Detection of Leptosphaeria Korrae with Pcr and Primers from the Ribosomal Internal Transcribed Spacers

Leptosphaeria korrae, the causal agent of necrotic ring spot, is a destructive patch disease of Kentucky bluegrass. To develop a rapid molecular test for the detection of this pathogen, an assay based on the polymerase chain reaction was developed utilizing the internal transcribed spacer region 1 (ITS 1) of L. korrae ribosomal DNA. DNA sequence comparison showed 94.8% similarity of the ITS 1 region among L. korrae isolates, and only 45-50% similarity between L. korrae and other fungal species. Based on ITS 1 sequence differences, a pair of oligonucleotide primers, LK17S and 5.8SC, were selected. With the polymerase chain reaction, the primers specifically amplified L. korrae DNA and did not amplify DNA isolated from 15 other fungal species or healthy Kentucky bluegrass. The assay could also specifically detect L. korrae in diseased turfgrass samples. INTRODUCTION Necrotic ring spot (NRS) is a destructive disease of turfgrass which severely affects Kentucky bluegrass (Poa pratensis L.). The causal agent, Leptosphaeria korrae Walker & Smith, was first identified in Australia causing spring dead spot, a disease of bermudagrass (Cynodon dactylon (L.) Pres.)(30). In North America, L. korrae was identified in 1984 along with Magnaporthe poae Landschoot & Jackson, as being separate components of the Fusarium blight complex (24). Necrotic ring spot on Kentucky bluegrass has been reported from many areas in North America, particularly in the American Great Lakes states and the Pacific Northwest (3, 6, 24, 31), but there has been few published reports of its occurrence in Canada (12, 15, 16). Leptosphaeria korrae produces arcs or rings of blighted grass as a foliar symptom. These symptoms are similar for a number of patch diseases of turfgrass such as NRS, take-all patch, summer patch, fusarium patch, yellow patch, pythium blight, rhizoctonia blight, and sclerotium blight (4). As a result, identifications based on macroscopic symptoms are difficult. If the disease is incorrectly identified, the resulting control measures may be ineffective due to the differing fungicide sensitivity of the various pathogens (25). Tentative identification of NRS can be made from microscopic examination of roots for dark ectotrophic hyphae (24). However, the diagnostic value of this symptom is limited because it can also be produced by a number of turf pathogens and saprophytes (25). Tentative identification can also be made based on growth rates and colony morphology (31), but sexual fruiting structures (pseudothecia) and ascospores are required for positive identification. Unfortunately, these sexual structures are only occasionally found in nature (26) and are difficult to induce in culture (8). Rapid and accurate diagnosis is very important in developing effective strategies for disease control, and several alternative diagnostic methods have been developed for identifying turfgrass pathogens. These include DNA restriction fragment length polymorphisms (13, 27), polyclonal and monoclonal antibodies (17, 20), isoelectric focusing (9) and cloned DNA probes (28). However, there remains a need for quicker pathogen diagnostic techniques for practical applications in turfgrass pathology. Recently the polymerase chain reaction (PCR) has been used to detect and differentiate closely related species of plant pathogens (10, 11, 19, 23, 32). The PCR approach not only requires less time to perform, but is also more sensitive than other molecular detection methods O’Gorman et al., Can. J. Bot. 2004 2 (7, 22). The objectives of this study were: (1) to sequence the internal transcribed spacer region 1 (ITS 1) of L. korrae ribosomal DNA; (2) to compare the DNA sequences with ITS 1 sequences of other species in order to design species-specific oligonucleotide primers; and (3) to test the primers for specific amplification of L. korrae DNA. METHODS AND MATERIALS Fungal Isolates: Isolates used in this study are listed in Table 1. Leptosphaeria korrae isolates 89-570, 90447-1, 90-447-2, 90-794, 90-796 and 90-797 were received from L. MacDonald, B.C. Ministry of Agriculture; M. poae isolates 73-1 and 73-15 were received from P. Landschoot, Pennsylvania State University; R. solani isolate RS1/T(AG1) was received from E.E. Butler, University of California, Davis. All other fungal isolates were from the University of Guelph. Stock cultures were maintained on slants of potato dextrose agar (PDA) stored at 4C or as inoculated chicken scratch stored at -10C. All stock cultures were subcultured onto PDA plates at 22C, except for Typhula incarnata which was grown at 15C. Plant Material: Field samples of Kentucky bluegrass showing symptoms of NRS were collected by removing a plug of turf 2x3 cm wide by 5 cm deep from the outer edge of necrotic patches. Fungal isolations were made by placing surface-sterilized root tips on 1/5 strength PDA amended with 30 μg/L streptomycin (8). After 10 days, colonies were transferred to full strength PDA. The remaining roots from each sample were washed free of soil under tap water, frozen in liquid nitrogen and stored at -20C for DNA extraction. Kentucky bluegrass was also grown in the greenhouse and inoculated with the L. korrae isolates listed in Table 1. Hard red spring wheat kernels infected with L. korrae were placed in sod within a 2 cm deep incision made in the centre of each pot. After 2 months when symptom development was obvious, small turf plugs were removed from the advancing edge of the patch. The plugs were washed under tap water, examined for characteristic runner hyphae, and frozen in liquid nitrogen for DNA extraction. DNA Extraction: DNA was isolated from fungal cultures grown on a 7 cm x 7 cm sterile cellulose membrane sheet (Flexel Inc., Atlanta, GA) overlaying 2% malt agar. The fungal mycelium was scraped from the cellulose membranes and placed into 1.5 mL microfuge tubes. DNA extractions were carried out by one of two methods. In the first method, plant and fungal DNA were extracted following the method of Rogers and Bendich (21) with minor alterations. Plant tissue or fungal mycelium was mixed with sterile sand and liquid nitrogen, and ground to a fine powder using a mortar and pestle. Approximately 300 mg of ground tissue were transferred to a 1.5 mL microfuge tube containing 500 μL of CTAB extraction buffer [2% CTAB (w/v)(hexadecyltrimethylamonium bromide, Sigma), 100 mM Tris-HCl (pH-8.0), 20 mM EDTA (pH 8.0), 1.4 M NaCl and 1% PVP]. Tissue was suspended by gentle vortexing and incubated for 30 min at 65C. The suspension was extracted with phenol/chloroform/isoamyl alcohol (25:24:1) followed by chloroform/isoamyl alcohol (24:1) until the interface was clear. The aqueous phase was transferred, and the DNA was precipitated with an equal volume 3 M NaOAc/isopropanol (1:10) (33). After centrifugation, the pellet was resuspended in buffer [10 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0), 1 M NaCl], and the DNA was then precipitated with 95% ethanol. The resuspended DNA was stored in 0.1 x TE buffer [1 mM Tris-HCl (pH 8.0), 0.1 mM EDTA (pH 8.0)]. For the second DNA extraction method, mycelium grown on cellulose membranes was placed in 1.5 mL microfuge tubes and washed first with 1 mL of 1 M NaCl and then distilled water. Sterile sand was added and the tissue was ground with a disposable pellet pestle (Kontes, Vinland, O’Gorman et al., Can. J. Bot. 2004 3 NJ). Following grinding, 750 μL of 10 mM Tris-HCl (pH 8.0) was added and the samples were boiled for 20-30 minutes (10). The samples were then centrifuged for 5 min at 8000 g. The supernatant was transferred to a new tube and stored at -20C. PCR, Sequencing and Primer Selection: Primers used for initial amplification and sequencing of L. korrae DNA were identified from conserved sequences of the 17S and 5.8S ribosomal DNA (rDNA) of Neurospora crassa and Saccharomyces carlsbergensis (2) and Thermomyces lanuginasus (18). The sequence of the 17S primer in the 17S rDNA was 5'-TCCCCGTTGGTGAACCAGCGG-3', and the anti-sense 5.8SC primer sequence in the 5.8S rDNA was 5'GCTGCGTTCTTCATCGATGC-3'. The DNA fragment flanked by the two primers contained the entire ITS 1 region including a portion of the 17S and 5.8S rDNA. Primers were synthesised with a Milligen Biosurge 8600 Synthesizer, and stored at -20C in 1 x TE buffer [10 mM Tris-HCl (pH 8.0), 1 mM EDTA]. Reaction conditions for the PCR amplification using the conserved 17S/5.8SC primers, and sequencing and analysis of the PCR products were performed according to Xue et al. (32). PCR and Pathogen Detection: PCR amplifications were carried out in 0.5 mL microfuge tubes with 25 μL reaction mixtures. The mixture contained either 1 ng of DNA extracted by the modified Rogers and Bendich method (21) or 3 μL of DNA extracted by boiling in Tris-HCl (pH 8.0), 200 μM of each dNTP, 0.5 μM of primers, 0.5 units of Vent DNA polymerase (New England Biolabs, Beverly, MA), 100 μg/mL BSA and 1 x DNA polymerase buffer [10 mM KCL, 10 mM (NH4)2SO4, 20 mM Tris-HCl (pH 8.8), 2 mM or 4 mM MgSO4, and 0.1 % Triton X-100). The reactions were overlaid with light mineral oil and amplifications were performed in a COY Tempcycler (Ann Arbor, Michigan) programmed for an initial cycle of 3 min at 94C, 2 min at 57C, and 2 min at 72C. This was followed by 35-40 cycles of 30 s at 94C, 50 s at 57C, 15 s at 72C, and a final extension cycle for 10 min at 72C. Amplification products were separated by gel electrophoresis in 2% agarose in 0.5 x TBE (45 mM Tris-borate and 1 mM EDTA). Gels were then stained in ethidium bromide and viewed under 260 nm UV light. RESULTS Sequencing and Primer Development: The primers, 17S and 5.8SC, selected from conserved regions of the 17S and 5.8S rDNA and used to amplify a single 249 bp PCR product from L. korrae DNA. The amplified fragments from two L. korrae isolates, 90-796 and LK-4, were sequenced (Fig. 1). Comparison of the DNA sequences with the GenBank sequence data base showed that they were similar to ITS 1 regions of other fungi, such as Leptosphaeria maculans (32) and N. crassa (2) (Fig. 1). The sequenced ITS 1 regions shared 94.8% similarity between the L. korrae isolates, and only 45-50% similarity between L. korrae and ITS 1 sequences of the other fungal isolates examined. Based on sequence differences found in the L. korrae ITS 1 region, a primer, LK17S, was designed. The LK17s primer is a 17-mer with the sequence, 5'-ACAAACTGCATGGGCGG-3' (bp 63-79, Fig. 1). LK17S was paired with the 5.8SC primer (bp 235-255, Fig. 1), in order to test for specific amplification of L. korrae. The single amplification product generated using the LK17S/5.8SC primer pair was a 193 bp fragment as predicted from the sequence (Fig. 2). Assay Specificity and Sensitivity: Amplification with the LK17S/5.8SC primer pair and DNA from pure cultures of 19 L. korrae isolates and 19 isolates chosen from 15 species of other common turfgrass pathogens, turfgrass saprophytes or related Leptosphaeria species showed that the assay was specific for L. korrae. Only DNA from the L. korrae isolates was successfully amplified, and there was no amplification from the DNA of any of the other 15 species of fungi tested (Table 1 and Fig. 2). In figure 2, the stained smears originating at the gel well for samples lacking the target sequence was a result of primer artifacts caused by the low DNA Km value which is a characteristic of Vent DNA polymerase (1). To ensure that the DNA could be amplified by PCR, amplification of the DNA from the 19 O’Gorman et al., Can. J. Bot. 2004 4 fungal test species was also done using the conserved 17S/5.8SC primers (Table 1). The simplicity of preparing DNA samples by boiling in Tris-HCl (pH 8.0) permitted fungal DNA to be prepared for PCR in less than one hour. This method also allowed DNA to be easily extracted from fungi such as Sclerotinia homoeocarpa which apparently produce large amounts of polysaccharides making DNA extraction difficult when using the modified Rogers and Bendich method. Sensitivity of the PCR-based assay using the LK17S/5.8SC primer pair was tested using 10-fold serial dilutions of L. korrae DNA. Amplification from L. korrae DNA was obvious with as little as 10 pg of DNA and was also observed with 1 pg of DNA. However, the reactions containing 1 pg of L. korrae DNA had only faint bands that are not readily apparent in Figure 3. There was no observable amplification detected at 0.1 pg of DNA (Fig. 3). Detection of Pathogen from Plant Tissue: Positive PCR results for L. korrae were achieved for both naturally infected and inoculated turfgrass samples. However, the PCR products amplified from infected plant tissue were usually not as strong as that from pure culture (Fig. 4). DISCUSSION Necrotic ring spot is one of the more recently recognized turfgrass patch diseases causing concern to the turfgrass industry throughout North America. Patch diseases are among the most difficult to diagnose due to the number of non-specific symptoms (4). The difficulties encountered when attempting to identify L. korrae isolated from diseased turfgrass samples has created a need for improved detection methods. PCR has been used successfully to specifically detect a number of fungal plant pathogens in pure culture and infected plant tissue (10, 11, 19, 23, 32). In this study we have demonstrated that PCR, when used with an apparently species-specific primer set, could detect L. korrae and differentiate it from other turfgrass pathogens. The specificity of this PCR assay is based on divergent sequences of the ITS-region of rRNA genes which exhibit considerable sequence differences between fungal species (2, 18, 32). The utilization of variable ITS regions to select primer sequences for differentiation of closely related fungal plant pathogens has proven successful (19, 32). Comparison of the ITS 1 sequence between L. korrae isolates revealed a very high degree of similarity. The differences found within this homothallic species may reflect the geographic isolation of the Ontario and B.C. isolates used in this study. Comparison of known fungal ITS 1 sequences showed much less similarity between L. korrae and other fungal species including Gaeumannomyces graminis and Magnaporthe poae (unpublished data). These major differences in ITS 1 sequences facilitated the design of a unique set of primers that specifically annealed to L. korrae DNA. The species-specific primer pair, LK17S/5.8SC, consistently amplified a 193 bp PCR product from DNA of L. korrae, and did not amplify DNA from related species or any of the commonly occurring fungi which inhabit turfgrass. High sensitivity of the PCR assay also allowed direct detection of L. korrae from infected plant tissue. However, the concentration of the 193 bp PCR product was consistently lower from infected plant tissue than from pure fungal culture. The lower intensity of the bands may be due to lower DNA concentrations or the presence of PCRinhibiting substances (14, 29). The presence of inhibitors was indicated by improved amplification following dilution of the DNA extracts prior to PCR. Although this has allowed us to circumvent the possible inhibition problem to a certain extent, other DNA purification techniques which remove any inhibitors may increase the intensity of the PCR products obtained from infected plant tissue samples. The utilization of PCR to specifically detect L. korrae permits reliable identification of suspected NRS samples within several hours. Previously, the time required to make a positive identification of this disease with conventional morphological techniques required 8 weeks or more. The application of PCR as a diagnostic tool for NRS should prove beneficial in disease O’Gorman et al., Can. J. Bot. 2004 5 management as well as accelerating further studies of the epidemiology of this disease. 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Pathotype identification of Leptosphaeria maculans with PCR and oligonucleotide primers from ribosomal internal transcribed spacer sequences. Physiol. Mol. Plant Pathol. 41:179-188. 33. Yoon, C.-S. D.A. Glawe, P.D. Shaw. 1991. A method for rapid small-scale preparation of fungal DNA. Mycologia 83:835-838. O’Gorman et al., Can. J. Bot. 2004 7 Table 1. Identity of fungal isolates, with PCR results using universal 17S and specific LK17S primers. PCR Species Isolate UN LK Leptosphaeria korrae LK-01 + + Leptosphaeria korrae LK-02 + + Leptosphaeria korrae LK-03 nt + Leptosphaeria korrae LK-04 + + Leptosphaeria korrae 89-570 nt + Leptosphaeria korrae 90-447-1 nt + Leptosphaeria korrae 90-447-2 nt + Leptosphaeria korrae 90-794 nt + Leptosphaeria korrae 90-796 + + Leptosphaeria korrae 90-797 nt + Leptosphaeria korrae LK-16 nt + Leptosphaeria korrae LK-19 nt + Leptosphaeria korrae LK-23 nt + Leptosphaeria korrae LK-25 nt + Leptosphaeria korrae LK-27 nt + Leptosphaeria korrae LK-28 nt + Leptosphaeria korrae LK-55 nt + Leptosphaeria korrae LK-57 nt + Leptosphaeria korrae LK-58 nt + Leptosphaeria maculans Unity + Leptosphaeria maculans Unity + Magnaporthe poae 73-1 + Magnaporthe poae 73-15 + Gaeumannomyces graminis Gg-1 + Gaeumannomyces graminis Gg-2 + Gaeumannomyces graminis Gg-3 + Cladosporium sp. Csp-40 + Cephalosporium sp. C.sp-40 + Drechslera poae Dp-1 + Pythium sp. P.sp 20 + Fusarium nivale FN-30 + Fusarium sp. Fsp-02 + Sclerotinia homoeocarpa SH-01 + Rhizoctonia solani RS1/T(AG1) + Typhula incarnata TI-17 + Trichothecium roseum TR-01 + Leucoagaricus naucina SM45b + Marasmius oreades SM27b + Poa pratensis NRS field sample LK-15 nt + NRS field sample LK-24 nt + inoculated LK-14 nt + inoculated LK-16 nt + inoculated LK-19 nt + inoculated LK-20 nt + inoculated LK-2 nt + inoculated LK-23 nt + inoculated LK-30 nt + inoculated LK-34 nt + -------------------------------------------------------------------a UN = universal 17S/5.8SC primers b LK = LK 17S/5.8SC primers nt = not tested O’Gorman et al., Can. J. Bot. 2004 8 90-796 -TCCCCGTTGGTGAACCAGCGGAAGG-ATCATTACAGAATA-------------GTAACAGG--CCCAAAGTG-CA 58 LK-4 -TCCCCGTTGGTGAACCAGCGGAAGG-ATCATTACACAGTATA-----------GTAACAGG--CCCAAAGTG-CA 60 UNITY -TCCCCGTTGGTGAACCAGCGGAAGG-ATCATTACCC-TTCTATCAGAGGATTGGTGTCAGGATTTCGGCCTTTGG 73 LEROY -TCCCCGTTGGTGAACCAGCGGAAGGGATCATTACCCATTT-TCAAAGCACTGCG------GGCCTCGATCAGTGG 68 NC -TCCCCGTTGGTGAACCAGCGGAAGGGATCATTACAGAGTTG-----------CAAAACTCCCACAAACCATCGCG 64 90-796 G---CACAAACT-GCATGGGCGGGTTATGTCTATTACCCTTGTTTAT--TGAGTAACCTA----------TGTTTC 118 LK-4 G---CACAAACT-GCATGGGCGGGTTATCTCTATTACCCTTGTTTATATTGAGTA-CCTA---------TTGTTTC 122 UNITY CTTACTTTCTGGCCCTTTCCTTTCTGATTC----TACCCATGTTTTTT--GCGTA--CTA--------TTTGTTTC 134 LEROY C---------GGCAGTCTACTT--TGATTC----TGCCCATGTTTTTT-GCGGTA--CTA-------TTTGGTTTC 119 NC AATCTTACCCGTACGGTTGCCT-CGGCG-CTGGCGGTCC-GGAA-----AG-GC---CTT-CGGGCCTCCCGGATC 127 90-796 CTT-GGTGGGCTTGCCTGCCAAAA-GGA----CACCCCATTGAACCT-ATTTA---TTTTT-AATCAG-CGTC--T 181 LK-4 CTT-GGTGGGCTTGCCTGCCAAAA-GGA----CACCCCATTGAACCT-ATTTA---TTTTT-AATCAG-CGTTC-T 185 UNITY CTTNGGTAGGCTTGCCTGCCAAAA-GGA--GGTACC-TTTTCCTACC-ACTT-GCAATTGC-AGTCAG-CGTCAGT 202 LEROY CTTGGGTGGGCTTGCCCGCCAAAAAATT--GGATCCCC-TAAA-ACCAACTT-GCAATTGC-AGTCAG-CGTCAGT 187 NC CTCGGGTCTCCC-GCTCGCGGCTGCCCGCCGGAGTGCCG-AAACT-AAACTCTTGATTTTT-ATGTCTCTCTGAGT 199 90-796 TGAA-T-AACAATAAT-AATTAC-AACTTTCAACAACGGNNCTCTTGGTTCTGGCATCGATGAAGAACGCAGC250 LK-4 TGAA-T-AACAATAATTAATAATGTAA-TTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGC255 UNITY TACACTGTA------TAAATT-ACTTCTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGC268 LEROY AACACTGTAA-----TAAATT-ACTTCTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGC254 NC AA-ACTTTTAA---AT-AAGTCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGC267 Fig. 1. Alignment of the ITS-1 sequences with flanking 17S and 5.8S rDNA from Leptosphaeria korrae LK-4 and 90-796, Leptosphaeria maculans Unity and Leroy, and Neurospora crassa NC. Position of the primers are underlined. LK17S primer position starts at base number 63 for LK-4 and base number 61 for 90-796. The 5.8SC primer position for both L.korrae isolates is shown at the 3' end of the sequence. The 17S and 5.8S rRNA sequences of N. crassa are described by Chambers et al. (2) and are represented within the shaded area and aligned with L. korrae and