A Novel Medium for Isolating Two Japanese Species in the Fusarium graminearum Species Complex and a Dipstick DNA Chromatography Assay for Species Identification and Trichothecene Typing

Members of the Fusarium graminearum species complex (Fg complex) are the primary pathogens that cause Fusarium head blight in wheat and barley. Fg complex members grow poorly on Fusarium oxysporum-selective media, such as Komada and Fo-G2, that have also been used for the isolation of other Fusarium species. Therefore, Komada medium was modified as FG medium for the isolation of Fg complex members. However, the production of pentachloronitrobenzene that is the most effective component of FG medium is discontinued and new media is required for the selective isolation of Fg complex members. In addition, the rapid diagnosis of isolated fungi is useful for the disease control. Novel tools have been developed for isolating and characterizing Fg complex members. FG21, a semi-selective medium for isolating Fg complex members, was developed using potato dextrose agar. Furthermore, a dipstick DNA chromatography assay was developed both to identify Fusarium graminearum sensu stricto and Fusarium asiaticum in the Fg complex and their trichothecene mycotoxin types. The easier isolation and characterization of Fg complex members in Japan was attained by the combined use of FG21 medium and the dipstick DNA chromatography assay.


Introduction
Fusarium head blight (FHB) is a devastating disease affecting cereal crops. It reduces yield, quality, contaminates the grain with mycotoxins, and affects the overall production of wheat and barley worldwide. In North America, the total loss of wheat by FHB during 1991-1997 was estimated to be USD 1.3 billion [1]. FHB causes the premature bleaching of part or the entire wheat ear. In some cases, salmon-pink sporodochia can be observed at glume edges. Flowering wheat heads are susceptible to airborne spore infection. Precipitation during the flowering stage of wheat promotes an FHB outbreak. FHB pathogens include Microdochium nivale and Fusarium spp., such as members of the Fusarium graminearum species complex (Fg complex), Fusarium culmorum, Fusarium avenaceum, Fusarium poae, and Fusarium pseudograminearum.
Mycotoxins produced by FHB pathogens cause further problems in the harvested grains. Trichothecene mycotoxins are produced by Fusarium spp. but not Microdochium nivale. Members of the Fg complex are the primary pathogens of FHB and are distributed worldwide. Phylogenetic analyses revealed that the Fg complex consists of at least 16 distinct

Strains and Growth Test
The strain information is summarized in Table 1. Strains of all Fg complex members and trichothecene types (F. graminearum s. str./3ADON and 15ADON, F. asiaticum/NIV, 3ADON, and 15ADON, and F. vorosii/15ADON) that have been detected in Japan [3,9] were used as the fungal strains to develop the FG21 medium and dipstick DNA chromatography assay. Other strains, including Fusarium spp., were used as reference fungi. Strains F1904008 and E1904019 were isolated from wheat seeds using prototype FG21 medium. Strains P1904014 and A1904015 were isolated from barley seeds. In these four strains, the internal transcribed spacer region nucleotide sequences of the nuclear ribosomal DNA were obtained by directly sequencing the PCR amplicons using the primer pair ITS1/ITS4 [22]. These results were used in a homology search at GenBank database for genus identification. The nucleotide sequences were deposited in the DDBJ/EMBL/GenBank database under the accession numbers LC719979-LC719982. For the medium growth test, strains precultured on PDA at 25 • C for 1 week were cut using a sterile cork borer with a 4 mm diameter. Four strains were transplanted onto the medium in a 90 mm Petri dish and incubated at 25 • C for 4 days.

Fungal Isolation from Plant Source
Seeds or wheat heads were sterilized by sequential washing with 70% ethanol, 1% sodium hypochlorite containing 0.02% Tween-20, and sterile distilled water. After removing the water, samples were placed on FG21 medium in a 90 mm Petri dish. Reddish colonies formed on the medium around the plant materials in 3 d at 25 • C. If no reddish colonies appeared, the Petri dish was turned over, removing the plant material, and incubated further for several days. Aerial mycelia from each reddish colony were collected and used for DNA extraction. For colony purification, colonies were subcultured on synthetic nutrient agar (SNA) under black light for sporulation. The spore suspension collected from the culture on SNA was spread onto PDA, and a single colony was used for DNA extraction.

DNA Extraction
Simple and rapid DNA extraction was performed using the Kaneka Easy DNA Extraction Kit, version 2 (Kaneka, Tokyo, Japan), according to the manufacturer's instructions with the following modifications: a crumb of a colony's aerial mycelia was sniped with tweezers and transferred to a microtube containing two small metal screws and 100 µL of Reagent A. The mycelia crumb was crushed by vortexing for several minutes. Fourteen microliters of Reagent B was added, and the metal screws were removed with a micropipette tip. After centrifugation at 17,800× g for 1 min, the supernatant was used as the DNA solution for multiplex PCR.

Primer Design and Multiplex PCR Conditions
The primers used are listed in Table 2 (Tohoku Bio-Array, Sendai, Japan). The HS976/HS1039 primer pair was designed for FGSG_17538, which encodes an aldehyde dehydrogenase that is present only in the genome of F. graminearum s. str. among the genomes of F. asiaticum, F. cortaderiae, and F. graminearum s. str. [26]. The HS974/HS1035 primer pair was designed for APS1, which encodes a non-ribosomal peptide synthetase involved in apicidin biosynthesis and was present only in the genome of F. asiaticum, among the three genomes [26]. Starkey et al. [3] designed 3CON, 3NA, 3D15A, and 3D3A primers on Tri3 for trichothecene typing using multiplex PCR. These primers were used in the current study, although the 5 termini of forward primers were labelled with five different tag-linker sequences and reverse primers labelled with biotin (Tohoku Bio-Array). Multiplex PCR for species identification was performed in 10 µL of a reaction mixture, containing 1 µL of 10× buffer, 1 µL of 2 mM dNTP mixture, 0.6 µL of 25 mM MgSO 4 , 2.0 µL of primer mix, 0.25 µL of 1 U/µL KOD-Plus-Neo DNA polymerase (Toyobo, Osaka, Japan), 4.15 µL of ultrapure water, and 1 µL of genomic DNA solution. Primer mix included 1.67 µM of HS970 and HS1039 and 0.83 µM of HS974 and HS1035. PCR was conducted using the T100 thermal cycler (Biorad, Hercules, CA, USA) under the following conditions: 94 • C for 2 min, 25 cycles of 94 • C for 30 s, and 66 • C for 30 s. Multiplex PCR for trichothecene typing was similarly performed, except with 3.2 µL of primer mix and 2.95 µL of ultrapure water. The primer mix included 1.25 µM of 3CON, 3D3A, 3D15A, and 2.5 µM of 3NA. A TaKaRa PCR Thermal Cycler TP600 (Takara, Otsu, Japan) was also used, with the cycle number increased to 30. For trichothecene typing, the multiplex PCR solution had 2.95 µL of ultrapure water replaced with primer mix.
Nucleotide sequences of PCR amplification region in FGSG_17538 and APS1 were used to search for homologous sequence in the Genbank genome database.

Dipstick DNA Chromatography Assay
The PCR amplicons were detected by dipstick DNA chromatography. For species identification, 5 µL of multiplex PCR solution was mixed with 5 µL of dipstick developing buffer and 1.5 µL of streptavidin-coated blue latex beads (Tohoku Bio-Array). The amplicons were bound to blue latex beads for 5 min by streptavidin-biotin association. The PCR solution for trichothecene typing was similarly prepared, then mixed with the species identification solution in a microtube. A dipstick (Tohoku Bio-Array) was inserted and left for 20 min for chromatography. The amplicon-tagged sequence hybridized to its corresponding anti-tag on the dipstick, and the positive reactions appeared as blue lines.

Development of the FG21 Medium
Fg complex members are known to grow relatively fast on PDA, which is a general medium for fungal culturing. Initially, the authors prepared a prototype FG21 medium based on PDA, noting that red pigmentation appearing on this medium, which can be an indicator of Fg complex members. F. graminearum s. str., F. asiaticum, and F. vorosii grew on the prototype FG21 medium, yet they grew more slowly than on PDA. Prototype FG21 medium effectiveness was evaluated by an isolation trial of Fg complex members from wheat and barley seeds. Several reddish colonies appeared, but alongside many other fungal colonies. Four strains (F1904008, E1904019, P1904014, and A1904015) that were not Fg complex members based on colony appearance were obtained as references for evaluating the final FG21 medium.
The same components of prototype FG21 were mixed at the same concentrations in oatmeal agar, cornmeal agar, Sabouraud dextrose broth agar, and malt extract agar. However, red pigmentation in colonies was not observed on any of the media.
To improve medium selectivity, Trifumin wettable powder was added to the prototype FG21 medium at 0.01% (w/v), 0.1%, or 1%. F. graminearum s. str., F. asiaticum, and F. vorosii showed no change in growth with 0.01% Trifumin, and growth reduction occurred with 0.1% and 1% Trifumin. Additionally, bacterial colonies were observed in the trial isolation of Fg complex members from wheat seeds using the prototype FG21 medium containing 0.01% Trifumin. These colonies were isolated, and their growth inhibition was measured using a range of chloramphenicol concentrations from 0.025% (w/v) to 0.125% in the prototype FG21 medium. Finally, the FG21 medium was established by adding 0.01% Trifumin and increasing the chloramphenicol concentration to 0.125%. To test its efficacy in isolating Fg complex members, the FG21 medium was compared with Komada, FG, and Fo-G2 media in a growth test using both Fg complex members and the reference fungal strains (Figure 1). F. graminearum s. str., F. asiaticum, and F. vorosii did not grow on Komada and Fo-G2 media; however, F. asiaticum Fa0239005 did grow on the Komada   Potato dextrose agar (PDA) is a general medium for fungal culture and is used as the foundation of FG21 medium. Komada [16] and Fo-G2 [17] are Fusarium oxysporum selective media. FG is a Fusarium graminearum-selective medium [20]. The strains were cultured at 25 °C for 4 d. Fusarium graminearum sensu stricto Fg0101020 (1), Fg0201201 (2), Fusarium asiaticum Fa0244004 (3), Fa0239005 (4), Fusarium vorosii Fv0301112 (5), Fv0301831 (6), Fusarium avenaceum MAFF101042 (7), MAFF235547 (8)

Development of the Dipstick DNA Chromatography Assay
The optimal multiplex PCR conditions for trichothecene typing and species identification were determined based on the specific target DNA amplification in all Fg complex members and trichothecene types that have been detected in Japan:  Potato dextrose agar (PDA) is a general medium for fungal culture and is used as the foundation of FG21 medium. Komada [16] and Fo-G2 [17] are Fusarium oxysporum selective media. FG is a Fusarium graminearum-selective medium [20]. The strains were cultured at 25 • C for 4 d.

Development of the Dipstick DNA Chromatography Assay
The optimal multiplex PCR conditions for trichothecene typing and species identification were determined based on the specific target DNA amplification in all Fg complex members and trichothecene types that have been detected in Japan: F. graminearum s. str./3ADON and 15ADON; F. asiaticum/NIV, 3ADON, 15ADON; F. vorosii/15ADON (Figure 2). These multiplex PCR conditions were also applied to other members of the Fg complex (Fusarium acaciae-mearnsii, Fusarium boothii, and Fusarium meridonale) and its related species (Fusarium lunulosporum and Fusarium pseudograminearum) to investigate the applicability of PCR; although unlike our chosen species, these have never been isolated in Japan (Figure 3). These multiplex PCR conditions were also applied to other members of the Fg complex (Fusarium acaciae-mearnsii, Fusarium boothii, and Fusarium meridonale) and its related species (Fusarium lunulosporum and Fusarium pseudograminearum) to investigate the applicability of PCR; although unlike our chosen species, these have never been isolated in Japan (Figure 3).
Target DNA amplification was performed in all strains tested by multiplex PCR for trichothecene typing. The identified trichothecene types matched with previously reported data [23]. Amplicons the same size as F. graminearum s. str. were observed in both strains of F. boothii using multiplex PCR for species identification. Additionally, the homologous nucleotide sequence of the amplified region of FGSG_17538 was detected in the genome database These multiplex PCR conditions were also applied to other members of the Fg complex (Fusarium acaciae-mearnsii, Fusarium boothii, and Fusarium meridonale) and its related species (Fusarium lunulosporum and Fusarium pseudograminearum) to investigate the applicability of PCR; although unlike our chosen species, these have never been isolated in Japan (Figure 3).  Target DNA amplification was performed in all strains tested by multiplex PCR for trichothecene typing. The identified trichothecene types matched with previously reported data [23]. Amplicons the same size as F. graminearum s. str. were observed in both strains of F. boothii using multiplex PCR for species identification. Additionally, the homologous nucleotide sequence of the amplified region of FGSG_17538 was detected in the genome database (whole-genome shotgun contigs) of A dipstick DNA chromatography assay was performed using a mixture of multiplex PCR for species identification and trichothecene typing. The tested PCR mixtures included all Fg complex members and trichothecene types that have been detected in Japan. The PCR amplicons used for trichothecene typing were designed to be bound on the lower three lines, and PCR amplicons for species identification were designed to be A dipstick DNA chromatography assay was performed using a mixture of multiplex PCR for species identification and trichothecene typing. The tested PCR mixtures included all Fg complex members and trichothecene types that have been detected in Japan. The PCR amplicons used for trichothecene typing were designed to be bound on the lower three lines, and PCR amplicons for species identification were designed to be bound on the two upper lines of the dipstick (Figure 4). In all the strains tested, positive lines appeared 20 min after chromatography development (Figure 4).

Practical Use of the FG21 Medium and the Dipstick DNA Chromatography Assay
Barley seeds were collected from fields where FHB symptoms were observed in 2022. Additionally, wheat heads with FHB symptoms were collected in 2022. They were used as sources for fungal isolation using the FG21 medium. Reddish colonies that were assumed to be Fg complex members appeared on the FG21 medium ( Figure 5). Figure 5. Isolation of species in the Fusarium graminearum species complex from wheat head or barley seeds on the FG21 medium. Wheat head or barley seeds were placed on FG21 medium after surface sterilization and kept at 25 °C for 3 days. Reddish colonies were identified as nivalenol type of Fusarium asiaticum using a dipstick DNA chromatography assay.

Practical Use of the FG21 Medium and the Dipstick DNA Chromatography Assay
Barley seeds were collected from fields where FHB symptoms were observed in 2022. Additionally, wheat heads with FHB symptoms were collected in 2022. They were used as sources for fungal isolation using the FG21 medium. Reddish colonies that were assumed to be Fg complex members appeared on the FG21 medium ( Figure 5).

Practical Use of the FG21 Medium and the Dipstick DNA Chromatography Assay
Barley seeds were collected from fields where FHB symptoms were observed in 2022. Additionally, wheat heads with FHB symptoms were collected in 2022. They were used as sources for fungal isolation using the FG21 medium. Reddish colonies that were assumed to be Fg complex members appeared on the FG21 medium ( Figure 5). Figure 5. Isolation of species in the Fusarium graminearum species complex from wheat head or barley seeds on the FG21 medium. Wheat head or barley seeds were placed on FG21 medium after surface sterilization and kept at 25 °C for 3 days. Reddish colonies were identified as nivalenol type of Fusarium asiaticum using a dipstick DNA chromatography assay. The barley seeds yielded 4 colonies, and the wheat heads yielded 19 colonies. Each colony was obtained from barley seeds equivalent to a 30 mL volume of different fields or a wheat head of different fields. DNA was extracted from aerial mycelia and used in the dipstick DNA chromatography assay. All the 23 colonies were identified as F. asiaticum (Table S1). Nine colonies were identified as NIV and thirteen colonies as 3ADON (Table S1). After single colony isolation, the dipstick DNA chromatography assay was performed again. The results were similar, except for one colony, Fgc22081501. Before single-colony isolation, this colony showed positivity for NIV and 3ADON and only NIV after singlecolony isolation (Table S1).

Discussion
In this study, FG21 medium was developed to be semi-selective for Fg complex members without requiring PCNB. In addition, a dipstick DNA chromatography assay was developed for faster and easier identification of Fg complex members (F. graminearum s. str. and F. asiaticum) and trichothecene typing (3ADON, 15ADON, and NIV) in Japan.
We developed the FG21 medium, owing to the lack of suitable media for isolation of Fg complex members. The FG medium was previously developed for F. graminearum isolation by modifying the Komada medium, but it used PCNB, which is now difficult to obtain [20]. PCNB is not included in the Fo-G2 medium; however, Fg complex members do not grow on this medium, which also contains the fungicide, iminoctadine triacetate, and the fungicide has been used for FHB control in Japan [21]. PDA was chosen as the basic medium for isolation of Fg complex members because the characteristic red pigmentation can be easily seen. The FG21 medium was developed for easy preparation; therefore, the medium included the minimum number of reagents. Chloramphenicol was the sole antibiotic used to inhibit bacterial growth in FG21 medium, and the pH was not adjusted. To improve bacterial growth suppression, adjusting the pH < 4 and/or supplementation with other antibiotics may be an option if bacterial growth disturbs the isolation of Fg complex members.
Because cryptic species have been discovered in the Fg complex, methods to determine species and trichothecene-type compositions have been developed in different countries [27]. In addition to biogeographical differences in the species, some species have host plant preferences: F. asiaticum for rice and F. boothii and F. meridonale for maize, although these preferences may be due to local conditions, such as climate, crop rotation, and presence/absence of competitors. The major FHB pathogen was F. culmorum before 2000 in the Netherlands, but it was replaced with F. graminearum s. str. in the following year [28,29]. Furthermore, the geographical alteration of trichothecene type within a species has been observed in Canada [12], the United States [11], and China [30]. These investigations demonstrate the importance of monitoring species and trichothecene-type compositions for risk assessment of members of the Fg complex, both as mycotoxin producers and plant pathogens.
PCR-based molecular tools to investigate species and trichothecene types have been developed for the Fg complex [3,31]. Niessen and Vogel [32] developed a loop-mediated isothermal amplification (LAMP) assay, based on the galactose oxidase gene (GaoA), to detect Fg complex members. The LAMP assay could uniquely detect all nine species of the Fg complex tested; however, each strain of F. asiaticum and F. cortaderiae were not detected. Knoll et al. [33] developed a method using DNA detection test strips to identify F. graminearum based on the PCR amplification of GaoA. Similar to the dipstick DNA chromatography assay, special instruments and agarose gel electrophoresis were not required. However, the detection target of the method was F. graminearum before division into 16 species, and it could not be used for species identification or trichothecene typing.
The current study demonstrated easier identification of Japanese species in the Fg complex using of FG21 medium and a dipstick DNA chromatography assay. The FG21 medium is convenient for mass screening of seeds, has high sensitivity, and lower investigational costs, compared to other DNA-based diagnostic procedures. Because visually assessing colonies cannot identify all species present, a dipstick DNA chromatography assay that can accurately identify two Japanese species in the Fg complex was developed. Fungal isolation by FG21 medium and identification by dipstick DNA chromatography assay were completed in approximately 3 days. The species of the Fg complex and the trichothecene type can be either simultaneously or separately identified with this assay.
A small amount of mycelia from a colony underwent DNA extraction, and two positive lines for trichothecene types were observed with the dipstick DNA chromatography assay (Table S1). This result suggests that multiple individuals may be present in a colony from the plant matter. If this possibility is eliminated, a single spore or single colony is essential prior to running the dipstick DNA chromatography assay.
F. asiaticum and F. graminearum s. str. were identified using the developed dipstick DNA chromatography assay. Because only the F. graminearum s. str. genome contains FGSG_17538, this was selected as its target gene [25]. Recently, the whole-genome sequences of all 16 species, including Fusarium sp. CBS 123663, was reported [34][35][36], and FGSG_17538 or its homolog was detected in nine species, constituting a subclade in the phylogenomic tree of the Fg complex [36]. Furthermore, positive amplification by PCR with HS976/HS1039 primers was observed for F. boothii, which belongs to the African clade ( Figure 3). Except for F. graminearum s. str., multiple Fg complex members with homologous FGSG_17538 have never been isolated in Japan. Therefore, species misidentification by the dipstick DNA chromatography assay using FGSG_17538 is unlikely to occur without new Fg complex member invasion in Japan. Although F. vorosii cannot be identified with the dipstick DNA chromatography assay, only two strains of F. vorosii have been detected in northern Japan. Positive amplifications for trichothecene typing were observed in Fg complex members, including F. vorosii and Fusarium spp., by multiplex PCR (Figure 3). However, DNA amplification for species identification was negative for F. vorosii and Fusarium spp. Therefore, F. vorosii and Fusarium spp. cannot be discriminated by the dipstick DNA chromatography assay currently. However, if genes that are uniquely present in a single species can be discovered, such as APS1 for F. asiaticum, these genes can be targeted to identify all species in the Fg complex.
The species and trichothecene-type composition of the Fg complex have been investigated in countries worldwide. Faster and easier molecular diagnostic tools are useful for monitoring the dynamic changes in Fg complex members.

Patents
Application number for the dipstick DNA chromatography assay is (2020) 27892.