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Molecular Diagnosis of Thiophanate-Methyl-Resistant Strains of Fusarium fujikuroi in Japan

    Affiliations
    Authors and Affiliations
    • Fang Jing Li1
    • Ryoji Komura2
    • Chiharu Nakashima2
    • Masafumi Shimizu3
    • Koji Kageyama4
    • Haruhisa Suga5
    1. 1United Graduate School of Agricultural Science, Gifu University, Gifu 501-1193, Japan
    2. 2Graduate School of Bioresources, Mie University, Tsu, Mie 514-8507, Japan
    3. 3Faculty of Applied Biological Sciences, Gifu University, Gifu 501-1193, Japan
    4. 4River Basin Research Center, Gifu University, Gifu 501-1193, Japan
    5. 5Institute for Glyco-core Research (iGCORE), Gifu University, Gifu, 501-1193, Japan

    Abstract

    Fusarium fujikuroi is the pathogen of rice bakanae disease and is subclassified into gibberellin and fumonisin groups (G and F groups). Thiophanate-methyl (TM), a benzimidazole fungicide, has been used extensively to control F. fujikuroi. Previous investigation showed that F-group strains are TM sensitive (TMS), whereas most G-group strains are TM resistant (TMR) in Japan. The minimum inhibitory concentration in TMS strains was 1 to 10 μg ml−1, whereas that in TMR strains was >100 μg ml−1. E198K and F200Y mutations in β2-tubulin were detected in TMR strains. A loop-mediated isothermal amplification-fluorescent loop primer method was developed for diagnosis of these mutations and applied to 37 TMR strains and 56 TMS strains. The results indicated that 100% of TMR strains were identified as having either the E198K mutation (41%) or the F200Y mutation (59%), whereas none of the TMS strains tested showed either mutation. We found one remarkable TMR strain in the F group that had an F200Y mutation. These results suggest that E198K and F200Y mutations in β2-tubulin contribute to TM resistance in F. fujikuroi.

    Rice is an important crop worldwide and is a food staple for more than half of the global population. Rice bakanae is a seedborne disease caused by Fusarium fujikuroi. This disease was first described more than 100 years ago and occurs in all major rice-producing regions worldwide (Ou 1985; Sun and Snyder 1981). Its typical symptoms are abnormal stem elongation and yellowish leaves (Sun and Snyder 1981). Infected rice plants do not produce edible grains because of infertility. This disease causes widely varying yield losses of 3.0 to 95.4% (Gupta et al. 2015) and threatens food production.

    The pathogen causing rice bakanae disease was formerly known as Fusarium moniliforme (Ou 1985). It was subsequently discovered that F. moniliforme comprises several distinct species and was renamed F. fujikuroi species complex (Aoki et al. 2014), which includes >50 phylogenetic species (Aoki et al. 2014) and 13 mating populations (Leslie 1995; Lima et al. 2012). Three mating populations have been frequently isolated from rice plants (Amatulli et al. 2010; Desjardins et al. 2000): mating population A (Fusarium verticillioides), mating population C (F. fujikuroi), and mating population D (Fusarium proliferatum). Among these, F. fujikuroi is considered the bakanae disease pathogen (Aoki et al. 2014). It produces phytohormone gibberellins, which contribute to symptoms of bakanae disease (Cerdá-Olmedo et al. 1994). In addition, F. fujikuroi is known to produce various mycotoxins such as fumonisin, fusaric acid, moniliformin, and beauvericin, posing a health risk to humans and animals (Morgavi and Riley 2007).

    Currently, the control of bakanae disease depends greatly on fungicide application because of the absence of rice cultivars highly resistant to the disease. Methyl benzimidazole carbamate (MBC) and sterol demethylation inhibitor fungicides have been widely used to control bakanae disease (Aurangzeb et al. 1998; Becher and Wirsel 2012). MBC, a systemic fungicide, contains many structurally diverse compounds: benomyl, carbendazim, furidazole, thiophanate-methyl (TM), and thiabendazole. MBC has been used to protect crops from F. fujikuroi for approximately 50 years in Japan (Ogawa and Takeda 1988).

    Frequent application of TM has led to the development of resistance. TM resistance in F. fujikuroi was observed in 1984 in Japan (Yoshino 1988). F. fujikuroi can be subclassified into gibberellin and fumonisin groups (G and F groups) based on phylogenetic and gibberellin and fumonisin production analyses (Suga et al. 2019). G-group strains produce a high amount of gibberellin and are fumonisin nonproducers, whereas F-group strains produce little or no gibberellin but a high amount of fumonisin (Suga et al. 2019). According to Suga et al. (2019), G-group strains induced typical bakanae symptoms in rice seedlings, but rice seedlings inoculated with F-group strains were symptomless. Interestingly, TM sensitivities of these two subgroups differ. Most G-group strains from Japan were resistant to TM, whereas no F-group strains were TMR (Suga et al. 2019).

    MBC fungicides have selective toxicity to fungi because of their binding affinity to fungal microtubules. This group of fungicides affects cellular processes, such as cell mitosis, cytoskeleton formation, and intracellular trafficking (Magnucka et al. 2007). Benomyl and carbendazim, the representative fungicides in MBC, can interfere with mitosis in fungal hyphae and cause chromosomal abnormalities (Richmond and Phillips 1975; Zutshi and Kaul 1975). TM destroys fungal cell structure and impairs nuclear division in cell mitosis. As a result, appressoria formation, mycelia growth, and spore germination are inhibited.

    Resistance to MBC fungicides has occurred in fungal pathogens, and its molecular mechanism has been investigated intensively. In most cases, resistance is correlated with a point mutation in a β-tubulin gene, which results in an amino acid change at the fungicide binding site (Ma and Michailides 2005). F. fujikuroi has β1-tubulin (gene ID: FFUJ04397 in the whole genome sequence of F. fujikuroi strain IMI58289) and β2-tubulin (FFUJ03388) (Wiemann et al. 2013). An E198V or F200Y mutation in β2-tubulin was discovered to be responsible for carbendazim resistance in Chinese F. fujikuroi (Chen et al. 2014).

    Molecular methods are useful in monitoring the resistance of plant pathogens to fungicides. One such method, loop-mediated isothermal amplification-fluorescent loop primer (LAMP-FLP), measures temperature during rehybridization after completion of the LAMP reaction, for the detection of a specific single nucleotide polymorphism (SNP). In recent years, LAMP-FLP has been applied to the detection of fungicide resistance in the Fusarium head blight phytopathogen (Komura et al. 2018).

    Clarification of the fungicide resistance mechanism and the molecular method of monitoring resistance based on the mechanism is important for the continuing application and development of new fungicide compounds. In this study, mutations in β2-tubulin that correlated with TM resistance were investigated in Japanese F. fujikuroi, and a LAMP-FLP method was developed for detection of TMR strains.

    Materials and Methods

    Fungal isolation and strains.

    Three rice plants with bakanae symptoms were used for fungal isolation. Pieces of the basal part of rice plants were cut out and sterilized with 70% ethanol and then 1% sodium hypochlorite. Surface-sterilized pieces were rinsed with sterile water and placed on the Komada medium, according to Suga et al. (2014). Fungal colonies grown from the rice pieces were recultured on potato dextrose agar (PDA). One colony was obtained from each plant. Conidia produced on PDA were spread on new PDA to obtain pure culture by single-colony isolation. Microconidial chains formed on synthetic nutrient agar under black light were confirmed by microscopy. They were identified as F. fujikuroi by species-specific PCR restriction fragment length polymorphism (PCR-RFLP) of translation elongation factor 1α (TEF) and histone H3 developed by Suga et al. (2014). The subgroup (G or F group) was identified by PCR-RFLP, based on the subgroup-specific SNP (T618G) in TEF (Suga et al. 2014).

    In total, five strains were isolated in this study (Table 1). First, G-group strain Gfc1524101 and F-group strains Gfc1524102 and Gfc1524502 were isolated with Komada medium without TM. Based on a previous study (Suga et al. 2019), we expected simultaneous infections of G- and F-group strains in a rice plant. However, there have been no reported recoveries of G-group strains from these types of infections. We tried to isolate a G-group strain from rice samples. We used Komada medium amended with 100 μg ml−1 of TM because most Japanese G-group strains exhibited TM resistance in a previous study (Suga et al. 2019). Two G-group strains were further isolated. G-group strain Gfc1524102Re was isolated from the rice plant from which F-group strain Gfc1524102 was first isolated. G-group strain Gfc1524502Re was isolated from the rice plant from which F-group strain Gfc1524502 was first isolated.

    Table 1. Phenotypic and molecular characteristics and thiophanate-methyl sensitivity of isolates of Fusarium fujikuroi recovered from rice plants

    In addition, we used 37 TMR strains and 56 thiophanate-methyl–sensitive (TMS) strains for LAMP-FLP analyses (Table 2). Their TM sensitivities were previously determined by possible (TMR) or impossible (TMS) growth on PDA containing 100 μg ml−1 of TM (Suga et al. 2019). Among them, G-group strains MAFF235949 and Gfc0801001 and F-group strain Gfc0825009 have been used as representative strains of F. fujikuroi subgroups in other studies (Bao et al. 2020; Suga et al. 2014; Sultana et al. 2019). One remarkable F-group strain, Gfc1521033, was also used because all F-group strains isolated so far were TMS (Suga et al. 2019). This Japanese strain was received from our collaborator as a nonpathogenic TMR F. fujikuroi strain.

    Table 2. Thiophanate-methyl resistance and sensitivity and corresponding amino acids at positions 198 and 200 in β2-tubulin as determined by loop-mediated isothermal amplification-fluorescent loop primer method in Fusarium fujikuroi

    Fungicide sensitivity testing.

    TM was provided as Topsin M water-soluble powder containing 70% TM (Nihon Noh-yaku, Tokyo, Japan). Topsin M powder was dissolved in pure water as a stock solution with a TM concentration of 3.57 mg ml−1 and was then added into PDA at final TM concentrations of 0, 0.25, 0.5, 0.75, 1, 10, and 100 μg ml−1. TM-containing PDA was sterilized by autoclave. A 4-mm mycelial plug from a F. fujikuroi strain precultured on PDA at 25°C for 7 days was placed at the center of the TM-containing PDA medium. Three repeats were conducted for each concentration. After 5 days of growth at 25°C, colony radius was measured. The minimum inhibitory concentration (MIC) was determined by the complete inhibition of growth on PDA containing a significant concentration of TM. The half-maximal effective concentration (EC50) was calculated via a least square regression with natural log value of the TM concentration and degree of inhibition (percentage) to growth on PDA without TM.

    Genomic DNA extraction.

    The strains were cultivated for 7 days on PDA and recultivated in potato dextrose broth for 3 to 4 days. A 5-mm2 mycelial mat was taken from potato dextrose broth. Genomic DNA extraction using potassium ethyl xanthogenate (PEX) solution (6.25 mM of PEX, 700 mM of NaCl, 100 mM of Tris-HCl, pH 7.5, and 10 mM of EDTA, pH 8.0) was performed as previously described (Suga et al. 2008). The DNA precipitation was resuspended in 400 μl of sterile ultra-pure water and kept in a freezer at −30°C until used.

    Sequencing of β1-tubulin and β2-tubulin genes.

    PCR amplification for β1-tubulin was carried out in a 40 μl reaction volume containing 0.2 μl of 1.25 U μl−1 Tks Gflex DNA polymerase (Takala Bio Inc., Shiga, Japan), 20 μl of 2× Gflex PCR buffer (Takala Bio Inc.), 4 μl of 5 μM HS838 primer, 4 μl of 5 μM HS839 primer (Table 3), 9.8 μl of sterile ultra-pure water, and 2 μl of genomic DNA. PCR parameters were as follows: 94°C for 1 min, followed by 30 cycles of 98°C for 10 s, 63°C for 15 s, and 68°C for 1 min, followed by 68°C for 10 min as final extension.

    Table 3. Primers and quencher probe used in this study

    PCR amplification of β2-tubulin was carried out in a 40 μl reaction volume containing 0.2 μl of 5 U μl−1 Taq DNA polymerase (Thermo Fisher Scientific, Waltham, MA), 4 μl of 10× PCR buffer (Thermo Fisher Scientific), 3.2 μl of 2.5 mM dNTPs mixture, 4 μl of 5 μM HS835 primer, 4 μl of 5 μM HS836 primer (Table 3), 22.6 μl of sterile ultra-pure water, and 2 μl of genomic DNA. PCR parameters were as follows: 94°C for 2 min, followed by 30 cycles of 98°C for 1min, 63°C for 2 min, 72°C for 3 min, and 72°C for 10 min as final extension. The PCR products of β1-tubulin and β2-tubulin were purified by Nucleospin Extract Kit II Gel (Macherey-Nagel, Düren, Germany) and sequenced by Big-Dye terminator V3.1 with cycle sequencing kits (Thermo Fisher Scientific) using the primers shown in Table 3. Products were run on a 3130 Genetic Analyzer (Thermo Fisher Scientific) as previously described (Suga et al. 2008). Nucleotide sequences were processed by ChromasPro (Technelysium Pty., Tewantin, Queensland, Australia) and Genetyx version 4.0 (Genetyx, Tokyo, Japan). The sequences of β1- and β2-tubulin obtained in this study were deposited to DDBJ/EMBL/GenBank (accession number LC529139-LC529156).

    Amplified fragment length polymorphism analysis.

    Amplified fragment length polymorphism (AFLP) analysis was performed according to Suga et al. (2019). An AFLP microbial fingerprinting kit (Thermo Fisher Scientific) was used. The samples were run on the 3130 Genetic Analyzer and data were processed using GeneMapper software (Thermo Fisher Scientific). A previous study determined an AFLP haplotype with 66 markers developed for F. fujikuroi (Suga et al. 2019).

    LAMP-FLP reaction.

    So as not to overlook a false-negative reaction in screening, the primer and probe sets for the LAMP-FLP (Komura et al. 2018) were designed for a TMS strain of F. fujikuroi in G group as a full match, which does not have a mutation in the β2-tubulin sequence. LAMP primers and probe sets were designed, including an FLP primer labeled with a carboxyfluorescein (FAM) and a remove fluorescent dye and a quencher probe (QP) primer specifically hybridizing to the SNP region labeled with a Dabcyl quencher (Table 3). The total volume of each LAMP reaction mixture was 25 µl, containing 1× buffer (Nippon Gene, Tokyo, Japan), 1.4 mM of dNTPs mixture (Nippon Gene), 1.6 µM of each forward inner primer (FIP) (Fusarium ID17 FIP) and backward inner primer (BIP) (Fusarium ID17 BIP), 0.2 µM of each forward outer primer (F3) (Fusarium ID17 F3) and backward outer primer (B3) (Fusarium ID17 B3), 0.8 µM of loop primer F (Fusarium ID17 LB90), 0.2 µM of FAM loop primer B (Fusarium ID17 FLF90), 0.5 µM of probe Dabcyl (Fusarium ID17 Prob Dab), 1 µl of the amplification enzyme (Nippon Gene), and 4 µl of genomic DNA. Sterile ultra-pure water was used as a negative control. The LAMP reaction was performed using an LF-8 detecting instrument (Nippon Gene) at 66°C for 35 min. DNA amplification was measured by the turbidity of the LAMP products. An annealing curve from 95° to 35°C based on the quenching time was then analyzed using LF-8 Manager software and the LF-8 instrument. The experiment was repeated twice for confirmation of result consistency.

    Results

    F. fujikuroi subgroup isolated from diseased rice.

    F. fujikuroi was isolated from rice plants expressing typical bakanae symptoms (Table 1). One strain (Gfc1524101) was identified as belonging to G group, and two strains (Gfc1524102 and Gfc1524502) were identified as belonging to F group, despite the F group being unable to induce bakanae symptoms (Suga et al. 2019). Further isolation detected G-group strains Gfc1524102Re and Gfc1524502Re from the rice piece after F-group strain isolation. All strains isolated in this study showed different AFLP haplotypes (Table 1; Supplementary Table S1). The genetic independence of each strain was therefore confirmed.

    TM sensitivity.

    The TM sensitivities of the strains isolated in this study were investigated, beginning with PDA amended with 100 μg ml−1 of TM according to Suga et al. (2019). Most G-group strains were determined as TMR based on their ability to grow on the medium. All F-group strains were determined as TMS. Subsequently, we confirmed MIC and EC50 of them with representative strains of G group and F group and a remarkable F-group strain, Gfc1521033, by using a series of TM concentrations (Table 1). Growths of the TMS strains were 100% reduced (average, n = 4) at 10.0 μg ml−1 of TM, whereas TMR strains showed 63 and 80% growth reductions (average, n = 5) at 10.0 μg ml−1 and 100.0 μg ml−1 of TM, respectively. Typical growth reductions dependent on TM concentrations are shown in Figure 1. The MICs of TMS strains were 1.0 to 10.0 μg ml−1 and were >100 μg ml−1 in TMR strains (Table 1). EC50 in TMS strains (3.3 to 4.1 μg ml−1) was lower than that in TMR strains (7.1 to 12.7 μg ml−1) (Table 1).

    Fig. 1.

    Fig. 1. Mycelial growth reduction of Fusarium fujikuroi by various concentrations of thiophanate-methyl (TM). Colony radius on potato dextrose agar (PDA) amended with various concentrations of TM was measured after 5 days of growth at 25°C. The gibberellin and fumonisin groups (G and F groups) are subgroups in F. fujikuroi (Suga et al. 2019). Strains were separated by possible (TMR, TM resistant) or impossible (TMS, TM sensitive) growth on PDA containing 100 μg ml−1 of TM. Error bars indicate the standard deviation of three repeats.

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    Mutation in β-tubulin.

    We compare the β1- and β2-tubulin sequences between TMS and TMR strains in Table 1. There was no amino acid substitution in β1-tubulin, and either E198K or F200Y mutation in β2-tubulin was detected in TMR strains (Fig. 2). In addition, N37T was detected in F-group strains except for Gfc1524102 compared with G-group strains (Fig. 2). Previously, to investigate E198K and F200Y mutations in F. fujikuroi strains, TM sensitivities of tested strains was determined (Suga et al. 2019). LAMP-FLP was developed for SNP diagnosis: E (GAG) 198K (AAG) and F (TTC) 200Y (TAC), according to Komura et al. (2018). The primer position used for LAMP-FLP is indicated in Figure 3. TMS strain MAFF235949 with no mutation and TMR strain Gfc0801001 with F200Y were correctly determined by the LAMP-FLP assay (Table 2). TMR strain Gfc0901009 was determined as E198K by the LAMP-FLP assay (Table 2), and E198K in this strain was confirmed by β2-tubulin sequencing. Therefore, accuracy of LAMP-FLP was verified. This method revealed that 40.5 and 59.5% of the 37 TMR strains have E198K and F200Y mutations, respectively, and that none of the 56 TMS strains has these mutations (Table 2).

    Fig. 2.

    Fig. 2. Nucleotide and amino substitutions in β2-tubulin in Fusarium fujikuroi. The gibberellin and fumonisin groups (G and F groups) are subgroups in F. fujikuroi (Suga et al. 2019). Positions of amino acid substitution are indicated on G-group strain MAFF235949. Putative exons are indicated as boxes. Strains were separated by possible (TMR, thiophanate-methyl resistant) or impossible (TMS, thiophanate-methyl sensitive) growth on potato dextrose agar containing 100 μg ml−1 of thiophanate-methyl. Amino acid substitutions specific to TMR (E198K and F200Y) are highlighted in gray. Nucleotides associated with amino acid substitution are underlined.

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    Fig. 3.

    Fig. 3. Design of primers and quencher probe for loop-mediated isothermal amplification-fluorescent loop primer (LAMP-FLP) method. The primer and probe for LAMP-FLP were designed as a full match for thiophanate-methyl– sensitive (impossible growth on potato dextrose agar containing 100 μg ml−1 of thiophanate-methyl) strain MAFF235949 of Fusarium fujikuroi, which has no substitution in the target position. The nucleotide sequence is indicated as double-strand DNA. Basic primers for LAMP reaction are forward and backward inner primers (FIP and BIP), forward and backward outer primers (F3 and B3), and loop primers (LB90 and FLP). FLP primer is labeled with a FAM fluorescent dye at the 5′-end. A quencher probe (QP) labeled with Dabcyl quencher is included. The target codons, E-(GAG) 198K-(AAG) and F-(TTC) 200Y-(TAC), that correlated with thiophanate-methyl resistance are boxed. The QP is located in the position including the target codons.

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    Discussion

    We determined that E198K or F200Y mutations in β2-tubulin correlated with TM resistance in Japanese F. fujikuroi. All TMR strains were identified by the LAMP-FLP method to have either E198K or F200Y. These results indicate that the LAMP-FLP developed in this study is useful for monitoring TM resistance in Japanese F. fujikuroi.

    TM resistance in other Fusarium species causing disease of other crop hosts has also been reported: Fusarium head blight caused by Fusarium graminearum (Yoshimatsu et al. 2006), potato dry rot caused by F. sambucinum (Kawchuk et al. 1994), and watermelon Fusarium wilt caused by F. oxysporum f. sp. niveum (Petkar et al. 2017). Moreover, TMR has been detected not only in Fusarium species but also in other fungal pathogens such as Botrytis cinerea and Monilinia fructicola (Amiri et al. 2014; Chen et al. 2013).

    Five types of tubulin genes have been found in fungi: α1-, α2-, β1-, β2-, and γ-tubulin. Trichoderma spp., F. graminearum, and F. fujikuroi have two types of β-tubulin (β1- and β2-tubulin) (Goldman et al. 1993; Wiemann et al. 2013), although most fungal pathogens carry only one type of β-tubulin (Goldman et al. 1993; Orbach et al. 1986). Previous research suggests that these two β-tubulins have different functions in biological characteristics (Liu et al. 2013). β1-tubulin functions on sexual reproduction, whereas β2-tubulin is important for vegetative growth (Liu et al. 2013; Zhao et al. 2014). Y50D mutation in β1-tubulin corresponded to benomyl resistance in F. verticillioides (Leslie and Dickman 1991; Yan and Dickman 1996), whereas nucleotide substitutions GAG (E) to GTG (V) at codon 198, TTC (F) to TAC (Y) at codon 200, and GGC (G) to GGT (G) at codon 235 in β2-tubulin corresponded to carbendazim resistance in Chinese F. fujikuroi (Chen et al. 2014). The current study also indicated that TM resistance is caused by mutations in β2-tubulin, possibly because of the importance of β2-tubulin in vegetative growth.

    In Japan, TM has been widely used to control bakanae disease. Therefore, bakanae disease controlled with TM for an extended period might impose TMR selection pressure on the G group as the bakanae pathogen. However, one anomalous TMR strain, Gfc1521033, was found in the F group. It suggests that TMR strains may become common in the F group if similar selection pressure is exerted on it.

    Multiple infections of pathogens are common in the field and are of major concern in food supply. Coinfection with different species, such as Ustilago maydis and F. verticillioides in maize, is known to occur (Jonkers et al. 2012), but coinfection with different strains of the same species is rarely proved in plant-pathogenic fungi. In the present study, we found coinfection with F-group and G-group strains of F. fujikuroi in a single rice plant. To our knowledge, this is the first report of coinfection with different strains of F. fujikuroi. Multiple infections with different strains of the same pathogenic species in a plant may occur frequently in the field but are unlikely to be detected because of difficulty identifying independent strains in isolation procedures.

    PCR-based techniques can monitor resistance faster than conventional methods, such as the mycelial growth inhibition assay on an agar medium. Allele-specific PCR, PCR-RFLP, primer-introduced restriction analysis PCR (PIRA-PCR), and quantitative allele-specific real-time PCR are successfully and widely used to diagnose MBC fungicide resistance (Banno et al. 2007; Silvestre and Humbert 2000; Zhang et al. 2015). PIRA-PCR was developed to determine carbendazim-resistant F. fujikuroi strains in China (Zhang et al. 2015). However, PIRA-PCR can identify only E198L and G235G substitution in β2-tubulin; it is unable to identify TMR strains in Japanese F. fujikuroi because they have either E198K or F200Y. Therefore, we developed a LAMP-FLP based on E-(GAG)198K-(AAG) and F-(TTC)200Y-(TAC) substitution (Fig. 2). LAMP-FLP has also been successfully used to detect F167Y, E198Q, and F200Y in β2-tubulin of F. graminearum that result in benzimidazole resistance (Komura et al. 2018).

    According to previous studies, the amino acid substitutions at codon 198 and 200 in β2-tubulin are responsible for MBC resistance in plant-pathogenic fungi (Chen et al. 2014; Koenraadt et al. 1992). In Japan, a part of F. fujikuroi TMR strains had E198K in β2-tubulin. This is the first observation of E198K in β2-tubulin associated with MBC resistance in F. fujikuroi. On the other hand, E198K responsible for MBC resistance was previously reported in B. cinerea, F. asiaticum, F. graminearum s. str., Lasiodiplodia theobromae, and Venturia inaequalis (Chen et al. 2020; Koenraadt et al. 1992; Yarden and Katan 1993). In addition, E198K, E198A, E198V, E198G, E198Q, and E198L at this position were also found to be associated with MBC resistance in various phytopathogens (Komura et al. 2018; Ziogas et al. 2009).

    F200Y was also found in some TMR strains in Japanese F. fujikuroi (Table 2). To date, F200Y in β2-tubulin of carbendazim-resistant strains of F. fujikuroi in China has been reported (Chen et al. 2014). In addition, F200Y associated with MBC resistance has been observed in other phytopathogens, such as B. cinerea, F. asiaticum, and Colletotrichum cereale (Suga et al. 2011; Yarden and Katan 1993; Young et al. 2010). Previous research suggested that the amino acid substitution at codon 198 or 200 in β-tubulin has been characterized as respectively highly or moderately resistant to MBC, based on EC50 values (Koenraadt et al. 1992; Yarden and Katan 1993). In various fungal pathogens, substitution at codon 198 in β-tubulin confers high resistance, whereas substitution at codon 200 resulted in moderate resistance to MBC (Koenraadt et al. 1992; Yarden and Katan 1993). In our study, EC50 of the TMR strains with E198K was slightly higher than the TMR strains with F200Y (Table 1). However, the association between the level of TM resistance and these substitution types in F. fujikuroi strains is uncertain because of the limited number of samples.

    PCR-RFLPs were developed for F. fujikuroi and its subgroup identification (Suga et al. 2014, 2019). We developed LAMP-FLP for diagnosis of TMR strains in F. fujikuroi in this study. In addition, 66 AFLP markers are available for identification of individual strains in F. fujikuroi (Suga et al. 2019). These assays are based on the SNPs in the genome. Therefore, once we obtained the genome of the target strain, information on the strain could be mined from the specific SNPs. Further development of more comprehensive, unitary, and easier methods for SNP analyses, such as microarrays, can advance F. fujikuroi population studies in future.

    Acknowledgments

    The authors thank Katsutoshi Kuroda (Mie Agricultural Research Center, Japan) for support in the collection of bakanae diseased rice, Hideki Watanabe (Gifu Prefecture Agricultural Technical Center, Japan) for providing the F-group TMR stain, and Tomomi Katsu (Gifu University, Japan) for technical support.

    The author(s) declare no conflict of interest.

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