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Article

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

1
Institute for Glyco-Core Research (iGCORE), Gifu University, Gifu 501-1193, Gifu, Japan
2
The National Agriculture and Food Research Organization, 3-1-1 Kannondai, Tsukuba 305-8517, Ibaraki, Japan
3
Miyagi Prefectural Furukawa Agricultural Experiment Station, 88 Fukoku, Ousaki, Furukawa 989-6227, Miyagi, Japan
4
Mie Prefecture Agricultural Research Institute, 530 Ureshinokawakita, Matsusaka 515-2316, Mie, Japan
5
Faculty of Applied Biological Sciences, Gifu University, Gifu 501-1193, Gifu, Japan
6
River Basin Research Center, Gifu University, Gifu 501-1193, Gifu, Japan
*
Author to whom correspondence should be addressed.
J. Fungi 2022, 8(10), 1048; https://doi.org/10.3390/jof8101048
Submission received: 30 August 2022 / Revised: 30 September 2022 / Accepted: 3 October 2022 / Published: 5 October 2022
(This article belongs to the Special Issue Mycotoxins and Toxigenic Fungi)

Abstract

:
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.

1. 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 species [2,3,4,5,6]. In addition to wheat and barley, these members can impact other grains. Members of the Fg complex have also been known to be the pathogens responsible for Gibberella ear rot in maize; Fusarium boothii, an Fg species complex member, was markedly associated with this disease in South Africa [7]. Fusarium asiaticum has a host preference for rice, suggested by its higher propensity for perithecial formation on rice straw, when compared to Fusarium graminearum sensu stricto (s. str.) and F. boothii [8]. In Japan, F. graminearum s. str., F. asiaticum, and Fusarium vorosii have been identified as members of the Fg complex, although F. vorosii has rarely been isolated [3,9].
Members of the Fg complex are known to produce mycotoxins, namely zearalenone and trichothecenes. Trichothecenes inhibit peptidyltransferase activity by binding to the 60S ribosomal subunit [10]. The strains of trichothecene-producing Fusarium spp. have been classified into three types based on the trichothecene produced: nivalenol (NIV), 3-acetyl deoxynivalenol (3ADON), and 15-acetyl deoxynivalenol (15ADON). Previous investigations have revealed that species and trichothecene-type compositions of the Fg complex vary geographically. The NIV type of F. graminearum s. str. has been detected in the United States [11], whereas no NIV type has been found in 50 strains of F. graminearum s. str. in Japan [9]. However, in the case of Japanese F. asiaticum, 173 (70%) out of 246 strains were of the NIV type [9].
Polymerase chain reaction (PCR) methods have previously been developed to identify the species and trichothecene types (NIV, 3ADON, or 15ADON) of the Fg complex. Ward et al. [12] developed a multilocus genotyping assay, using multiplex PCR to identify 11 species and 3 trichothecene types. This assay produced a comprehensive and accurate assessment of Fg complex members using DNA polymorphisms, but it required allele-specific primer extension reactions, fluorescent microspheres, and equipment for signal detection. Starkey et al. [3] developed a multiplex PCR assay targeting TRI3, encoding the trichothecene 15-O-acetyltransferase, and TRI12, encoding trichothecene efflux pump, for trichothecene typing. Suga et al. [9] developed PCR-RFLPs to identify F. graminearum s. str. and F. asiaticum, based on a species-specific single-nucleotide polymorphism in the histone H3 gene. Suzuki et al. [13] developed a multiplex PCR assay for the simultaneous identification of the species and trichothecene types in F. asiaticum and F. graminearum s. str. However, PCR-based methods generally require agarose gel electrophoresis to detect species-identifying amplicons. In contrast, the dipstick DNA chromatography assay is an alternative, quick, and simple detection method for PCR amplicons [14,15].
A selective medium, on which only specific microorganisms can grow, is critical for both microorganism detection and identification. Both Komada and Fo-G2 media have been developed to be Fusarium oxysporum-selective [16,17]. Komada medium has also been used for the isolation of other Fusarium spp., such as members of the Fusarium solani and Fusarium incarnatumequiseti species complexes [18,19]. However, Komada medium cannot be used to isolate Fg complex members because they grow poorly on this medium [20]. In addition, Fo-G2 medium contains Befran (iminoctadine triacetate), which has been employed for FHB control in Japan, making this medium’s applicability to Fg complex unlikely due to its toxicity [21]. Togawa [20] modified the Komada medium for Fg complex members, naming it FG medium. One of the most effective components in the Komada and FG media for the selective growth of Fusarium spp. is pentachloronitrobenzene (PCNB). However, PCNB production is discontinued because it has not been a registered pesticide in Japan since 2000. Therefore, new media without PCNB is required for the selective isolation of Fg complex members.
In this study, the authors developed a semi-selective FG21 medium without PCNB for isolating Fg complex members and used a dipstick DNA chromatography assay to identify two species in the Fg complex and their trichothecene types. The amplicons in the multiplex PCR assay were visually detected on the dipstick within 20 min. Thus, the authors used this simpler method to isolate and characterize Fg complex members with both FG21 medium and dipstick DNA chromatography.

2. Materials and Methods

2.1. Media

FG21 medium was prepared as follows: potato dextrose broth (PDA; Difco Laboratories, Detroit, MI, USA) with 2% agar was autoclaved. Before PDA solidification, fungicide, antibiotics, and detergents were mixed; 0.015% (w/v) Rizolex wettable powder (Sumitomo Chemical Co., Ltd., Takarazuka, Japan), 0.01% (w/v) Trifumin wettable powder (Nippon Soda Co. Ltd., Tokyo, Japan), 0.125% (w/v) chloramphenicol, 0.05% (v/v) tergitol type NP-10, and 0.1% (w/v) sodium cholate were added. The prototype FG21 medium had 0.025% (w/v) chloramphenicol and lacked Trifumin but had the same composition as the final FG21 medium.
Oatmeal agar (Difco Laboratories) and cornmeal agar (Nissui, Tokyo, Japan) were prepared according to the manufacturers’ instructions. Sabouraud dextrose broth agar (Biokar Diagnostics, Beauvais, France) was prepared, as according to the manufacturer’s instructions, with addition of 2% (w/v) agar. Malt extract agar contained 2% (w/v) malt extract powder (Nacalai Tesque, Inc., Kyoto, Japan), 2% (w/v) D-(+)-glucose, 1% (w/v) Bacto Peptone (Difco Laboratories), and 2% (w/v) agar.

2.2. 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.

2.3. 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.

2.4. 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.

2.5. 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 5ʹ 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 MgSO4, 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.

2.6. 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.

3. Results

3.1. 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 medium. On the prototype FG21 and final FG21 medium, all strains grew similarly, including Fg complex members, except Fusarium sp. F1904008.

3.2. 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).
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 Fusarium austroamericanum (accession VSSV01000012.1), F. boothii (JAGDVE010000010.1), Fusarium brasilicum (JAGDVC010000008.1), F. cortaderiae (JAGDVB010000002.1), Fusarium gerlachii (JAGDUY010000005.1), Fusarium louisianense (JAGDUV010000021.1), Fusarium mesoamericanum (JAGDUS010000059.1), and Fusarium sp. CBS 123663 (JAGDUJ010000036.1) in the Fg complex with E-values from 3e−163 to 3e−152 and F. pseudograminearum (JTGC01000409.1), with an E-value of 1e−144, but not in the genome database of F. culmorum. A significant homologous nucleotide sequence of the amplified region in APS1 was only found in the genome sequence of F. asiaticum.
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).

3.3. 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).
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 single-colony isolation (Table S1).

4. 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.

5. Patents

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

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/jof8101048/s1, Table S1: Colonies isolated from plant source in the field.

Author Contributions

Conceptualization, H.S.; methodology, M.H., N.M., H.I. and K.N.; investigation, H.S.; resources, M.K., T.K., T.T. and T.N.; writing—original draft preparation, H.S.; writing—review and editing, H.S.; supervision, M.S. and K.K.; funding acquisition, H.S. and M.K. All authors have read and agreed to the published version of the manuscript.

Funding

This research was partly funded by the Ministry of Agriculture, Forestry and Fisheries of Japan, Regulatory research projects for food safety, animal health and plant protection (JPJ008617.18072043) and JSPS KAKENHI Grant Number 22K05647.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

The authors thank T. Katsu (Gifu University, Japan) and A. Usui (Gifu University, Japan) for technical support. NRRL strains were kindly provided from K. O’Donnell (USDA).

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Figure 1. Fungal growth comparison of the FG21 medium and previously developed media. 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), Fusarium culmorum MAFF101144 (9), NRRL3288 (10), Fusarium cerealis NRRL25805 (11), NRRL13721 (12), Fusarium poae MAFF236500 (13), MAFF236648 (14), Microdochium nivale MAFF235834 (15), MAFF236681 (16), Fusarium sp. F1904008 (17), Pestalotiopsis sp. P1904014 (18), Alternaria sp. A1904015. (19), Epicoccum sp. E1904019 (20), Fusarium sporotrichioides MAFF236638 (21), MAFF236639 (22), Fusarium fujikuroi Gfc0801001 (23), and Gfc0825009 (24).
Figure 1. Fungal growth comparison of the FG21 medium and previously developed media. 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), Fusarium culmorum MAFF101144 (9), NRRL3288 (10), Fusarium cerealis NRRL25805 (11), NRRL13721 (12), Fusarium poae MAFF236500 (13), MAFF236648 (14), Microdochium nivale MAFF235834 (15), MAFF236681 (16), Fusarium sp. F1904008 (17), Pestalotiopsis sp. P1904014 (18), Alternaria sp. A1904015. (19), Epicoccum sp. E1904019 (20), Fusarium sporotrichioides MAFF236638 (21), MAFF236639 (22), Fusarium fujikuroi Gfc0801001 (23), and Gfc0825009 (24).
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Figure 2. Multiplex PCR for species identification and trichothecene typing of the Fusarium graminearum species complex. Multiplex PCR with the primers 3CON, 3D3A, 3D15A, and 3NA for trichothecene typing. Expected amplicon sizes were 3-acetyl deoxynivalenol (3ADON): 243 bp; 15-acetyl deoxynivalenol (15ADON): 610 bp; and nivalenol (NIV): 840 bp. Multiplex PCR with primers HS974, HS976, HS1035, and HS1039 for species identification. Expected amplicon sizes for Fusarium graminearum sensu stricto (s. str.): 317 bp; Fusarium asiaticum: 332 bp. PCR amplicons were subjected to 1% agarose gel electrophoresis. Lane SM, 100 bp-size marker; lanes 1 to 7, multiplex PCR for trichothecene typing (lane 1, Fg0101020/3ADON; lane 2, Fg0201201/15ADON; lane 3, Fa0244004/3ADON; lane 4, Fa0239005/NIV; lane 5, Fa0343747/15ADON; lane 6, Fv0301112/15ADON; lane 7, water control); lanes 8 to 14, multiplex PCR for species identification (lane 8, Fg0101020/F. graminearum s. str.; lane 9, Fg0201201/F. graminearum s. str.; lane 10, Fa0244004/F. asiaticum; lane 11, Fa0239005/F. asiaticum; lane 12, Fa0343747/F. asiaticum; lane 13, Fv0301112/Fusarium vorosii; lane 14, water control).
Figure 2. Multiplex PCR for species identification and trichothecene typing of the Fusarium graminearum species complex. Multiplex PCR with the primers 3CON, 3D3A, 3D15A, and 3NA for trichothecene typing. Expected amplicon sizes were 3-acetyl deoxynivalenol (3ADON): 243 bp; 15-acetyl deoxynivalenol (15ADON): 610 bp; and nivalenol (NIV): 840 bp. Multiplex PCR with primers HS974, HS976, HS1035, and HS1039 for species identification. Expected amplicon sizes for Fusarium graminearum sensu stricto (s. str.): 317 bp; Fusarium asiaticum: 332 bp. PCR amplicons were subjected to 1% agarose gel electrophoresis. Lane SM, 100 bp-size marker; lanes 1 to 7, multiplex PCR for trichothecene typing (lane 1, Fg0101020/3ADON; lane 2, Fg0201201/15ADON; lane 3, Fa0244004/3ADON; lane 4, Fa0239005/NIV; lane 5, Fa0343747/15ADON; lane 6, Fv0301112/15ADON; lane 7, water control); lanes 8 to 14, multiplex PCR for species identification (lane 8, Fg0101020/F. graminearum s. str.; lane 9, Fg0201201/F. graminearum s. str.; lane 10, Fa0244004/F. asiaticum; lane 11, Fa0239005/F. asiaticum; lane 12, Fa0343747/F. asiaticum; lane 13, Fv0301112/Fusarium vorosii; lane 14, water control).
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Figure 3. Multiplex PCR for trichothecene typing and species identification of Fusarium spp. Multiplex PCR with primers, 3CON, 3D3A, 3D15A, and 3NA for trichothecene typing (a). Expected amplicon size for 3-acetyl deoxynivalenol (3ADON): 243 bp; 15-acetyl deoxynivalenol (15ADON): 610 bp; nivalenol (NIV): 840 bp. Multiplex PCR with primers HS974, HS976, HS1035, and HS1039 for species identification (b). Expected amplicon size for Fusarium graminearum sensu stricto (s. str.): 317 bp; Fusarium asiaticum: 332 bp. PCR amplicons were subjected to 1% agarose gel electrophoresis. Lane SM, 100 bp size marker; lane 1, Fg0101020 (F. graminearum s. str./3ADON); lane 2, Fg0201201 (F. graminearum s. str./15ADON); lane 3, Fa0239005 (F. asiaticum/NIV); lane 4, NRRL26755 (Fusarium acaciae-mearnsii/unknown); lane 5, NRRL34207 (F. acaciae-mearnsii/unknown); lane 6, NRRL26916 (Fusarium boothii/15ADON); lane 7, NRRL29105 (F. boothii/15ADON); lane 8, NRRL29010 (Fusarium meridonale/NIV); lane 9, NRRL28721 (F. meridonale/NIV); lane 10, NRRL3288 (Fusarium culmorum/3ADON); lane 11, NRRL25475 (F. culmorum/3ADON); lane 12, NRRL28062 (Fusarium pseudograminearum/3ADON); lane 13, NRRL28334 (F. pseudograminearum/unknown); lane 14, NRRL13393 (Fusarium lunulosporum/NIV).
Figure 3. Multiplex PCR for trichothecene typing and species identification of Fusarium spp. Multiplex PCR with primers, 3CON, 3D3A, 3D15A, and 3NA for trichothecene typing (a). Expected amplicon size for 3-acetyl deoxynivalenol (3ADON): 243 bp; 15-acetyl deoxynivalenol (15ADON): 610 bp; nivalenol (NIV): 840 bp. Multiplex PCR with primers HS974, HS976, HS1035, and HS1039 for species identification (b). Expected amplicon size for Fusarium graminearum sensu stricto (s. str.): 317 bp; Fusarium asiaticum: 332 bp. PCR amplicons were subjected to 1% agarose gel electrophoresis. Lane SM, 100 bp size marker; lane 1, Fg0101020 (F. graminearum s. str./3ADON); lane 2, Fg0201201 (F. graminearum s. str./15ADON); lane 3, Fa0239005 (F. asiaticum/NIV); lane 4, NRRL26755 (Fusarium acaciae-mearnsii/unknown); lane 5, NRRL34207 (F. acaciae-mearnsii/unknown); lane 6, NRRL26916 (Fusarium boothii/15ADON); lane 7, NRRL29105 (F. boothii/15ADON); lane 8, NRRL29010 (Fusarium meridonale/NIV); lane 9, NRRL28721 (F. meridonale/NIV); lane 10, NRRL3288 (Fusarium culmorum/3ADON); lane 11, NRRL25475 (F. culmorum/3ADON); lane 12, NRRL28062 (Fusarium pseudograminearum/3ADON); lane 13, NRRL28334 (F. pseudograminearum/unknown); lane 14, NRRL13393 (Fusarium lunulosporum/NIV).
Jof 08 01048 g003aJof 08 01048 g003b
Figure 4. The dipstick DNA chromatography assay for species identification and trichothecene typing of the Fusarium graminearum species complex. Multiplex PCR solutions for species identification (Fusarium graminearum sensu stricto [s. str.] and Fusarium asiaticum) and trichothecene typing (3-acetyl deoxynivalenol, 3ADON; 15-acetyl deoxynivalenol, 15ADON; and nivalenol, NIV) were mixed and developed using dipstick DNA chromatography. Lane 1, Fg0101020 (F. graminearum s. str./3ADON); lane 2, Fg0201201 (F. graminearum s. str./15ADON); lane 3, Fa0244004 (F. asiaticum/3ADON); lane 4, Fa0239005 (F. asiaticum/NIV); lane 5, Fa0343747 (F. asiaticum/15ADON); lane 6, Fv0301112 (Fusarim vorosii/15ADON); lane 7, water control.
Figure 4. The dipstick DNA chromatography assay for species identification and trichothecene typing of the Fusarium graminearum species complex. Multiplex PCR solutions for species identification (Fusarium graminearum sensu stricto [s. str.] and Fusarium asiaticum) and trichothecene typing (3-acetyl deoxynivalenol, 3ADON; 15-acetyl deoxynivalenol, 15ADON; and nivalenol, NIV) were mixed and developed using dipstick DNA chromatography. Lane 1, Fg0101020 (F. graminearum s. str./3ADON); lane 2, Fg0201201 (F. graminearum s. str./15ADON); lane 3, Fa0244004 (F. asiaticum/3ADON); lane 4, Fa0239005 (F. asiaticum/NIV); lane 5, Fa0343747 (F. asiaticum/15ADON); lane 6, Fv0301112 (Fusarim vorosii/15ADON); lane 7, water control.
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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.
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.
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Table 1. Strains used in the current study.
Table 1. Strains used in the current study.
SpeciesStrain aApplicationTrichothecene Type bReference
Fusarium graminearum sensu strictoFg0101020FG21 medium, Multiplex PCR, dipstick DNA chromatography assay3ADON[9]
Fg0201201FG21 medium, Multiplex PCR, dipstick DNA chromatography assay15ADON[9]
Fusarium asiaticumFa0244004FG21 medium, Multiplex PCR, dipstick DNA chromatography assay3ADON[9]
Fa0239005FG21 medium, Multiplex PCR, dipstick DNA chromatography assayNIV[9]
Fa0343747FG21 medium, Multiplex PCR, dipstick DNA chromatography assay15ADON[9]
Fusarium vorosiiFv0301112 cFG21 medium, Multiplex PCR, dipstick DNA chromatography assay15ADON[3,9]
Fv0301831 dFG21 medium
Fusarium acaciae-mearnsiiNRRL26755Multiplex PCRNIV *
NRRL34207Multiplex PCRNIV *
Fusarium boothiiNRRL26916Multiplex PCR15ADON[23]
NRRL29105Multiplex PCR15ADON[23]
Fusarium meridonaleNRRL29010 Multiplex PCRNIV[23]
NRRL28721 Multiplex PCRNIV[23]
Fusarium cerealisNRRL25805FG21 medium
Fusarium culmorumMAFF101144FG21 medium
NRRL3288FG21 medium, Multiplex PCR3ADON[23]
NRRL25475 Multiplex PCR3ADON[23]
Fusarium fujikuroiGfc0801001FG21 medium [24,25]
Gfc0825009FG21 medium [24,25]
Fusarium lunulosporumNRRL13393Multiplex PCRNIV[23]
Fusarium poaeMAFF236500FG21 medium
MAFF236648FG21 medium
Fusarium pseudograminearumNRRL28062 Multiplex PCR3ADON[23]
NRRL28334 Multiplex PCR3ADON *
Fusarium sporotrichioidesMAFF236638FG21 medium
MAFF236639FG21 medium
Microdochium nivaleMAFF235834FG21 medium
MAFF236681FG21 medium
Fusarium sp. eF1904008FG21 medium
Pestalotiopsis sp. eP1904014FG21 medium
Alternaria sp. eA1904015FG21 medium
Epicoccum sp. eE1904019FG21 medium
a MAFF: The Research Center of Genetic Resources, National Agriculture and Food Research Organization (NARO), Tsukuba, Japan; NRRL: Agriculture Research Service Culture Collection, National Center for Agricultural Utilization Research, USDA/ARS, Peoria, IL, United States. b * shows that trichothecene type (15-acetyl deoxynivalenol: 15ADON; 3-acetyl deoxynivalenol: 3ADON; and nivalenol: NIV) was newly identified by multiplex PCR in this study. Other parameters have been previously determined in the literature. c Equivalent strain names are NRRL38207 and MAFF241221. d Equivalent strain name is MAFF241222. e The genus was identified by a homological search of the nucleotide sequences of the internal transcribed spacer region of the nuclear ribosomal DNA.
Table 2. Primers used in the current study.
Table 2. Primers used in the current study.
ApplicationPrimer5′-   -3′Target GeneFunctionExpected Amplification Size (bp)Reference
Multiplex PCR for trichothecene typing3CONBiotin-TGGCAAAGACTGGTTCACTri3Reverse for all three trichothecene types [3]
3D15ATagX-ACTGACCCAAGCTGCCATCTri315-acetyl deoxynivalenol (15ADON) type specific forward610[3]
3D3ATagY-CGCATTGGCTAACACATGTri33-acetyl deoxynivalenol (3ADON) type specific forward243[3]
3NATagZ-GTGCACAGAATATACGAGCTri3Nivalenol (NIV) type specific forward840[3]
Multiplex PCR for species identificationHS974TagV-GCTGGCATGGTTGATATGGGAPS1Fusarium asiaticum specific forward332
HS1035Biotin-GTGGGCCCGTACATGTTGAAPS1F. asiaticum specific reverse
HS976TagW-CAAGTCAAGTCTTCCACCGTFGSG_17538Fusarium graminearum sensu stricto specific forward317
HS1039Biotin-GCACCCAGGCCATATTCAGTFGSG_17538F. graminearum sensu stricto specific reverse
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Suga, H.; Hayashi, M.; Kushiro, M.; Miyano, N.; Inoue, H.; Nakajima, K.; Kawakami, T.; Tonooka, T.; Nakajima, T.; Shimizu, M.; et al. 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. J. Fungi 2022, 8, 1048. https://doi.org/10.3390/jof8101048

AMA Style

Suga H, Hayashi M, Kushiro M, Miyano N, Inoue H, Nakajima K, Kawakami T, Tonooka T, Nakajima T, Shimizu M, et al. 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. Journal of Fungi. 2022; 8(10):1048. https://doi.org/10.3390/jof8101048

Chicago/Turabian Style

Suga, Haruhisa, Masahiro Hayashi, Masayo Kushiro, Norichika Miyano, Hiroyoshi Inoue, Kaori Nakajima, Taku Kawakami, Takuji Tonooka, Takashi Nakajima, Masafumi Shimizu, and et al. 2022. "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" Journal of Fungi 8, no. 10: 1048. https://doi.org/10.3390/jof8101048

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