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Development of SCAR Markers for the Identification of Phytophthora katsurae Causing Chestnut Ink Disease in Korea

Sequence characterized amplified region (SCAR) markers are one of the most effective and accurate tools for microbial identification. In this study, we applied SCAR markers for the rapid and accurate detection of Phytophthora katsurae, the casual agent of chestnut ink disease in Korea. In this study, we developed seven SCAR markers specific to P. katsurae using random amplified polymorphic DNA (RAPD), and assessed the potential of the SCAR markers to serve as tools for identifying P. katsurae. Seven primer pairs (SOPC 1F/SOPC 1R, SOPC 1-1F/SOPC 1-1R, SOPC 3F/SOPC 3R, SOPC 4F/SOPC 4R, SOPC 4F/SOPC 4-1R, SOPD 9F/SOPD 9R, and SOPD 10F/SOPD 10R) from a sequence derived from RAPD fragments were designed for the analysis of the SCAR markers. To evaluate the specificity and sensitivity of the SCAR markers, the genomic DNA of P. katsurae was serially diluted 10-fold to final concentrations from 1 mg/mL to 1 pg/mL. The limit of detection using the SCAR markers ranged from 100 µg/mL to 100 ng/mL. To identify the limit for detecting P. katsurae zoospores, each suspension of zoospores was serially diluted 10-fold to final concentrations from 10 × 105 to 10 × 101 zoospores/mL, and then extracted. The limit of detection by SCAR markers was approximately 10 × 101 zoospores/mL. PCR detection with SCAR markers was specific for P. katsurae, and did not produce any P. katsurae-specific PCR amplicons from 16 other Phytophthora species used as controls. This study shows that SCAR markers are a useful tool for the rapid and effective detection of P. katsurae.

Since 2005, damage to chestnut cultivation by chestnut ink disease has been reported in the southern chestnut cultivation area of Korea [1]. The pathogen behind this disease has been identified as the soil-borne Phytophthora katsurae. P. katsurae has multiple hosts and can infect them in multiple regions [2]. Consequently, this pathogen may serve as a limiting factor for chestnut cultivation [3]. The initial symptoms of chestnut ink disease are the extrusion of black liquid and necrosis in the stem. The damaged tree eventually dies, displaying crown dieback and releasing immature fruit [2]. To date, four species of the genus Phytophthora have been reported to cause chestnut ink disease: P. cambivora, P. cinnamomi, P. cryptogea, and P. katsurae. Of these four species, all except for P. katsurae have been reported to cause severe damage to chestnut plantations throughout Europe, the United States, and southern Australia. The American chestnut [Castanea dentata (Marsh.)], most of which is planted in most regions of Europe and the United States, is susceptible to these pathogenic fungi [2]. In contrast, the Japanese chestnut (Castanea crenata), most of which is widely planted and cultivated in Korea, is susceptible to P. katsurae, and this fungal pathogen has severely damaged chestnut cultivation. To prevent the spread of this pathogen to healthy hosts and disease-free regions, a rapid, simple and accurate or reliable identification system of P. katsurae will be required for effective disease management because of its wide host range, broad distribution and economic impact. Identification by morphological features has limitations, including laborious work, a high time consumption, requirement for microscopic techniques and a comprehensive knowledge of fungal pathogens. Molecular-based tools, such as a DNA-based system, can quickly and easily achieve an adequate database of reference strains [4,5]. Due to genotype-specific variation, the internal transcribed spacer (ITS) region has been widely used in the design of species-specific PCR primers, and has proven useful in the development of diagnostic tests for the detection of these pathogenic species [5,6]. However, in the case of the genus Phytophthora, some studies have found that using the ITS region for diagnostic purposes is not always appropriate, as in some cases it is still difficult to differentiate among closely related species using the ITS sequences [5,7,8]. This difficulty in designing specific primers is the result of insufficient variation in the ITS sequence of Phytophthora species [9,10]. Although some authors have reported other multicopy genes, including the mitochondrial cytochrome oxidase (cox) gene and the Ypt1 gene as alternatives to ITS regions, in some cases, it is difficult to distinguish hybrid species and closely related Phytophthora species e.g., P. cactorum/P. idaei, and P. iliciss/P. nemerosa [11,12]. An alternative approach would be to develop specific primers using random amplified polymorphic DNA (RAPD) since RAPD can be applied for the design of species or strain-specific markers from randomly amplified sequences [13,14]. Thus, sequence characterized amplified region (SCAR) markers specific to fungal pathogens can be derived from the sequence of a specific strain or gene, or from sequences randomly amplified by decamers [15]. In this study, we aimed to develop specific SCAR markers for P. katsurae and to apply them for the identification of this fungal pathogen.

MATERIALS AND METHODS
Fungal isolates and inoculation. All fungal isolates used in this study, ten isolates of P. katsurae and 16 isolates of other Phytophthora species, including destructive plant or tree pathogens causing severe damage in chestnut trees (as well as a wide range of economically important cultivated plants), were obtained from the Tree Pathology and Mycology Lab (Kangwon National University, Chuncheon, Korea) and the Korean Agricultural Culture Collection (Suwon, Korea). However, in the case of P. katsurae, only isolates from Korea were included for this study, as low variance between these isolates of P. katsurae has been proven by Oh [1]. The obtained fungal strains were cultured on V8 medium [16], a Phytophthora-selective medium covered with a sterilized cellulose membrane (Cellu-Sep T4; Membrane Filtration Products, Inc., Segiun, TX, USA) to facilitate the collection of mycelium and stored at 25 o C in the dark [3]. The list of fungal isolates is shown in Table 1.
Genomic DNA extraction. To extract genomic DNA from P. katsurae, 1 mg of mycelium was scraped from a cellulose membrane after 1 wk of incubation at 25 o C, and freeze-dried in a chamber at −20 o C for further study. Genomic DNA was extracted using a DNeasy Plant mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer' s instructions. Genomic DNA from P. katsurae zoospores was extracted using a Power Soil DNA isolation kit (MoBio Laboratories, Carlsbad, CA, USA) according to the manufacturer's instructions, with slight modifications. To gather zoospores of P. katsurae at the bottom of a 1.5 mL microtube, 1 mL of zoospore suspension was vigorously vortexed for 1 min. Each sample was then immediately transferred to a 2.0 mL Power Soil bead tube.

Development and detection of P. katsurae zoospores.
To produce zoosporangium, agar discs with actively growing mycelium at the margin of the culture colony were cut off using an 8 mm cork borer and transferred into a 90-mmdiameter Petri dish containing soil extraction solution [17]. P. katsurae zoospores were released according to Lee et al. [3] with slight modifications. The culture was incubated in a growth chamber at 25 o C for 10~14 days with light (1,000 lux). The culture was chilled at −4 o C for 1 hr in the dark after transplanting the small pieces into a 35-mm-diameter Petri dish containing soil extraction solution. This dish was then incubated at 25 o C for 2 hr. Zoospore release was achieved by incubating the dish at 25 o C for 2 hr in the dark. The zoospore concentration was determined using a hemacytometer, and the zoospore suspension was serially diluted 10-fold to final concentrations ranging from 10 × 10 5 to 10 × 10 1 zoospores/mL.
Amplification conditions for RAPD analysis. RAPD amplification was performed in a total reaction volume of 50 µL containing 2 µL of genomic DNA, 1 µL (10 pM) of random primer (Operon, Inc., Huntsville, AL, USA), 2.5 units of Taq polymerase (Takara Shuzou Co. Ltd., Otsu, Japan), 4 µL (2.5 mM) of the dNTP mixture, and 5 µL of 10× Ex Taq buffer (+20 mM Mg 2+ ). The amplification reaction was conducted on a My Cycler thermal cycler system (BioRad, Hercules, CA, USA). The cycling profile for RAPD analysis consisted of an initial denaturation at 94 o C for 3 min, followed by 40 cycles of denaturation at 94 o C for 30 sec, annealing at 37 o C for 30 sec, and extension at 72 o C for 1 min. A final extension was performed at 72 o C for 10 min. This experiment was conducted at least three times to ensure the reproducibility of results. The amplification products were analyzed by electrophoresis (Mupid-21. Cosmo Bio Ltd., Tokyo, Japan) on a 1.5% (w/v) agarose gel in 1× TAE buffer (40 mM Tris, 20 mM acetic acid, and 1 mM EDTA at pH 8.0), and then visualized using a UV transilluminator (Gel Documentation System; Bio-Rad) after staining with ethidium bromide (10 µg/mL) for 30 min.
RAPD analysis. RAPD analysis of the total DNA extracted from P. katsurae was conducted using 40 random decamer primers (Operon, Inc., Huntsville, AL, USA). PCR amplification was performed with the OPC and OPD primer sets (Operon, Inc., Huntsville, AL, USA) as described above. RAPD analysis of two P. katsurae isolates, TPML06522 and TPML08001, and 16 other Phytophthora species was conducted. The RAPD profiles were specific to each Phytophthora species, and seven DNA fragments distinguishing P. katsurae from the other 16 Phytophthora species were screened. The P. katsurae-specific DNA fragments were directly eluted from the agarose gel using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA) and ligated into a pGEM-T easy vector (Promega, Madison, WI, USA) according to the manufacturer's instructions. The plasmids were used to transform Escherichia coli strain JM 109 competent cells (Promega, Madison, WI, USA) and the recombinant colonies were identified by blue-white color selection after 15 hr of growth at 37 o C on Luria broth (LB) agar medium (1.8% trypton-NaCl, 0.5% yeast extract, 1.6% agar) containing ampicillin, 20% IPTG (isopropyl b-D-1thiogalactopyranoside), and 2% X-Gal (5-bromo-4-chloro-3indolyl b-D-galactopyranoside) following the manufacturer's recommendations. Plasmids were purified from LB/ampicillin liquid cultures of the selected colonies using a Wizard Miniprep DNA Purification System (Promega, Madison, WI, USA). Purified plasmid DNA was sequenced using an ABI PRISM 3730XL Analyzer Sequencer (Applied Biosystems, Foster City, CA, USA). All sequences obtained from this study were deposited in GenBank with the following the accession numbers, JQ796817 to JQ796822. The underlined sequence represents the original random decamer primer sequence used in the random amplified polymorphic DNA reaction.

Identification of P. katsurae Using SCAR Markers 89
Design of SCAR markers and the SCAR reaction. SCAR markers derived from RAPD markers specific to P. katsurae were designed by conversion of the selected RAPD markers into SCAR markers. Seven sets of the SCAR markers designed and used in this study are listed in Table 2. The SCAR reaction was carried out in a total volume of 25 µL containing 1 µL of genomic DNA, 1 µL of upstream primer (20 pM), 1 µL of downstream primer (20 pM), 5 µL of 10× Taq buffer, 0.5 µL of 10 mM dNTPs, and 2.5 units of Taq polymerase (Promega, Madison, WI, USA). A PCR assay with the SCAR markers was performed according to the method described in Table 3.
Primer specificity and sensitivity. To evaluate the specificity of the designed SCAR markers, seven sets of the SCAR markers, SOPC 1F/SOPC1R, SOPC 1-1F/SOPC 1-1R, SOPC 3F/SOPC 3R, SOPC 4F/SOPC 4R, SOPC 4F/ SOPC 4-1R, SOPD 9F/SOPD 9R, and SOPD 10F/SOPD 10R designed in this study were tested against two isolates of P. katsurae and 16 isolates of other Phytophthora species as the controls. To assess the sensitivity of the specific markers, all genomic DNA extracted from a pure mycelium of P. katsurae was serially diluted 10-fold to final concentrations ranging from 1 mg/mL to 1 pg/mL.

RESULTS AND DISCUSSION
RAPD analysis. RAPD is an easy and rapid method widely used [18,19] for the analysis of genetic diversity among species of Phytophthora, as well as for discriminating genotypes among different species of Phythophthora, because this method does not require any information on the sequence data of fungal pathogens. Moreover, Hadrys et al. [20] demonstrated that this method might be useful for the identification of fungal isolates and applications to molecular ecology. However, in the case of Phytophthora species, the design of species-specific primers based on the rDNA ITS region may be unprofitable because of interspecies variation in this part of the sequence [21,22]. However, RAPD facilitates the confirmation of variation because it can be applied to the entire genome when comparing genetic variation among fungal pathogens at the species or genera level. Thus, RAPD should be a reliable alternative for constructing species-specific primers, including SCAR makers. This method was thus applied in our study for developing specific SCAR markers. Forty random decamer primers (Operon, Inc., Huntsville, AL, USA) were evaluated using P. katsurae isolates in PCR reactions. Among the 40 random decamer primers tested in this study, five (OPC 02, OPC 05, OPC 11,OPD 18,and OPD 20) produced band patterns specific to P. katsurae (data not shown). PCR amplification with these five random decamer primers was carried out using DNA from two isolates of P. katsurae and 16 isolates of other Phytophthora species. Each random decamer primer produced approximately 20~25 amplicons (data not shown). The RAPD bands shown to be unique to P. katsurae, with sizes of 1,209 bp (OPC 02), 1,120 bp (OPC 05), 2,100 bp (OPC 11), 1,424 bp (OPD 18), and 1,718 bp (OPD20), were selected and used to design SCAR markers. All SCAR markers designed in this study are listed in Table 2.   Specificity of the SCAR markers for P. katsurae. PCR amplification of ten isolates of P. katsurae (Fig. 1) and 16 isolates of other Phytophthora species as controls (Fig. 2) was performed with SCAR markers. All SCAR markers produced amplicons specific to P. katsurae with sizes of 1,209 bp (SOPC 1F/1R), 1,100 bp (SOPC 1-1F/1-1R), 1,120 bp (SOPC 3F/3R), 2,100 bp (SOPC 4F/4R), 2,100 bp (SOPC 4F/ 4-1R), 1,424 bp (SOPD 9F/9R), and 1,718 bp (SOPD 10F/ 10R). Furthermore, when PCR products amplified by SCAR markers were sequenced and compared with the sequence used to design SCAR markers, the DNA sequences were identical to each other. In contrast, PCR amplification with SCAR markers did not produce amplicons for the 16 isolates of other Phytophthora species. In addition, bands non-specific to P. katsurae (Fig. 2C~F) did not correspond to any P. katsurae sequence. These results suggest that the detection of P. katsurae using SCAR markers is rapid and accurate. The detection and identification of fungal pathogens based on molecular methods using primers derived from the ITS region of rDNA have been widely used in recent years [5,23]. However, in some cases, species-specific primers based on the ITS sequences are unable to differentiate closely related species. Gomez-Alpizar et al. [24] and Kroon et al. [25] reported that P. infestans, P. phaseoli, P. ipomoeae, P. sp. 'andina' , and P. mirabilis are closely related species and have a 99.9% homology in ITS sequence. The same situation has been shown between P. katsurae and P. heveae, which belong to the same clade [4], and have up to 99.9% similarity in ITS sequence. In addition, Schena and Cooke [26] have also shown that P. katsurae is closely related with P. quercina and P. palmivora when analyzing phylogenetic relationships using DNA sequence data from four spacers between mitochondrial genes. Due to these limitations in identification using the ITS sequences, SCAR markers might be an alternative method for overcoming these restrictions. In addition, the isolation and identification of fungal pathogens using morphology-based methods is inefficient due to the long growth periods and the requirement of comprehensive knowledge about fungi. To overcome these limitations, alternative methods for constructing PCR primers have been developed and applied to detect Phytophthora species in recent years [13,15], and some authors have reported that SCAR markers could be used as reliable and efficient tools for the differentiation of closely related species [15,27].
Sensitivity of SCAR markers. All SCAR markers designed for this study were evaluated using genomic DNA extracted from P. katsurae mycelia ranging in concentration from 1 mg/mL to 1 pg/mL. The limit of detection of the SCAR markers was approximately 1 µg/mL, with two exceptions (Fig. 3). One exception was the PCR assay using the SOPC 4F/4-1R marker, which had a sensitivity of 100 ng/mL (Fig.  3C). The second exception was the PCR assay using the SOPC 10F/10R marker, which had a sensitivity of 100 µg/ mL (Fig. 3G). The best result was obtained using a PCR assay with SOPC 3F/3R, suggesting that SOPC 3F/3R may be a reliable diagnostic tool for the detection of fungal pathogens in the field. However, even though the sensitivity of other primer sets, SOPC 1F/1R, SOPC 1-1F/1-1R, SOPC 4F/4R, SOPC 4F/4-1R, SOPD 9F/9R, and SOPD 10F/10R is lower than that of SOPC 3F/3R, these primer sets should be useful for the identification of P. katsurae using genomic DNA extracted from pure mycelium in vitro. SCAR markers have been successfully used for the diagnosis and detection of Phytophthora species and other fungal pathogens, and have the high sensitivity of PCR [21,28]. Although the sensitivity of PCR using the SCAR markers in this study was lower than previously reported results, the PCR protocol applied in this study was still applicable, as 100 ng/mL of template DNA can be easily extracted from the pure mycelium of P. katsurae, and can produce clear and discernible bands after PCR. Thus, this technique can provide a possible solution for designing specific primers for closely related species.