Morphological Characterization and Genetic Diversity of Rice Blast Fungus, Pyricularia Oryzae, from Thailand Using ISSR and SRAP Markers

Journal of Fungi

Article Morphological Characterization and Genetic Diversity of Rice Blast Fungus, Pyricularia oryzae, from Thailand Using ISSR and SRAP Markers

Apinya Longya 1 , Sucheela Talumphai 2 and Chatchawan Jantasuriyarat 1,3,* 1 Department of Genetics, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand; [email protected] 2 Major Biology, Department of Science and Technology, Faculty of Liberal Arts and Science Roi Et Rajabhat University, Roi Et 45120, Thailand; [email protected] 3 Center for Advanced Studies in Tropical Natural Resources, National Research University-Kasetsart University (CASTNAR, NRU-KU), Kasetsart University, Bangkok 10900, Thailand * Correspondence: [email protected]

Received: 6 March 2020; Accepted: 18 March 2020; Published: 19 March 2020

Abstract: Rice blast disease is caused by the ascomycete fungus Pyricularia oryzae and is one of the most destructive rice diseases in the world. The objectives of this study were investigating various fungal morphological characteristics and performing a phylogenetic analysis. Inter-simple sequence repeat (ISSR) and sequence-related amplified polymorphism (SRAP) markers were used to examine the genetic variation of 59 rice blast fungus strains, including 57 strains collected from different fields in Thailand and two reference strains, 70-15 and Guy11. All isolates used in this study were determined to be P. oryzae by internal transcribed spacer (ITS) sequence confirmation. A total of 14 ISSR primers and 17 pairs of SRAP primers, which produced clear and polymorphic bands, were selected for assessing genetic diversity. A total of 123 polymorphic bands were generated. The similarity index value for the strains ranged from 0.25 to 0.95. The results showed that the blast fungus population in Thailand has both morphological and genetic variations. A high level of genetic variation, or genome adaptation, is one of the fungal mechanisms that could overcome host resistance to avoid host recognition. Results from this research study could bring substantial benefits and ultimately help to understand the blast fungal pathogen genome and the population structure in Thai blast fungus.

Keywords: ascomycete; genetic diversity; ISSR marker; Pyricularia oryzae; rice blast; SRAP marker

1. Introduction The ascomycete fungus Pyricularia oryzae (teleomorph: Magnaporthe oryzae), is one of the most damaging rice diseases in the world and is able to cause a yield loss of up to 30% and infect more than 50 species of grasses, including wheat, barley, oat, and millet [1]. Resistant rice cultivars have been used to control blast disease, but they usually lose resistance within a few years of release because of fungal genome adaptation that escape plant immunity [2]. The genetic diversity of the pathogen has limited the effectiveness of this approach. Understanding the structure of the blast population and genetic diversity are important for managing the disease. DNA profiling techniques that have been successfully used to assess genetic diversity include inter-simple sequence repeat (ISSR), random-amplified polymorphic DNA (RAPD), and amplified fragment length polymorphism (AFLP) [3]. They are dominant markers, which use universal primers for anonymous regions to extensively analyze the genetic diversity [4]. More recently, sequence-related amplified polymorphism (SRAP) markers have been developed, which are used to amplify coding

J. Fungi 2020, 6, 38; doi:10.3390/jof6010038 www.mdpi.com/journal/jof J. Fungi 2020, 6, 38 2 of 13 regions with primers targeting open reading frames (ORFs) [5]. The molecular markers diﬀer in cost, speed, complication, labor, degree of polymorphism, and repeatability. ISSR and SRAP marker techniques are fast and low cost, they do not require sequence information, have high repeatability, and use only one-step PCR. They provide highly discriminating information with good reproducibility and are relatively abundant, while AFLP and RAPD are more labor intensive and time consuming [4,6]. We characterized blast fungal morphology, performed DNA sequence analyses of the internal transcribed spacer (ITS), and used 20 ISSR and 30 SRAP markers to assess the genetic diversity of 59 rice blast strains, including 57 strains collected from Thai rice-growing areas and two reference strains, 70-15 and Guy11. The information obtained from this study will help to understand the population structure and evolution of rice blast fungus in Thailand.

2. Materials and Methods

2.1. Rice Blast Materials A total of 59 rice blast strains were used, including 57 strains collected from Thai rice-growing areas representing central, northern, and north-eastern parts of Thailand. Two rice blast strains, 70-15 and Guy11, were used as reference strains (Table1). The stock of each blast strain as in ﬁlter paper was cultured and DNA was extracted using the cetyl-trimethyl-ammonium-bromide (CTAB) method described by Longya et al. [7].

Table 1. Fungal morphological characteristics of 59 rice blast strains and their internal transcribed spacer (ITS) region.

Melanin Conidia Isolate Origin (a) Year Mycelium Features (b) ITS (e) Pigment (c) Shape (d) 10100 Srakaew 2006 Cream, Fluffy, Densely + C MT126183 10301 Sisaket 2006 White, Flat, Densely ++++ C MT126184 10302 Sisaket 2006 White, Flat, Densely + B MT126185 10459 Lampang 2006 Grey, Flat, Densely ++++ C MT126186 10551 Nakhon Ratchasima 2006 White, Flat, Densely ++++ C MT126187 10552 Nakhon Ratchasima 2006 White, Fluffy, Densely + C MT126188 10576 Kalasin 2006 Cream, Fluffy, Densely ++++ C MT126189 10577 Nakhon Ratchasima 2006 White, Flat, Densely - D MT126190 10578 Nakhon Ratchasima 2006 Cream, Flat, Densely + C MT126191 10581 Nakhon Ratchasima 2006 White, Fluffy, Densely +++ C MT126192 10652 Maha Sarakham 2006 Cream, Fluffy, Densely +++ C MT126193 10681 Roi Et 2006 White, Flat, Densely ++ B MT126194 10694 Nong Khai 2006 Cream, Flat, Densely + B MT126195 10732 Kamphaeng Phet 2006 White, Flat, Densely - C MT126196 10760 Kamphaeng Phet 2006 White, Fluffy, Densely - D MT126197 10812 Chiang Rai 2006 White, Flat, Densely - D MT126198 10837 Surin 2006 White, Flat, Densely ++ B MT126199 10873 Mae Hong Son 2006 Cream, Fluffy, Highly dense ++ C MT126200 10926 Phayao 2006 White, Flat, Densely - C MT126201 10927 Phayao 2006 White, Flat, Densely - C MT126202 10941 Lampang 2006 White, Flat, Densely - C MT126203 10971 Nan 2006 White, Flat, Densely - C MT126204 10985 Sisaket 2006 Cream, Flat, Densely + C MT126205 10993 Buriram 2006 White, Fluffy, Densely + C MT126206 J. Fungi 2020, 6, 38 3 of 13

Table 1. Cont.

Melanin Conidia Isolate Origin (a) Year Mycelium Features (b) ITS (e) Pigment (c) Shape (d) 11100 Udon Thani 2006 White, Fluffy, Densely + C MT126207 11108 Ubon Ratchathani 2006 White, Flat, Densely ++++ C MT126208 11109 Ubon Ratchathani 2006 Cream, Fluffy, Densely + C MT126209 BAG1.2 Phitsanulok 2006 White, Fluffy, Densely + C MT126210 BAG4.6 Phitsanulok 2006 White, Flat, Densely +++ D MT126211 40.3 n/a n/a Grey, Fluffy, Densely ++ C MT126212 SRN54001 Surin 2011 White, Flat, Densely +++ D MT126213 SRN54002 Surin 2011 White, Fluffy, Densely ++ D MT126214 SRN54005 Surin 2011 White, Flat, Densely ++ D MT126215 SRN54006 Surin 2011 White, Fluffy, Densely ++ C MT126216 SRN54007 Surin 2011 Cream, Flat, Densely ++++ C MT126217 SRN54009 Surin 2011 White, Flat, Densely +++ C MT126218 CCO56001 Chachoengsao 2013 Cream, Fluffy, Densely ++ C MT126219 CCO56002 Chachoengsao 2013 White, Flat, Densely ++ C MT126220 CCO56003 Chachoengsao 2013 Cream, Fluffy, Densely ++++ C MT126221 CCO56004 Chachoengsao 2013 White, Fluffy, Densely + C MT126222 CPM55001 Chaiyaphum 2012 Cream, Flat, Densely + C MT126223 CPM55002 Chaiyaphum 2012 Grey, Flat, Densely ++++ C MT126224 CPM55003 Chaiyaphum 2012 White, Fluffy, Densely ++++ C MT126225 NYK55001 Nakhon Nayok 2011 Grey, Flat, Densely ++ C MT126226 BKK55001 Bangkok 2011 White, Fluffy, Densely +++ C MT126227 BKK55002 Bangkok 2011 White, Flat, Densely + D MT126228 BKK55003 Bangkok 2011 Grey, Fluffy, Densely +++ C MT126229 RBR55001 Ratchaburi 2011 Cream, Fluffy, Highly dense ++++ A MT126230 RBR55002 Ratchaburi 2011 White, Flat, Densely - C MT126231 RBR55003 Ratchaburi 2011 White, Fluffy, Densely ++ C MT126232 B1-2 Ubon Ratchathani 2001 White, Fluffy, Densely ++++ C MT126233 TH196031 Ubon Ratchathani 2001 White, Fluffy, Densely ++ C MT126234 TH196036 Ubon Ratchathani 2001 White, Fluffy, Densely ++ C MT126235 TRG1 Nong Khai 2013 Grey, Fluffy, Densely + C MT126236 TRG2 Nong Khai 2013 White, Flat, Densely - C MT126237 TRG3 Nong Khai 2013 Grey, Fluffy, Densely ++ C MT126238 TRG4 Nong Khai 2013 Cream, Flat, Densely - C MT126239 70-15 Laboratory strain 1988 Grey, Flat, Densely - C MT126240 Guy11 French Guyana 1988 Grey, Fluffy, Densely - C MT126241 (a) Name of Thai province or the origin of blast sampling. (b) Fungal mycelia characteristics on rice flour agar (RFA), including color and mycelial appearance. (c) Melanin pigment production was observed at the ventral view of the culturing plate, scoring is as follows: No melanin pigment (-), melanin pigment production in less than 20% of the mycelia area (+), melanin production in 20%–50% of the mycelia area (++), melanin production in 50%–80% of the mycelia area (+++), and melanin production in more than 80% of the mycelia area (++++). (d) Conidial structure under light microscope. (e) GenBank accession number. A: Oval conidia, B: Short pyriform conidia, C: Pyriform conidia, and D: Long pyriform conidia.

2.2. Fungal Morphology Characterization

Each blast fungal strains as in ﬁlter paper was cultured on rice ﬂour agar (RFA) at 28 ◦C for 5 days, then a cork borer was used to cut a hole of the activated mycelium and was transferred to a new RFA plate, followed by incubation at 28 ◦C. Mycelium radii were measured at 4, 6, 8, and 10 days after incubation and each series was repeated 3 times. Mycelial characteristics on agar plates, including appearance, color, and amount of melanin pigment produced, were recorded. Conidia production was stimulated by scraping the surface of the fungal mycelium with a sterile spreader and incubating under ultraviolet light (black light) for 2 days. Conidia were harvested with sterile water. J. Fungi 2020, 6, 38 4 of 13

2.3. ITS Amplification and Sequencing ITS regions (18S rDNA, ITS1, 5.8S rDNA, and ITS2) were amplified using the universal primers ITS5 (forward primer; 50-GGAAGTAAAAGTCGTAACAAGG-30) and ITS4 (reverse primer; 50-TCCTCCGCTTATTGATATGC-30). Polymerase chain reaction (PCR) amplification was conducted in a 25 µL reaction volume containing 100 ng of DNA template, 2.5 µL of 10 PCR buffer (200 mM × Tris-HCl, 500 mM KCl), 2 mM of MgCl2, 0.2 mM of each dNTP, 0.5 µM of each primer, and 1 unit of Taq DNA polymerase (Vivantis, Shah Alam, Malaysia). PCR was performed using a Mastercycler nexus (Eppendorf, Hamburg, Germany) under the following conditions: Initial denaturation at 95 ◦C for 5 min; 35 cycles of denaturation at 95 ◦C for 30 s, annealing at 55 ◦C for 30 s, and elongation at 72 ◦C for 1 min, as well as a final extension at 72 ◦C for 5 min. The amplification products were resolved in 1% agarose gel using 0.5 TBE buffer at 100 V for 45 min followed by DNA staining × with EtBr and analysis using a Gel Doc XR+ with Image Lab software (Bio-Rad, Hercules, CA USA). PCR products were purified using the GF-1 PCR Clean-Up Kit (Vivantis, Shah Alam, Malaysia) based on the manufacturer’s instructions. The purified PCR products were sequenced with the ITS4 primer by a commercial sequencing service provider (U2Bio, Bangkok, Thailand). The resulting sequences of each isolate were refined using a BioEdit sequence alignment editor. To analyze relationships between isolates, all 59 sequences from this study and sequences of 10 reference Pyricularia oryzae isolates from difference hosts (obtained from the GenBank) were aligned using the MEGA X (Version 10.1.7) with Pyricularia pennisetigena (MN947526) used as an outgroup.

2.4. ISSR Analysis A total of 20 ISSR primers (Table2) were used for PCR. PCR reactions were performed in a total volume of 25 µL containing 1 PCR buﬀer, 0.25 mM dNTPs, 5 U Taq polymerase (Vivantis, Shah Alam, × Malaysia), 0.8 µmol ISSR primer, and 100 ng DNA template. PCR was performed using a Mastercycler nexus (Eppendorf, Hamburg, Germany) under the following conditions: Initial activation at 94 ◦C for 5 min; 35 cycles of denaturation at 94 ◦C for 1 min, annealing at 55 ◦C for 1 min, and extension at 72 ◦C for 2 min, as well as ﬁnal incubation at 72 ◦C for 10 min. PCR products were visualized by separation in a 1.5% agarose gel followed by DNA staining with EtBr and analysis using a Gel Doc XR+ with Image Lab software (Bio-Rad, Hercules, CA USA). All primers were repeated at least twice.

Table 2. Polymorphism in 59 rice blast strains revealed by inter-simple sequence repeat (ISSR) primers.

Primer ID Primer Name (a) Total Bands Polymorphic Bands % Polymorphic PIC (b) ISSR1 UBC-807 12 4 33.33 0.39 ISSR2 UBC-808 14 8 57.14 0.15 ISSR3 UBC-809 8 2 25.00 0.49 ISSR4 UBC-811 8 3 37.50 0.16 ISSR5 UBC-812 11 4 36.36 0.49 ISSR6 UBC-813 3 3 100.0 0.49 ISSR7 UBC-814 0 ISSR8 UBC-815 0 ISSR9 UBC-817 6 2 33.33 0.26 ISSR10 UBC-818 5 1 20.00 0.38 ISSR11 UBC-820 0 ISSR12 UBC-823 2 2 100.0 0.32 ISSR13 UBC-824 0 ISSR14 UBC-825 8 4 50.00 0.50 ISSR15 UBC-826 10 9 90.00 0.50 ISSR16 UBC-827 0 ISSR17 UBC-841 16 14 87.50 0.50 ISSR18 UBC-847 4 1 25.00 0.07 J. Fungi 2020, 6, 38 5 of 13

Table 2. Cont.

Primer ID Primer Name (a) Total Bands Polymorphic Bands % Polymorphic PIC (b) ISSR19 UBC-848 11 5 45.45 0.33 ISSR20 UBC-849 0 Total 118 62 Average 8.43 4.43 52.90 0.36 (a) ISSR primers set from the University of British Columbia (Microsatellite UBC primer set 9, University of British Columbia, Vancouver, Canada). (b) The polymorphism information content (PIC) value was used to measure the informativeness of a genetic marker. Grey rows of the table denote primers that were unable to amplify the blast fungal genome.

2.5. SRAP Analysis A total of 5 forward and 6 reverse primers of SRAP markers (for a total of 30 primer combinations) were used (Table3). The PCR reaction was in a total volume of 25 µl containing 1 PCR buﬀer, × 0.5 mM dNTPs, 1.5 U Taq polymerase (Vivantis, Shah Alam, Malaysia), 0.2 µmol forward primer and reverse primer, and 50 ng DNA template. PCR was performed using a Mastercycler nexus (Eppendorf, Hamburg, Germany) under the following conditions: Initial activation at 94 ◦C for 5 min; 6 cycles of denaturation at 94 ◦C for 1 min, annealing at 35 ◦C for 40 s, and extension at 72 ◦C for 2 min; 32 cycles of denaturation at 94 ◦C for 1 min, annealing at 46 ◦C for 40 s, and extension at 72 ◦C for 2 min; and ﬁnal incubation at 72 ◦C for 10 min. PCR products were visualized by separation in a 1.5% agarose gel followed by DNA staining with EtBr and analysis using a Gel Doc XR+ with Image Lab software (Bio-Rad, Hercules, CA USA). All primers were repeated at least twice.

Table 3. Polymorphism in 59 rice blast strains revealed by sequence-related ampliﬁed polymorphism (SRAP) primer combinations.

Primer ID Primer Names (a) Total Bands Polymorphic Bands % Polymorphic PIC (b) SRAP1 me1/em1 5 4 80.00 0.50 SRAP2 me1/em2 0 SRAP3 me1/em3 5 5 100.0 0.49 SRAP4 me1/em4 0 SRAP5 me1/em5 9 6 66.67 0.47 SRAP6 me1/em6 10 5 50.00 0.50 SRAP7 me2/em1 0 SRAP8 me2/em2 11 4 36.36 0.50 SRAP9 me2/em3 0 SRAP10 me2/em4 14 6 42.86 0.48 SRAP11 me2/em5 1 1 100.0 0.32 SRAP12 me2/em6 9 3 33.33 0.40 SRAP13 me3/em1 6 5 83.33 0.37 SRAP14 me3/em2 6 4 66.67 0.49 SRAP15 me3/em3 6 5 83.33 0.44 SRAP16 me3/em4 3 2 66.67 0.41 SRAP17 me3/em5 0 SRAP18 me3/em6 6 4 66.67 0.38 SRAP19 me4/em1 2 2 100.0 0.39 SRAP20 me4/em2 5 1 20.00 0.03 SRAP21 me4/em3 0 SRAP22 me4/em4 0 SRAP23 me4/em5 0 SRAP24 me4/em6 2 2 100.0 0.50 SRAP25 me5/em1 0 SRAP26 me5/em2 0 SRAP27 me5/em3 0 SRAP28 me5/em4 1 1 100.0 0.34 SRAP29 me5/em5 2 1 50.00 0.03 SRAP30 me5/em6 0 Total 103 61 Average 5.72 3.39 69.22 0.39 (a) SRAP markers were designed by Li and Quiros in 2001 to speciﬁcally amplify coding regions of genomes by targeting GC-rich exon regions (forward primers, named as me) and AT-rich intron regions (reverse primers, named as em). (b) The polymorphism information content (PIC) value was used to measure the informativeness of a genetic marker. Grey rows of the table denote primers that were unable to amplify the blast fungal genome. J. Fungi 2019, 5, x FOR PEER REVIEW 7 of 14

used to measure the informativeness of a genetic marker. Grey rows of the table denote primers that J. Fungiwere2020, unable6, 38 to amplify the blast fungal genome. 6 of 13

2.6. Data Analysis 2.6. Data Analysis Amplification products were scored as present (1), absent (0), or missing data (9). The level of Ampliﬁcation products were scored as present (1), absent (0), or missing data (9). The level of polymorphism for each primer was represented by the percentage of polymorphic variable loci polymorphism for each primer was represented by the percentage of polymorphic variable loci relative relative to all the loci analyzed. Dendrograms were constructed using the unweighted pair-group to all the loci analyzed. Dendrograms were constructed using the unweighted pair-group method method with arithmetic means (UPGMA) for clustering. For each of the dendrograms obtained from with arithmetic means (UPGMA) for clustering. For each of the dendrograms obtained from the ISSR, the ISSR, SRAP, and ISSR+SRAP combination data, a cophenetic matrix was generated using SRAP, and ISSR+SRAP combination data, a cophenetic matrix was generated using NTSYSpc version NTSYSpc version 2.01. The polymorphism information content (PIC) of an individual locus for 2.01. The polymorphism information content (PIC) of an individual locus for dominant markers (ISSR dominant markers (ISSR and SRAP markers) was calculated as: and SRAP markers) was calculated as: 2ƒi(12ƒi(1-ƒi),–ƒi), (1) (1)

f i wherewhere fii indicatesindicates thethe frequencyfrequency of of bands bands presence presence for for locus locus i[ 8[8].]. 3. Results 3. Results 3.1. Fungal Morphology Variation 3.1. Fungal Morphology Variation The morphological characteristics of 59 rice blast strains were examined. Fungal filaments on RFA platesThe were m observed,orphological and characteristics each strain showed of 59 di ricefferent blast characteristics strains were in examined. terms of appearance Fungal filament and color.s on ThreeRFA plate colorss were recorded:observed, White,and each cream, strain and showed grey. Flat differen or flutff characteristicsy filaments and in filament terms of density appearance were observedand color for. Three filamentous colors were pattern recorded (Table1: andWhite, Figure cream1)., After and grey. 10 days Flat of or culture, fluffy melaninfilament pigments and filament was observeddensity were from observeda ventral view for filam of theentous RFA plate pattern and (Table each strain 1 and was Figure classified 1). After based 10 on days their of melanin culture, productionmelanin pigment capability was and observed given onefrom of a fiveventral scores, view - to of++++ the R,FA depending plate and on each the strain amount was of classified melanin theybased produced on their (Table melanin1 and production Figure2). Fungalcapability growth and wasgiven measured one of five by thescores mycelial, - to ++++ radius, depend at four,ing six, on eight,the amount and 10 of days melanin after incubationthey produce (Supplementald (Table 1 and Data Figure S1). 2). The Fungal average growth radius was of fungal measured mycelium by the atmyceli four,al six, radius eight, at and four 10, six days, eight, were and 8.70, 10 15.36, days 21.78,after incubation and 26.94 mm(Supplement with standardal Data deviations 1). The average of 0.6, 1.13,radius 1.76, of andfungal 2.34 mycelium mm, respectively. at four, six, The eight, conidia and shape10 days was were observed 8.70, 15.36, under 21.78 a microscope, and 26.94 andmm waswith classifiedstandard asdevia onetion of fours of patterns:0.6, 1.13, Oval1.76, shapeand 2.34 (pattern mm, A,respectively. Figure3A), The short conidia pyriform shape shape was (pattern observed B, Figureunder3 aB), microscope pyriform shapeand was (pattern classified C, Figure as one3C), of andfour long patterns: pyriform Oval shape shape (pattern (pattern D, A Figure, Figure3D). 3A), Mostshortconidia pyriform were shape pyriform (pattern shape B, Figure (77.97%, 3B), 46pyriform/59). Four shape of 59 (pattern (6.78%) C of, Figure the blast 3C) fungal, and lon isolatesg pyriform had shortshape pyriform (pattern conidia,D, Figure eight 3D). isolates Most conidia (13.60%) were were pyriform long pyriform shape conidia,(77.97%, and 46/59). one Four strain of (RBR55001) 59 (6.78%) (1.69%)of the blast produced fungal oval isolates shape had conidia short (Table pyriform1). conidia, eight isolates (13.60%) were long pyriform conidia, and one strain (RBR55001) (1.69%) produced oval shape conidia (Table 1).

Figure 1. Variation in morphology of Pyricularia oryzae mycelia on the rice ﬂour agar (RFA) medium, showingFigure 1. di Variationﬀerences in morphology colors and mycelial of Pyricularia growth oryzae patterns. mycelia on the rice flour agar (RFA) medium, showing differences in colors and mycelial growth patterns.

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Figure 2. Variation in Pyricularia oryzae melanin production on ventral view of the rice flour agar Figure 2. Variation in Pyricularia oryzae melanin production on ventral view of the rice ﬂour agar (RFA) (RFA) medium, showing differences in melanin production ability. Scoring: No melanin pigment (-), medium,Figure 2. showing Variation di ﬀinerences Pyricularia in melanin oryzaeproduction melanin production ability. Scoring: on ventral No melanin view of pigment the rice (-), flour melanin agar melanin pigment production in less than 20% of the mycelia area (+), melanin production in 20%–50% pigment(RFA) medium production, showing in less differences than 20% ofin themelanin mycelia production area (+), ability melanin. Scoring production: No melanin in 20%–50% pigment of the (-), of the mycelia area (++), melanin production in 50%–80% of the mycelia area (+++), and melanin myceliamelanin area pigment (++), production melanin production in less than in 50%–80%20% of the of mycelia the mycelia area area(+), melanin (+++), and production melanin in production 20%–50% production in more than 80% of the mycelia area (++++). inof more the mycelia than 80% area of the(++) mycelia, melanin area production (++++). in 50%–80% of the mycelia area (+++), and melanin production in more than 80% of the mycelia area (++++).

Figure 3. Variation in conidia morphology shape under a light microscope. A: Pattern A, oval conidia;Figure 3. B: Variation Pattern B, in short conidia pyriform morphology conidia; shape C: Pattern under a C, light pyriform microscope conidia;. A: andPattern D: PatternA, oval D,conidia long; pyriformB: Pattern conidia. B, short pyriform conidia; C: Pattern C, pyriform conidia; and D: Pattern D, long pyriform Figure 3. Variation in conidia morphology shape under a light microscope. A: Pattern A, oval conidia; conidia. 3.2. ITSB: P Sequenceattern B, Analysisshort pyriform conidia; C: Pattern C, pyriform conidia; and D: Pattern D, long pyriform conidia. 3.2. ITSThe Sequence ITS gene Analysis regionswere successfully amplified and were 567 bp long, of which 497 bp contained an3.2. 18S ITSThe ribosomal Sequence ITS gene Analysis RNA regions partial were sequence successfully and complete amplified sequences and were of internal 567 bp transcribed long, of which spacer 497 1, the bp 5.8Scontained ribosomal an 18S RNA ribosomal gene, and RNA internal partial transcribed sequence spacer and 2.complete BLASTn sequence queries indicateds of internal that transcribed all isolates The ITS gene regions were successfully amplified and were 567 bp long, of which 497 bp usedspacer in this1, the study 5.8S wereribosomal 100% identicalRNA gene to,P. and oryzae internalin GenBank. transcribed 57 of spacer 59 isolates 2. BLASTn had the queries same nucleotide indicated contained an 18S ribosomal RNA partial sequence and complete sequences of internal transcribed sequencethat all isolates as the used rice blastin this reference study were isolate 100% UPM-PO identical (accession to P. oryzae no. in KT693184). GenBank. 57 Two of 59 isolates, isolates B1-2 had spacer 1, the 5.8S ribosomal RNA gene, and internal transcribed spacer 2. BLASTn queries indicated andthe 70-15,same nucleotide had a 99.80% sequence identity as with the rice most blast isolates reference with oneisolate nucleotide UPM-PO substitution (accession atno position. KT693184 403). that all isolates used in this study were 100% identical to P. oryzae in GenBank. 57 of 59 isolates had (TTwo/G), whichisolates, is located B1-2 and in the 70-15 ITS2, had region. a 99.80% However, identity both isolates with most were isolate 100%s identical with one with nucleotide another the same nucleotide sequence as the rice blast reference isolate UPM-PO (accession no. KT693184). ricesubstitution blast reference at position isolate, 403 Gongzhuling01 (T/G), which is (accessionlocated in no.the ITS2 AM180561). region. H Allowever isolates, both were isolate depositeds were Two isolates, B1-2 and 70-15, had a 99.80% identity with most isolates with one nucleotide in100% GenBank identical with with accession another numbers rice blast MT126183–MT126241 reference isolate, Gongzhuling01 (Table1). Interestingly, (accession isolates no. AM180561). B1-2 and substitution at position 403 (T/G), which is located in the ITS2 region. However, both isolates were 70-15All isolates had mating were type deposited locus MAT1-1, in GenBank while with the accession rest of the numbers isolates hadMT126183 mating–MT126241 type locus ( MAT1-2.Table 1). 100% identical with another rice blast reference isolate, Gongzhuling01 (accession no. AM180561). AInterestingly, dendrogram isolates depicted B1 by-2 and UPGMA 70-15 analysishad mating was type constructed locus MAT1 using-1, thewhile Thai the rice rest blast of the isolates isolates with had All isolates were deposited in GenBank with accession numbers MT126183–MT126241 (Table 1). eightmatingP. oryzaetype locusfrom MAT1 difference-2. A hosts.dendrogram The dendrogram depicted by showed UPGMA that analysis all Thai was rice constructed blast isolates using were the Interestingly, isolates B1-2 and 70-15 had mating type locus MAT1-1, while the rest of the isolates had inThai the rice same blast clade isolates with thewith reference eight P. rice oryzae blast from isolate differenc UPM-POe hosts. (accession The dendrogram no. KT693184) showed and isolatethat all mating type locus MAT1-2. A dendrogram depicted by UPGMA analysis was constructed using the Gongzhuling01Thai rice blast isolates (accession were no. in the AM180561) same clad ande with were theseparated reference rice from blast blast isolate isolates UPM from-PO other (accession host Thai rice blast isolates with eight P. oryzae from difference hosts. The dendrogram showed that all species.no. KT693184) The isolate and isolate SLSsh051 Gongzhuling01 (host: Stenotaphrum (accession secundatum no. AM180561)) and isolate and were w11-124 separated (host: fromPanicum blast Thai rice blast isolates were in the same clade with the reference rice blast isolate UPM-PO (accession miliaceumisolates from) were oth iner the host same species. clade The (Figure isolate4), andSLSsh051 another (host: clade Stenotaphrum comprise blast secundatum pathogens) and from isolate six no. KT693184) and isolate Gongzhuling01 (accession no. AM180561) and were separated from blast diw11fferent-124 hosts:(host: IsolatesPanicum BrTae miliaceum (host:) wereTriticum in the aestivum same clade), CBch004 (Figure (host: 4), andBromus another catharticus clade comprise), 19M7 (host: blast isolates from other host species. The isolate SLSsh051 (host: Stenotaphrum secundatum) and isolate xpathogen Triticosecales from), 19M5 six different (host: Hordeum hosts: I vulgaresolates), BrTae MZ5-1-6 (host: (host: TriticumEleusine aestivum coracana), CBch004), and IAsh017 (host: Bromus (host: w11-124 (host: Panicum miliaceum) were in the same clade (Figure 4), and another clade comprise blast Avenacatharticus strigosa), 19M7). The results (host: x confirmed Triticosecale that), all 19M5 isolates (host: used Hordeum in this study vulgare belonged), MZ5- to1-6 the (host:P. oryzae Eleusinerice blastpathogen pathogen.s from six different hosts: Isolates BrTae (host: Triticum aestivum), CBch004 (host: Bromus catharticus), 19M7 (host: x Triticosecale), 19M5 (host: Hordeum vulgare), MZ5-1-6 (host: Eleusine

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J. Fungi 2020, 6, 38 8 of 13 coracana), and IAsh017 (host: Avena strigosa). The results confirmed that all isolates used in this study belonged to the P. oryzae rice blast pathogen.

FFigureigure 4 4.. DendrogramDendrogram based onon thethe internal internal transcribed transcribed spacer spacer (ITS) (ITS) of theof the 59 isolates59 isolates used used in this in studythis studycompared compared to the to reference the referencePyricularia Pyricularia oryzae sequences oryzae sequences from di ff fromerent different hosts obtained hosts obtained from GenBank from GenBankbased on UPGMA based on cluster UPGMA analysis. cluster Hosts analysis. are specified Hosts inare parentheses, specified andin parentheses GenBank accession, and GenBank numbers accessionare provided numbers following are provide the colon.d following the colon. 3.3. Genetic Polymorphism from ISSR Marker 3.3. Genetic Polymorphism from ISSR Marker From the 20 ISSR primers, 14 primers showed reproducible and polymorphic DNA amplification From the 20 ISSR primers, 14 primers showed reproducible and polymorphic DNA patterns. 118 DNA bands were obtained with an average of 8.43 DNA bands per primer, and 62 DNA amplification patterns. 118 DNA bands were obtained with an average of 8.43 DNA bands per bands (52.90%) were polymorphic bands with an average of 4.43 polymorphic DNA bands per primer primer, and 62 DNA bands (52.90%) were polymorphic bands with an average of 4.43 polymorphic (Table2). The vast majority of ISSR primers showed a moderate level of polymorphism. PIC scores of DNA bands per primer (Table 2). The vast majority of ISSR primers showed a moderate level of each primer ranged from 0.07 (ISSR18) to 0.50 (ISSR14, ISSR15, and ISSR17), with an average of 0.36. polymorphism. PIC scores of each primer ranged from 0.07 (ISSR18) to 0.50 (ISSR14, ISSR15, and A genetic similarity ranged from 0.27–1.00. A dendrogram and two-dimensional principal component ISSR17), with an average of 0.36. A genetic similarity ranged from 0.27–1.00. A dendrogram and two- analysis (PCA) presenting the relationship from ISSR markers based on UPGMA cluster analysis dimensional principal component analysis (PCA) presenting the relationship from ISSR markers showed that the rice blast population in Thailand is a diverse, sparse arrangement that does not classify based on UPGMA cluster analysis showed that the rice blast population in Thailand is a diverse, into a group (Figure5A,B). sparse arrangement that does not classify into a group (Figure 5A and Figure 5B).

J. Fungi 2019, 5, x FOR PEER REVIEW 9 of 14

Eleusine coracana), and IAsh017 (host: Avena strigosa). The results confirmed that all isolates used in this study belonged to the P. oryzae rice blast pathogen.

Figure 4. Dendrogram based on the internal transcribed spacer (ITS) of the 59 isolates used in this study compared to the reference Pyricularia oryzae sequences from different hosts obtained from GenBank based on UPGMA cluster analysis. Hosts are specified in parentheses, and GenBank accession numbers are provided following the colon.

3.3. Genetic Polymorphism from ISSR Marker From the 20 ISSR primers, 14 primers showed reproducible and polymorphic DNA amplification patterns. 118 DNA bands were obtained with an average of 8.43 DNA bands per primer, and 62 DNA bands (52.90%) were polymorphic bands with an average of 4.43 polymorphic DNA bands per primer (Table 2). The vast majority of ISSR primers showed a moderate level of polymorphism. PIC scores of each primer ranged from 0.07 (ISSR18) to 0.50 (ISSR14, ISSR15, and ISSR17), with an average of 0.36. A genetic similarity ranged from 0.27–1.00. A dendrogram and two- dimensional principal component analysis (PCA) presenting the relationship from ISSR markers J. Fungi 2020, 6, 38 9 of 13 based on UPGMA cluster analysis showed that the rice blast population in Thailand is a diverse, sparse arrangement that does not classify into a group (Figure 5A,B).

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Figure 5. A., Dendrogram presenting the relationship from ISSR markers based on unweighted pair-

group method with arithmetic means (UPGMA) cluster analysis. B., 2D principal component analysis (PCA)Figure based 5. (A ),on Dendrogram genetic relationships presenting from the ISSR relationship data using from the ISSRDECENTER markers and based EIGEN on unweighted features of NTSYSpair-grouppc. method with arithmetic means (UPGMA) cluster analysis. (B), 2D principal component analysis (PCA) based on genetic relationships from ISSR data using the DECENTER and EIGEN features 3.4. Geneticof NTSYSpc. Polymorphism from SRAP Marker 3.4. GeneticFrom the Polymorphism 30 SRAP primerfrom SRAP combinations, Marker 17 combinations showed clear reproducible band patternsFrom and the exhibited 30 SRAP polymorphism. primer combinations, The results 17 combinationsshowed 103 DNA showed bands clear with reproducible 61 polymorphic band bandspatterns (68.22%). and exhibited The average polymorphism. number Theof generated results showed DNA 103bands DNA and bands polymorphic with 61 polymorphic DNA bands bands per primer(68.22%). were The 5.72 average and number3.38, respectively of generated (Table DNA 3). bands The vast and majority polymorphic of SRAP DNA primers bands per showed primer a weremoderate 5.72 andlevel 3.38, of polymorphism. respectively (Table PIC3 scores). The vastof ea majoritych primer of ranged SRAP primersfrom 0.03 showed (SRAP29) a moderate to 0.50 (SRAP1,level of polymorphism. SRAP6, SRAP8, PIC and scores SRAP24), of each with primer an average ranged of from 0.39. 0.03 The (SRAP29) coefficients to 0.50of similarity (SRAP1, ranged SRAP6, fromSRAP8, 0.29 and–0.97. SRAP24), A dendrogram with an averageand two of-dimensional 0.39. The coe PCAﬃcients presenting of similarity the relationship ranged from from 0.29–0.97. SRAP Amarkers dendrogram based and on UPGMA two-dimensional cluster analysis PCA presenting showed the that relationship the rice blast from population SRAP markers in Thailand based onis diverseUPGMA (Fig clusterure 6 analysisA,B). showed that the rice blast population in Thailand is diverse (Figure6A,B).

Figure 6. A(A.,), Dendrogram Dendrogram presenting presenting the the relationship relationship from from SRAP SRAP based based on on UPGMA UPGMA cluster cluster analysis analysis.. B B( .,), 2D 2D principal principal component component analysis analysis (PCA) (PCA) based based on on genetic genetic relat relationshipsionships from from SRAP SRAP data data using using the DECENTER and EIGEN features of NTSYSpc. DECENTER and EIGEN features of NTSYSpc. 3.5. Genetic Polymorphism from ISSR and SRAP Markers Combination 3.5. Genetic Polymorphism from ISSR and SRAP Markers Combination A total of 123 polymorphic DNA bands from the combined data of ISSR and SRAP markers were usedA to total construct of 123 a polymorphic dendrogram. DNA The coe bandsﬃcients from of the similarity combined ranged data fromof ISSR 0.28–0.95. and SRAP The markers dendrogram were usedand two-dimensional to construct a PCAdendrog presentingram. the The relationship coefficients from of ISSR similarity and SRAP ranged markers from based 0.28 on–0.95. UPGMA The dendrogram and two-dimensional PCA presenting the relationship from ISSR and SRAP markers based on UPGMA cluster analysis showed that most rice blast strains were clustered together with only a few strains separated from the main group (Figure 7A,B).

J. Fungi 2020, 6, 38 10 of 13 cluster analysis showed that most rice blast strains were clustered together with only a few strains J.separated Fungi 2019, 5 from, x FOR the PEER main REVIEW group (Figure7A,B). 11 of 14

FigureFigure 7. 7. A(A., ),Dendrogram Dendrogram presenting presenting the the relationship relationship from ISSRISSR markersmarkers and and SRAP SRAP markers markers based based on onUPGMA UPGMA cluster cluster analysis. analysis (B),. 2D B. principal, 2D principal component component analysis (PCA)analysis based (PCA) on genetic based relationships on genetic relationshipsfrom ISSR and from SRAP ISSR data and using SRAP the data DECENTER using the DECENTER and EIGEN featuresand EIGEN of NTSYSpc. features of NTSYSpc. 4. Discussion 4. Discussion 4.1. Morphological Characteristics of Rice Blast Fungus Strains in Thailand are Diverse 4.1. Morphological Characteristics of Rice Blast Fungus Strains in Thailand are Diverse Studies on the morphological characteristics of different rice blast fungus strains in Thailand revealedStudies variations on the morphological with respect to characteristics mycelium color, of different morphology, rice blast and conidiafungus shape.strains Colonyin Thailand color revealedvaried from variation whites to with grey. respect Some strainsto mycelium were unable color, to morphology produce melanin, and pigment,conidia shape. while mostColony showed color a variedrange offrom production white to capabilities. grey. Some The strains colony were radii unable of different to produce isolates were melanin recorded, pigment and, while the standard most showeddeviations a range at day of four, production six, eight, capabilities and 10 were. The 0.6, colony 1.13, 1.76, radii and of 2.34different mm, respectively. isolates were At recorded, the beginning, and theall standard strains grew deviation at similars at rates,day four but, theysix, eight, eventually and 10 began were growing0.6, 1.13, at1.76 diff, anderent 2.34 rates mm and, respectively. with unique Apatterns.t the beginning This led, toall the strains production grew at of similar different rate myceliums, but they morphologies. eventually Isolatebegan 10873,growing due at to different its highly ratedenses and mycelium, with unique showed pattern slowers. myceliumThis led to growth the production when compared of different with the mycelium others, which morphologies is a similar. Isolateresult to10873 a previous, due to reportits highly by Sonahdense etmycelium al. [9] in, thatshowed both slower color and mycelium mycelium growth grown when pattern compare variedd withgreatly the withother strainss, which on i oats a similar meal agar. result The to Magnaporthaceae a previous report are by distinguishedSonah et al. [9 from] in that the both Pyriculariaceae color and myceliumby their asexual grown morphs, pattern whichvaried are greatly Phialophora- with strains or Harpophora-like. on oat meal agar. Additionally, The Magnaporthaceae Magnaporthaceae are distinguishedhave falcate, versicolored from the Pyriculariaceae conidia on brown, by erect their conidiophores, asexual morphs, while whichPyricularia are orPhialophora Pyricularia-like- or Harpophoraspecies are characterized-like. Additionally, by pyriform Magnapo two-septaterthaceae have conidia, falcate three-celled, versicolored with conidia difference. on brown, The size erect and conidiophores,shape of spores while are an importantPyricularia criteria or Pyricularia for the classification-like species and are identification characterized of Pyricularia by pyriformspecies two [9-]. septateStrains conidia, significantly three varied-celled inwith spore difference. morphology The size depending and shape on specific of spores hosts. are Somean important isolates criteria derived forfrom the non-riceclassification hosts and also identification showed abnormal of Pyriculari sporea morphology species [9]. Strains as spores significantly were longer, varied cylindrical, in spore morphologyand obpyriform depend [9].ing In on our specific results, host wes found. Some varied isolates conidial derived shapes, from non from-rice oval hosts to long also pyriform.showed abnormalMost conidia spore were morphology a pyriform as shape spores (46 were/59). longer, Only one cyli strainndrical, (RBR55001) and obpyriform produced [9] oval. In our conidia result ands, wethis found was di variedfferent conidial from other shape strainss, from as well oval as to from long previous pyriform. reports. Most Manyconidia epidemiological were a pyriform studies shape on (46/59).the relationship Only one between strain (RBR55001) the formations, produced dispersal, oval and conidia infection and this behavior was different of spores from and environmental other strains asfactors well as have from been previous reported report [10].s. TheMany fungal epidemiological growth and studies its morphology on the relationship are affected between by different the formations,environmental dispersal factors,, and such infection as temperature, behavior pH,of spore moisture,s and degreeenvironmental of aeration, factors and have amount been and reported type of [10nutrients]. The fungal [11]. Furthermore, growth and its mycovirus morphology infection are affected in P. oryzae by differentcauses morphological environmental changes, factors including, such as temperature,changes in their pH, growth moisture, on degree agar, melanin of aeration, biosynthesis, and amount and formationand type of of nutrients conidia and [11]. appressoria Furthermore [12,]. mycovirusDue toinfection the overlap in P. oforyzae some causes morphological morphological characteristics changes, andincluding host-specific changes confirmation, in their growth ITS onDNA agar sequencing, melanin biosynthesis, has been employed and formation to identify of conidia fungal pathogens.and appressoria ITS regions [12]. have previously been usedDue for theto the detection, overlap identification, of some morphological and classification characteristics of Pyricularia and hostisolates-specific [13]. confirmation, A previous study ITS DNAconfirmed sequencing that the has ITS been gene employed regions could to identify identify fungalPyricularia pathogensat the. speciesITS regions level, have and previously sometimes lowerbeen used for the detection, identification, and classification of Pyricularia isolates [13]. A previous study confirmed that the ITS gene regions could identify Pyricularia at the species level, and sometimes lower than the species level [13,14]. In this study, we used the universal primers ITS5 and ITS4 to amplify the ITS region, because the ITS1 and ITS4 primer pair showed very poor success in our

J. Fungi 2020, 6, 38 11 of 13 than the species level [13,14]. In this study, we used the universal primers ITS5 and ITS4 to amplify the ITS region, because the ITS1 and ITS4 primer pair showed very poor success in our samples (the ITS1 and ITS4 primers can give rise to very poor ampliﬁcation in some species [15]). Our results conﬁrmed that all isolates used in this study belonged to the P. oryzae rice blast pathogen.

4.2. Genetic Diversity of Pyricularia oryzae Using SRAP and ISSR Markers The analysis of genetic variation in plant pathogen populations is an important prerequisite for understanding coevolution in the plant pathosystem [16]. In the present study, based on ISSR and SRAP markers, SRAP markers produced a higher percentage of polymorphic bands (69.22%) than ISSR markers (52.90%). However, the average number of polymorphic bands from different markers detected by ISSR primers was 4.43, which was higher than that of SRAP primers (3.39). The dendrograms from the ISSR, SRAP, and ISSR+SRAP combined data showed slightly different groupings. We were unable to group the fungal strains when using ISSR data alone, while the SRAP dendrogram and PCA showed that most of the population tended to cluster together, with only 11 strains separated from the main group (10926, 10927, BKK55002, RBR55001, CCO56002, BKK55001, BKK55003, Guy11, 40.3, 70-15, and B1-2). Sequence-related amplified polymorphism (SRAP) markers amplify the ORF, so the dendrogram may relate to gene variation. The range of similarity in ISSR, SRAP, and ISSR+SRAP analyses were 0.27–1.00, 0.29–0.97, and 0.25–0.95, respectively. The fact that the similarity coefficients in different marker systems were comparable suggests that both markers were equally effective. Mixed multiple marker systems provide more complete genome information about both ORFs and microsatellites. Molecular phylogenetic grouping obtained by ISSR and SRAP analyses did not correlate with morphological characteristics, but morphology may affect conidia germination or pathogenicity. Ngernmuen et al. [17] developed SSR markers from whole genome sequences of rice blast fungus strains and evaluated the genetic diversity of rice blast strains from Thailand. The results showed a high level of genetic diversity, and blast strains could be clustered based on geographic distribution and collection time. Silva et al. [18] used rep-PCR analysis with two primer sequences from Pot2 to assess genetic diversity of rice blast strains from Brazil. Xu et al. [19] used RAPD, REMAP, rep-PCR markers, and AVR gene sequences to examine the genetic diversity of rice blast fungus strains from China. The results revealed that the genetic structure of the P. oryzae population changed significantly over 3 years. Our results indicated that the rice blast population in Thailand does not explicitly group into different clades by site or year of sampling. The results from ISSR and SRAP data show low bootstrap values, suggesting clonal populations in our samples. The genetic variation supports the hypothesis that the main mechanism by which P. oryzae overcomes host resistant in natural conditions might be genome adaptation to avoid host recognition. Two main mechanisms for genome adaptation in fungi are sexual mating and parasexual recombination [20], but sexual reproduction in rice blast fungus has not been observed in the field and sexual spores have been produced only in laboratory settings. Therefore, parasexual recombination could most likely be the major mechanism for the exchange of DNA fragments [21] that causes genetic variation in the rice blast population. The genetic diversity and pathogen population structure from this study furthers our understanding of the rice blast fungus and could be useful for selecting rice cultivars or improving rice cultivars in the future.

Supplementary Materials: The following are available online: http://www.mdpi.com/2309-608X/6/1/38/s1, Data S1: Fungal growth on rice ﬂour agar (RFA) after 4, 6, 8, and 10 days after incubation; fungal mycelium were measured as the radius from the origin in millimeters. #1, 2, and 3 indicate individual replicates. #1 indicates replication 1, #2 indicates replication 2, and #3 indicates replication 3. Author Contributions: Each author contributed to the production of this manuscript: Conceptualization, C.J.; methodology, A.L.; software, A.L.; validation, A.L. and C.J.; formal analysis, A.L. and C.J.; investigation, C.J.; resources, A.L. and C.J.; data curation, A.L. and C.J.; writing—original draft preparation, A.L.; writing—review and editing, S.T. and C.J.; visualization, A.L., S.T., and C.J.; supervision, C.J.; project administration, C.J.; funding acquisition, A.L. and C.J. All authors have read and agreed to the published version of the manuscript. J. Fungi 2020, 6, 38 12 of 13

Funding: This work was supported by funding from Faculty of Science, Kasetsart University and the Royal Golden Jubilee Ph.D. Program (RGJ-Ph.D. Program) Grant No. PHD/0152/2556. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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