 μM for the forward outer primer (F3) and the reverse outer primer (B3), more particularly 0.4 μM; and 0.4-0.8 μM for the forward loop primer (LF) and / or the reverse loop primer (LB), more particularly 0.8 μM, which primer concentrations can be used to accelerate the amplification of nucleic acids present in lawn samples and reduce the detection time of any target fungal DNA that may be present in such lawn samples.

[0110] Suitable buffer systems for reactions that can be used for LAMP assays include:

[0111] 1X Isothermal Amplification Buffer Set from New England Biolabs

[0112] 20mM Tris-HCl

[0113] 10mM(NH4)2SO4

[0114] 50mM KCl

[0115] 2mM MgSO4

[0116] 0.1% 20

[0117] (at 25°C, pH 8.8)

[0118] 1X Isothermal Amplification Buffer II Set from New England Biolabs

[0119] 20mM Tris-HCl

[0120] 10mM(NH4)2SO4

[0121] 150mM KCl

[0122] 2mM MgSO4

[0123] 0.1% 20

[0124] (at 25°C, pH 8.8)

[0125] Suitable enzyme systems (DNA polymerase, etc.) that can be used for the reaction of the LAMP assay include:

[0126]

[0127] In one embodiment, the LAMP reaction is carried out on a Genie instrument (Optigene) in a test strip with a dry reagent (Optigene). In one embodiment, these strips have eight 150 μl holes (2 controls and 6 for testing). Real-time LAMP determination is carried out in a 25 μl reaction mixture containing 15 μl of an isothermal premix (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 of Table 1. Before adding the template, the freeze-dried reaction strip is resuspended in 22 μl of resuspension buffer (Optigene). All test strips include a negative control and a positive plant control primer set provided by Optigene. For all assays, 3 μl templates are added to each reaction and hole. The reaction is kept at 64°C for 30-55min, followed by an annealing procedure. The temperature profile of the annealing program was determined at a ramp rate of -0.1°C / sec during cooling from 98°C to 65°C.

[0128] The isothermal master mix contains a fluorescent double-stranded DNA binding dye to allow real-time detection of amplicons. The assay was optimized with respect to reaction time, temperature, and volume of DNA added per reaction.

[0129] Fluorescence data obtained during the amplification phase at 64°C are reported as the amplification time. Fluorescence extrapolated data obtained during the annealing phase are reported as the annealing temperature.

[0130] Alternatively, LAMP assay reaction does not include annealing procedure, in which case, pH sensitive indicator dye can be used to assess the presence of target fungal DNA. In some instances, pH sensitive indicator dye is a colored dye detectable under visible light. In a special instance, the colored dye comprises cresol red, phenol red, metacresol purple, bromocresol purple, neutral red, naphtholphthalein, thymol blue or naphtholphthalein. In other instances, pH sensitive indicator dye is a fluorescent indicator dye. In a special instance, the fluorescent dye comprises 2', 7'-bis-(2-carboxyethyl)-5 (6)-carboxyfluorescein, 5 (6)-carboxy-2', 7'-dichlorofluorescein, 5 (6)-carboxyfluorescein, 3,6-diacetoxyphthalonitrile, 6,8-dihydroxy-l,3-pyrene disulfonic acid or 5-(and-6)-carboxyl seminaphthol rhodan fluorescein (seminaphthorhodafluor).

[0131] Following the above procedure, detection of the presence of fungal pathogen DNA (Table 3) in turfgrass samples can indicate the presence of turfgrass fungal pathogens that may cause relevant turfgrass diseases, including, for example, anthracnose, take-all, summer spot, snow rot, pythium wilt, brown spot, and dollar spot. Early and effective detection allows for appropriate turfgrass management decisions to be made.

Claims

1. A method for detecting fungal DNA in a turfgrass sample using a loop-mediated isothermal amplification (LAMP) assay containing primers for fungal DNA of at least one turfgrass pathogenic fungus selected from the group consisting of: Sclerotinia sclerotiorum, Rhizoctonia solani species, Pythium aphanidermatum, Aspergillus graminearum species, Pythium nivale species, Psoralea corylifolia, Colletotrichum graminearum, Colletotrichum graminearum, and Pythium ultimum ulminum var. ultimum, the method comprising: The lawn sample is subjected to a LAMP reaction, wherein the LAMP reaction uses a primer set of four or more nucleic acid sequences, each primer in the set has 15 to 50 nucleic acids, and wherein the set of primers comprises at least one primer set selected from the group consisting of: (a) a primer set for detecting Sclerotinia sclerotiorum DNA, the Sclerotinia sclerotiorum DNA comprising or selected from primers each having a sequence at least 90% identical to SEQ ID NO: 14, 15, 16 and 17, respectively; (b) a primer set for detecting Rhizoctonia solani DNA, the primer set comprising or being selected from primers each having a sequence at least 90% identical to SEQ ID NOs: 20, 21, 22 and 23, respectively, or comprising or being 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 Pseudomonas aeruginosa var. nivalis DNA, the primer set comprising or selected from primers each having a sequence at least 90% identical to SEQ ID NO: 26, 27, 28 and 29, respectively; (d) a primer set for detecting Pythium aphanidermatum DNA, the primer set comprising or selected from primers each having a sequence at least 90% identical to SEQ ID NO: 32, 33, 36 and 37, respectively; (e) a primer set for detecting Sordella graminearum var. avenae DNA, the primer set comprising or selected from primers each having a sequence at least 90% identical to SEQ ID NO: 38, 39, 42 and 43, respectively; (f) a primer set for detecting Pseudomonas nivalis var. megasporus DNA, the primer set comprising or selected from primers each having a sequence at least 90% identical to SEQ ID NO: 44, 45, 48 and 49, respectively; (g) a primer set for detecting DNA of summer leaf spot pathogen, the primer set comprising or selected from primers each having a sequence at least 90% identical to SEQ ID NO: 50, 51, 54 and 55; (h) a primer set for detecting Colletotrichum graminearum DNA, the primer set comprising or selected from primers each having a sequence at least 90% identical to SEQ ID NO: 74 75, 78 and 79, respectively; (i) a primer set for detecting corn anthracnose DNA, the primer set comprising or selected from primers each having a sequence at least 90% identical to SEQ ID NO: 80, 81, 84 and 85, respectively; as well as (j) A primer set for detecting Pythium ultimum var. ultimum DNA, the primer set comprising or selected from primers each having a sequence at least 90% identical to SEQ ID NO: 86, 87, 90 and 91, respectively.

2. The method according to claim 1, wherein: The primer set for detecting Sclerotinia sclerotiorum DNA comprises or is selected from primers each having a sequence at least 90% identical to SEQ ID NO: 14, 15, 16, 17, 18 and 19, respectively.

3. The method according to 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 NO: 62, 63, 64, 65, 66 and 67, respectively.

4. The method according to 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 NO: 20, 21, 22, 23, 24 and 25, respectively.

5. The method according to claim 1, wherein: The primer set for detecting the DNA of Pseudomonas aeruginosa var. nivalis 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. The method according to 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 NO: 32, 33, 34, 35, 36 and 37.

7. The method according to 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 NO: 68, 69, 70, 71, 72 and 73, respectively.

8. The method according to claim 1, wherein: The primer set for detecting the S. graminis var. avenae DNA comprises or is selected from primers each having a sequence at least 90% identical to SEQ ID NO: 38, 39, 40, 41, 42 and 43, respectively.

9. The method according to claim 1, wherein: The primer set for detecting the S. graminis var. avenae DNA comprises or is selected from primers each having a sequence at least 90% identical to SEQ ID NO: 56, 57, 58, 59, 60 and 61, respectively.

10. The method according to claim 1, wherein: The primer set for detecting Pseudomonas nivalis var. megasporus DNA comprises or is selected from primers each having a sequence at least 90% identical to SEQ ID NO: 44, 45, 46, 47, 48 and 49, respectively.

11. The method according to claim 1, wherein: The primer set for detecting DNA of summer leaf spot pathogen comprises or is selected from primers each having a sequence at least 90% identical to SEQ ID NO: 50, 51, 52, 53, 54 and 55.

12. The method according to claim 1, wherein: The primer set for detecting Colletotrichum graminearum DNA comprises or is selected from primers each having a sequence at least 90% identical to SEQ ID NO: 74, 75, 76, 77, 78 and 79, respectively.

13. The method according to claim 1, wherein: The primer set for detecting corn anthracnose DNA comprises or is selected from primers each having a sequence at least 90% identical to SEQ ID No 80, 81, 82, 83, 84 and 85, respectively.

14. The method according to 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 NO: 86, 87, 88, 89, 90 and 91, respectively.

15. A kit for detecting fungal DNA in a turfgrass sample using a LAMP assay, the kit comprising one or more than one primer set identified in claims 1-14. The kit according to claim 15 , further comprising a buffer and a DNA polymerase.

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