Fusarium head blight (FHB) is a notable fungal plant disease that reduces crop yields and causes mycotoxin contamination (Moonjely et al., 2023). The pathogens causing this disease are mostly members of the Fusarium graminearum species complex (FGSC), and at least 16 species have been identified as major FHB pathogens worldwide (van der Lee et al., 2015). The FGSC includes five recently designated species: Fusarium vorosii (Fv) (Lee et al., 2016b), Fusarium gerlachii (Starkey et al., 2007), Fusarium ussurianum (Yli-Mattila et al., 2009), Fusarium louisianense, and Fusarium nepalense (Sarver et al., 2011). And these five species of FGSC have been reported as pathogens to various crops causing FHB (Choi et al., 2023; Shin et al., 2018). FGSC is a member of the bigger group called the Fusarium sambucinum species complex (FSAMSC), which also includes other FHB-associated species such as Fusarium culmorum and Fusarium poae (Cerón-Bustamante et al., 2018). In Korea, FHB outbreaks caused mainly by Fusarium asiaticum and Fusarium graminearum have been reported in cereal crops such as rice, wheat, and corn (Jang et al., 2019; Jeong et al., 2023; Ryu et al., 2011; Shin et al., 2018).
Most of FSAMSC species produce toxic secondary metabolites, such as trichothecenes, which are considered virulence factors for FHB infection (Laraba et al., 2021; Mielniczuk and Skwaryło-Bednarz, 2020). Trichothecenes are divided into two structural types based on the absence (type A) or presence (type B) of the carbonyl group at the C-8 position of the trichothecene ring (Ibáñez-Vea et al., 2011). FGSC members generally produce type B trichothecenes and can be classified into three chemotypes (Ward et al., 2002). The NIV type produces nivalenol (NIV) and its acetylated derivative 4-acetyl-nivalenol (4ANIV), the 15-acetyl-deoxynivalenol (15ADON) type produces deoxynivalenol (DON) and its acetylated derivative 15ADON, and the 3-acetyl-deoxynivalenol (3ADON) type produces DON and its acetylated derivative 3ADON. The F. asiaticum-NIV type is a major pathogen causing FHB in rice-growing areas throughout Asia (Ahn et al., 2022; Dong et al., 2020; Jang et al., 2019), whereas the F. graminearum-15ADON type is known to cause FHB in wheat (Leplat et al., 2012; Xu et al., 2021; Zhu et al., 2019). Since chemotypes are highly correlated with pathogenicity for host crops, identifying chemotypes of FHB-causing pathogenic fungi belonging to the FGSC group is important in the study of FHB infection and mycotoxin production.
Most of the fungal species associated with FHB in the FSAMSC group contain a about 26 kb-long trichothecene biosynthetic gene cluster consisting of 13-16 TRI genes on their genomes (Gil-Serna et al., 2020; Proctor et al., 2022). Among the genes in the cluster, especially the TRI3-TRI14 genes, are known to be core genes encoding enzymes that catalyze the trichothecene biosynthetic pathway (Brown et al., 2004; Kimura et al., 2007; Villafana et al., 2019). Previous studies have demonstrated that nucleotide mutations in the 12 core TRI genes cause chemotype differences among FGSC strains (Lu et al., 2021; McCormick et al., 2011). In particular, it has been reported that NIV and DON chemotypes in F. graminearum and F. asiaticum strains are determined by deletion or disruption of the TRI7 and TRI13 genes (Kimura et al., 2007; Lee et al., 2014). Also, the polymorphisms of the TRI12 gene in F. graminearum were able to discriminate between 3ADON and 15ADON types (Alexander et al., 2011; Pasquali et al., 2011).
Fv is a relatively understudied species among FGSC members, with only four previous reports worldwide (Aoki et al., 2012; Lee et al., 2016b; Obradović et al., 2022; Yli-Mattila et al., 2009). Fv was first described as FHB pathogen in Hungary (Starkey et al., 2007), but other studies have reported that Fv belongs to the Asian clade within the FGSC, which also includes F. asiaticum, F. ussurianum, and F. nepalense (Fernández-Ortuño et al., 2013; Molnár et al., 2024; Valverde-Bogantes et al., 2020). Fv was also found during the harvest season of rice and other cereal crops in Korea, but its prevalence was low, accounting for only 1% of all Fusarium isolates (Lee et al., 2016b). In another study, Fv strains caused FHB in rice, wheat, and barley and rot disease on maize in Korea (Choi et al., 2023). However, little information has been known on the mycological or genomic characteristics of Fv as a causative agent of FHB.
In this study, we aimed to compare the mycological and genomic characteristics of Fv strains isolated from the field samples in Korea with those of Fv, a major pathogen of FHB in the Asia region. We determined the chemotypes of Fv and investigated sexual and asexual sporulation, growth rate, trichothecene production, and pathogenicity. In addition, we assembled complete genomes of two Fv strains at the chromosome level, providing reference genome sequences. The fully assembled genome sequences of Fv strains will be useful for studies comparing genetic diversity within the FGSC.
Materials and Methods
Fungal isolation, molecular species identification, and trichothecene genotyping
Fv strains were isolated from rice, maize, barley, and gramineous weed samples collected in South Korea in October 2020. Fungal isolation was performed as previously described (Ahn et al., 2022). A total of 12 Fv strains were isolated and kept at −80°C in 20% glycerol. DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) was used to extract genomic DNA from 0.05 g of lyophilized mycelium. The TEF-1α and TRI12 genes were used to identify species and trichothecene genotype, respectively (O’Donnell et al., 1998; Ward et al., 2002).
The TEF-1α sequences of 12 strains of Fv and 16 representative species of FGSC were used in a phylogenetic study using the MEGA X software and the neighbor joining technique. TEF-1α sequences of each F. ussurianum 29813 (MG989747), F. nepalense CBS 127943 (KM889630), Fusarium acacia-mearnsii CBS 110255 (MW233086), Fusarium aethiopicum CBS 122858 (MW233126), F. boothii CBS 316.73 (MW233088), F. louisianense CBS 127525 (MW233134), F. graminearum PH-1 (23555797), Fusarium mesoamericanum CBS 415.86 (MW233083), F. gerlachii CBS 119176 (MW233118), Fusarium meridionale CBS 110249 (AF212436), Fusarium austroamericanum CBS 10246 (AF212440), F. cortaderiae CBS 119183 (MW233098), Fusarium brasilicum NRRL 31238 (MW233104), and F. culmorum Class2-1B (MF807227) were obtained from GenBank database. The TEF-1α sequences of Fv CBS 119177 (GCA_017656615.1), Fv CBS 119178 (GCA_017656575.1), and F. asiaticum KCTC 16664 (GCA_025258505.1) were derived from their chromosomal sequences.
Mycelial growth, conidiation, and sexual reproduction
To compare the mycological characteristics of Fv and F. asiaticum, 10 strains of F. asiaticum-NIV type were randomly selected from previously isolated isolates from FHB-infected rice samples (unpublished). The mycelial growth, asexual sporulation, and sexual reproduction of 12 Fv strains and 10 F. asiaticum strains were investigated according to our previous report (Ahn et al., 2022). All strains were cultured on potato dextrose agar (PDA) medium at 25°C for 7 days and used as inoculum for the following three tests. To measure the mycelial growth rate, one mycelial plug (4 mm in diameter) was inoculated into the center of a 180-mm-long custom-made test tube containing PDA medium. The tubes were incubated at 25°C and the mycelial growth was measured after 7 days. After inoculating five mycelial plugs of each strain, macroconidia were produced in 20 ml of carboxymethyl cellulose liquid medium. After incubation for 7 days at 25°C on a rotary shaker at 150 rpm, the number of conidia was counted using a hemocytometer. The size of each conidium was examined using imaging software (NIS-Elements BR3.0, Nikon Instruments Inc., Tokyo, Japan). To investigate sexual reproduction, one mycelial plug was inoculated onto carrot agar medium in a 60-mm diameter petri dish to induce perithecia formation. After 7 days of incubation at 25°C, 1 ml of 2.5% Tween 60 was added, and aerial mycelia were knocked down with a glass rod. After continuous incubation under near-UV light at 25°C for 7 days, the number and size of perithecium were observed. All three tests were performed in triplicate for each strain.
Chemical analysis of trichothecenes
Trichothecene production was measured by inoculating five 7-day-old mycelial plugs of Fv (12) and F. asiaticum (10) onto white rice solid medium. White rice solid medium was prepared by mixing 50 g of Indica variety white rice with 30 ml of deionized sterile water and then autoclaving twice. The rice solid medium inoculated with each strain was incubated at 25°C for 21 days. The rice cultures were harvested, dried in a ventilation hood for 3 days, and then ground. Five grams of ground rice culture were mixed with 8.5 ml of deionized sterile water. Trichothecenes were extracted from the mixture using Q-Sep QuEChERS extraction salts (Restek, Lisses, France) and cleaned using Q-Sep QuEChERS dSPE tubes (Restek). The extracted trichothecenes were quantified using high-performance liquid chromatography (HPLC) with a variable wavelength detector and C18 column (4.6 × 250 nm, 5 μm, Restek). The mobile phase consisted of acetonitrile and water, and the gradient and flow rate were adjusted as previously reported (Ahn et al., 2022). Standard toxins of the type B trichothecene, including NIV, 4ANIV, DON, 15ADON, and 3ADON, were purchased from Romer Labs (Union, MO, USA).
Disease assessment
The pathogenicity of a total of 22 strains of Fv (12) and F. asiaticum (10) was investigated on rice and wheat. Pathogenicity tests were performed by inoculating rice panicles and wheat spikelets with spore suspensions of each strain during the flowering period in a greenhouse at 25°C under a 13-h photoperiod. Spore suspension (105 conidia/ml in 0.05% Tween 20) of each strain was spray-inoculated on 15 panicles of rice, and point-inoculated on 15 wheat spikelets. To keep humidity of inoculated plants, they were covered with plastic bags for 72 h and stayed within the greenhouse for 21 days. Rice and wheat grains infected with FHB symptoms were counted at 7, 14, and 21 days after inoculation, and the area under the disease-progress curve (AUDPC) (Jeger and Viljanen-Rollinson, 2001) was calculated to determine the degree of pathogenicity.
Inoculated rice and wheat were harvested after 21 days, dried in a hood for 3 days, ground, and used for the determination of mycotoxin occurrence. Five grams of ground samples were mixed with 8.5 ml of ultrapure water. The type B trichothecenes in the mixture were extracted using Q-Sep QuEChERS extraction salts (Restek) and then cleaned with Q-Sep QuEChERS dSPE tubes (Restek). The amount of trichothecenes was determined using HPLC.
Whole genome sequencing
Whole genome sequencing of Fv-NIV type RN1 (FvRN1) and Fv-15ADON type W15A1 (FvW15A1), which have different chemotypes and are the most pathogenic in rice and wheat, was performed using the PacBio Sequel II platform at National Instrumentation Center for Environmental Management (Seoul National University, Seoul, Korea). Funannotate pipeline v1.8.9 (Palmer and Stajich, 2020) was used for gene annotation, and the quality of genome assembly and gene annotations was assessed using BUSCO v5.2.2 (Simão et al., 2015). We used TANTAN v49 (Frith, 2011) to make soft-masked genome sequences and Augustus v3.3.3 (Stanke and Waack, 2003), GeneMark-ES v4.38 (Ter-Hovhannisyan et al., 2008), and GlimmerHMM v3.0.4 (Majoros et al., 2004) to predict genes. We generated evidence-based gene models by aligning the soft-masked genome sequences with the combined protein sequence (UniProtKB) database using DIAMOND v2.1.8 (Buchfink et al., 2015), and then polished them using Exonerate v2.4.0 (Slater and Birney, 2005). We utilized EVidenceModeler v1.1.1 (Haas et al., 2008) and its weighting algorithm, integrated into the Funannotate pipeline, to choose consensus models from the ab initio and evidence-based gene sets. We performed functional annotation of the consensus models by performing sequence similarity searches against the Pfam v36.0 (El-Gebali et al., 2019), InterPro v97.0 (Mitchell et al., 2019), BUSCO v5.2.2 (Simão et al., 2015), EggNOG v2.1.2 (Ferrés and Iraola, 2018), MEROPS v12.0 (Rawlings et al., 2018), and CAZyme v12.0 (Terrapon et al., 2017) databases, as well as using the SignalP secretome prediction program v4.1 (Armenteros et al., 2019). We identified the tRNA genes using tRNAscan-SE v2.0.12 (Lowe and Eddy, 1997).
OrthoANI (Lee et al., 2016a) was used to calculate average nucleotide identity (ANI) values for 18 genomes of FGSC members, including FvRN1, FvW15A1, and 16 strains (15 species) (Kulik et al., 2015). Whole genome sequences were obtained from GenBank for Fv CBS 119177 (GCA_017656615.1), Fv CBS 119178 (GCA_ 017656575.1), F. aethiopicum CBS 122858 (GCA_ 017657045.1), F. acacia-mearnsii CBS 110255 (GCA_ 017657105.1), F. ussurianum 29813 (GCA_017656725.1), F. asiaticum KCTC 16664 (GCA_025258505.1), F. nepalense CBS 127943 (GCA_017656675.1), F. cortaderiae CBS 119183 (GCA_017656915.1), F. austroamericanum CBS 10246 (GCA_017657035.1), F. meridionale CBS 110249 (GCA_017656785.1), F. mesoamericanum CBS 415.86 (GCA_017656745.1), F. graminearum PH-1 (GCA_900044135.1), F. gerlachii CBS 119176 (GCA_017656835.1), F. louisianense CBS 127525 (GCA_017656825.1), F. boothii CBS 316.73 (GCA_017656985.1), and F. culmorum Class2-1B (GCA_016952355.1). F. brasilicum is not included since its whole genome sequence is not registered in National Center for Biotechnology Information (NCBI). The phylogenetic analysis based on ANI values of 18 genomes was carried out using the R program’s hierarchical clustering function.
Secondary metabolite gene cluster and TRI gene cluster comparison
To define the secondary metabolite gene clusters (SMGCs) in the genomes of FvRN1 and FvW15A1, we performed antiSMASH analyses (Blin et al., 2021; Weber et al., 2015) and manually curated based on the previous results (Adpressa et al., 2019; Hansen et al., 2015; Sieber et al., 2014). For comparison, SMGCs in the genomes of three strains, Fv CBS 119177, Fv CBS 119178, and F. asiaticum KCTC 16664, were also analyzed using the same method.
To compare the TRI gene cluster of Fv strains, we analyzed 12 core TRI genes from 10 strains, including FvRN1 and FvW15A1 obtained in this study, two trains registered in NCBI, and six strains with various chemotypes from a previous study (Jeong et al., 2023). Based on the annotation information of FvRN1, the sequence and annotation information of the TRI gene cluster region (TRI8-TRI14) were extracted from 10 genomes by BlastN analysis (Altschul et al., 1990). Multiple alignment and guide tree analyses were carried out using the default option of the Mafft program (Kuraku et al., 2013).
Data availability
The complete genomic data of two Fv strains have been deposited to the NCBI GenBank database under the accession numbers CP104255-CP104258 (FvRN1) and CP104259-CP104262 (FvW15A1). Genome sequences of two strains were also deposited to the National Agricultural Biotechnology Information Center (NABIC) under accession numbers NG-1543-000001 to NG-1543-000004 (FvRN1) and NG-1545-000001 to NG-1545-000004 (FvW15A1).
Results
The isolation of Fv from FHB-infected cereal crops and gramineous weeds
A total of 12 Fv strains were isolated from FHB symptomatic rice (2 isolates), corn (4 isolates), barley (one isolate), and gramineous weed samples (5 isolates) collected in 2020. The 12 isolates were identified as Fv by molecular species identification using sequence analysis of the translation elongation factor-1α gene (TEF-1α). Phylogenetic analysis using TEF-1α sequence from 16 reference species belonging to the FGSC showed that the Fv strains isolated in this study were closely related to previously known Fv (99.5-100.0%), F. asiaticum (98.9%), and F. graminearum (98.5%) (Fig. 1). Trichothecene genotyping of 12 Fv isolates revealed that 10 strains were of the NIV genotype and 2 strains were of the 15ADON genotype (Supplementary Fig. 1).
Mycological characteristics of the Fv strains
Fv strains formed carmine red and yellowish colony on PDA medium, showing hyphal growth similar to that of F. asiaticum (Fig. 2). The mycelial growth rate of Fv strains was slightly higher than that of F. asiaticum strains, but there was no significant difference (P = 0.152) (Table 1). The conidial development of Fv and F. asiaticum did not differ significantly in terms of morphology, length, width, and the number of septa (Table 1, Fig. 2). In addition, both species did not form perithecia on carrot agar medium, inducing sexual reproduction, indicating that they rarely reproduced sexually. In conclusion, Fv strains isolated from the field had very similar mycological characteristics to those of F. asiaticum, whereas only their conidia production ability was significantly different (Table 1). Regardless of the trichothecene genotype, all of the Fv strains except one (about 104 spores/ml) produced conidia greater than 106 spores/ml, which was 100- to 1,000-fold more than the F. asiaticum strains (P = 0.0016).
Type B trichothecene production of the Fv strains
Production of the type B trichothecene by 12 Fv strains was determined by HPLC. The NIV type strains produced NIV and 4ANIV, and the 15ADON type produced DON and 15ADON (Table 2). Nine of the Fv-NIV type strains produced NIV in the range of 0 to 61.5 μg/g, which was almost three times lower than the production of 4ANIV (3.6 to 129.3 μg/g). Trichothecene production of the Fv-NIV type was significantly reduced (less than 10 times) compared to that of the F. asiaticum-NIV type (P = 0.037 for NIV production, P = 0.005 for 4ANIV production). In two strains of Fv-15ADON type, DON production was significantly increased compared to 15ADON production.
Higher aggressiveness of the Fv-NIV type on rice than wheat
The 12 Fv strains obtained in this study were confirmed to be pathogenic on rice and wheat (Table 3). Ten Fv-NIV type strains showed AUDPC values of 85 on rice and 77 on wheat, indicating that they were slightly more aggressive on rice than on wheat (P = 0.594). However, the AUDPC of Fv-NIV type was only about half of that of F. asiaticum-NIV type both in rice and wheat (P = 0.007 for AUDPC on rice, P = 0.036 for AUDPC on wheat). Two Fv-15ADON type strains showed AUDPC values of 103 on rice and 112 on wheat, indicating that they were more aggressive on wheat than on rice.
Twenty-one days after inoculation, the occurrence of type B trichothecene in inoculated rice and wheat was examined using HPLC (Table 3). Only 4ANIV was detected in rice and wheat inoculated with Fv-NIV type and F. asiaticum-NIV type, while NIV was not detected. The amounts of 4ANIV produced by Fv-NIV type and F. asiaticum-NIV type in rice (P = 0.009) were slightly higher than those in wheat (P = 0.019). DON and 15ADON were detected in rice and wheat inoculated with the Fv-15ADON type strain, and a higher amount of trichothecenes was detected in wheat than in rice.
The complete assembly of FvRN1 and FvW15A1 genome sequences
The PacBio Sequel II platform generated 684,258 HiFi reads in FvRN1 (about 255-fold coverage) and 662,555 HiFi reads in FvW15A1 (about 275-fold coverage). We used the HiCanu assembly (Koren et al., 2017; Nurk et al., 2020) for de novo assembly, generating 90 contigs for FvRN1 and 115 contigs for FvW15A1. These sequences were subsequently assembled into four complete chromosomes from telomere to telomere without gaps by de novo assembly (Table 4). The assembled genome size of FvRN1 was 38.9 Mb (N50, 9.6 Mb; GC content, 48.5%), and that of FvW15A1 was 38.4 Mb (N50, 9.5 Mb; GC content, 48.5%). Gene annotation revealed that FvRN1 and FvW15A1 have 12,616 and 12,562 protein-coding genes, respectively. They have about 500 more genes than Fv CBS 119177 and Fv CBS 119178 and approximately 200 more genes than F. asiaticum KCTC 16664.
The ANI values between the genome sequences of FvRN1, FvW15A1, and 15 representative FGSC species (16 strains) were calculated. The ANI values between the 15 representative strains and FvRN1 and FvW15A1 were found to range from 95.98% to 99.38% and 95.98% to 99.58%, respectively. The phylogenetic tree using ANI showed that FvRN1 and FvW15A1 had the highest similarity to Fv among the 15 representative species, with an average ANI value of 99.48% (Fig. 3).
Comparison of SMGCs and TRI gene clusters of three chemotypes
The number of SMGCs in the genomes of FvRN1 and FvW15A1 was found to be 78 and 79, respectively, and 23 of the SMGCs were associated with known SMs (Supplementary Table 1). A total of 82 SMGCs were discovered across 4 genomes of Fv and 1 genome of F. asiaticum. Of the 82 SMGCs found in the five genomes, six SMGCs were distributed differently between species or strains. The newly discovered SMGC, C89 cluster, was only present in FvW15A1 and Fv CBS 119178. Two SMGCs, an unknown secondary metabolite (SM) biosynthetic cluster (C62) and the apicidin biosynthetic gene cluster (C77), were found only in F. asiaticum. The remaining three SMGCs (C32, C67, and C87) were strain-specific.
Twelve core TRI genes within the TRI gene cluster were compared in the genomes of Fv species, including FvRN1, FvW15A1, two Fv strains registered in NCBI, and six strains with diverse trichothecene genotypes from a previous study (Jeong et al., 2023). Regardless of the species, the approximately 1.4-kb TRI7 gene and the 1.8-kb TRI13 gene existed only in NIV type strains, and were pseudo-formed or deleted in the other 15ADON type and 3ADON type strains (Fig. 4). Strains of the same trichothecene genotype had closer ANI values among the 12-core TRI genes than the species. The ANI of the 12-core TRI genes of the NIV type strains ranged from 96.8% to 98.7%. In contrast, the average ANI of the 12-core TRI genes between the NIV type strains and either the 15ADON or the 3ADON type strains was about 90.3%.
Discussion
Members of the FGSCs are fungal pathogens causing FHB, which have been reported in areas where cereal crops such as wheat, barley, and rice are grown (Martínez et al., 2021; Przemieniecki et al., 2014; Qiu et al., 2014). The cropping system of these crops is one of several factors that influence the distribution of FGSC (Chiotta et al., 2021; Gomes et al., 2014; Xu et al., 2021). These factors have made F. asiaticum the predominant pathogen in rice-growing areas and F. graminearum in wheat-growing areas (Dong et al., 2020; Karugia et al., 2009; Mielniczuk and Skwaryło-Bednarz, 2020). In Korea, the most common FGSC member isolated from crops with FHB symptoms is F. asiaticum, which is highly pathogenic and toxigenic to rice and is known as a representative species of the Asian clade among FGSC (Jang et al., 2019; Przemieniecki et al., 2014). Since 2018, we have collected Fusarium isolates from rice, maize, and barley with FHB symptoms and gramineous weeds surrounding rice fields, collecting approximately 850 FGSC isolates and strains of other Fusarium species complexes (unpublished). Consistent with previous studies (Ahn et al., 2022; Xu et al., 2021), F. asiaticum accounted for about 75% of the Fusarium isolates, and another Asian clade species, Fv was also isolated from FHB-infected cereal crops (Fernández-Ortuño et al., 2013; Sarver et al., 2011). Fv has also been isolated from gramineous weeds and is likely to play an important role in the epidemiology of FHB in rice and other cereals, confirming that gramineous weeds are potential reservoirs of the FHB causative pathogen (Ahn et al., 2022; Dong et al., 2020). The occurrence of 12 Fv strains obtained in this study (Fig. 1) accounted for less than 1% of the total isolated Fusarium strains, similar to the occurrence ratio of Fv in Korea previously reported (Lee et al., 2016b). Our results showed that Fv strains had similar mycelial growth and sexual reproduction to F. asiaticum (Fig. 2). However, 11 out of the 12 Fv strains showed a conidia production that was 100- to 1,000-fold higher than that of the F. asiaticum strains (Table 1). This propensity of Fv strains to produce a large number of fungal spores, which are the principal source of infection in the early stages of plant disease epidemiology, may increase the possibility of pathogen spreading and thus infection outbreak in nature (Nicholson and Epstein, 2013; Osborne and Stein, 2007).
FGSC members produce type B trichothecenes, such as NIV, DON, and their acetylated derivatives (Aoki et al., 2012), and are classified into three chemotypes, NIV, 15ADON, and 3ADON, depending on the trichothecenes they synthesize (Lee et al., 2002). Strains of the 15ADON and 3ADON types synthesize 15ADON and 3ADON, respectively, and then deacetylate them into the final metabolite DON, whereas the NIV type synthesizes 4ANIV and deacetylates it to produce the final metabolite NIV (McCormick et al., 2011). F. asiaticum, a representative species of the Asian clade within the FGSC, is known to be almost exclusively NIV type, whereas F. graminearum is known to be mostly 15ADON type (Przemieniecki et al., 2014; Zingales et al., 2021). In this study, the chemotype of the isolated Fv strains was mostly NIV type, accounting for 10 out of 12 strains. In terms of trichothecene analysis, the Fv-NIV type strains produced significantly less NIV (P = 0.037) and 4ANIV (P = 0.005) than the F. asiaticum-NIV type strains (Table 2). In addition, similar to the F. asiaticum-NIV type strains, they produced about 3 times more 4ANIV than NIV. The two strains of the Fv 15ADON type produced about 10 times less 15ADON than DON, similar to the F. graminearum 15ADON type, showing similar results to a previous study (Ahn et al., 2022). In previous studies, it has been reported that the NIV type strain produces more precursor (4ANIV) than the final metabolite (NIV) (Fang et al., 2022; Maeda et al., 2020), whereas the DON type strain is known to deacetylate the precursors such as 15ADON or 3ADON to produce more final metabolite (DON) (Lee et al., 2014; Wu et al., 2020). Likewise, our results confirmed that Fv-NIV type strains produce more of the NIV precursor, whereas the 15ADON type strains produce more of the final metabolite DON (Table 2).
Although trichothecene production is regarded as an important factor in the ability of FHB infection (Boutigny et al., 2008), previous studies have shown that the ability of FGSC members to cause FHB varies strain-specifically (Goswami and Kistler, 2005; Laraba et al., 2021). The pathogenicity and toxin production results in this study confirmed that Fv strains have sufficient ability to infect FHB in wheat and rice and are potential pathogens with low natural occurrence frequency (Table 3). In rice, the Fv-NIV type strains showed about 40% less pathogenicity than the F. asiaticum-NIV type strains, while 4ANIV occurred at similar concentrations in inoculated rice. This suggests that Fv-NIV type strains can produce 4ANIV at similar concentrations to F. asiaticum-NIV type strains in host plants despite a statistically significant difference in pathogenicity (Table 3). Meanwhile, Fv-15ADON type strains showed lower pathogenicity than F. asiaticum-NIV type strains in rice and similar pathogenicity in wheat. In addition, as in other previous reports, the pathogenicity of Fv-15ADON type strains was slightly higher in wheat in rice, similar to that of F. graminearum-15ADON type strains (Ahn et al., 2022; Mielniczuk and Skwaryło-Bednarz, 2020). Toxin production in the host inoculated with Fv-15ADON type strains was investigated to be only 15ADON in rice and both DON and 15ADON toxins in wheat (Table 3). Unlike their toxin production in culture media (Table 2), these strains were found to synthesize more 15ADON than DON in the inoculated rice and wheat. Taken together, the results of this study suggest that Fv strains can contaminate and cause FHB in hosts such as rice and wheat by synthesizing B type trichothecenes. In particular, Fv produced high concentrations of acetylated derivatives, 4ANIV or 15ADON, in rice and wheat, suggesting that the deacetylation process was low during the trichothecenes biosynthesis in the host crops.
Among the Fv strains obtained in this study, two strains with different chemotypes with high pathogenicity and toxin production were selected to obtain genome information and assembled at the reference genome level (Table 4). Comparing the genomes of the Fv strains, which has high potential as a pathogen, and that of F. asiaticum, a representative pathogen in the Asian region, through genome information and additional analysis will enable in-depth research on the interaction and mechanism between the pathogen itself and the host (Möller and Stukenbrock, 2017; Urban et al., 2015). However, since Fv is not a major pathogenic fungus, there are only two genomes registered in the NCBI database (GCA_017656615.1, GCA_017656575.1), and there are no completely assembled genome sequences of Fv at this time. In particular, no genome sequence of the Fv-NIV type strain has been published. In this study, we report the first complete genome sequences of FvRN1 (CP104255-CP104258) and FvW15A1 (CP104259-CP104262) (Table 4) at the chromosome level. The genome sizes of the FvRN1 and FvW15A1 are 38.90 Mb and 38.80 Mb, respectively, which are about 3 Mb bigger than those of previously described Fv strains. In addition, both strains were found to have approximately 500 more protein-coding genes than previously described. BUSCO analysis revealed that the sequences of the two strains were 99.5% complete, which allows for a wider evolutionary range of comparative analysis within the genus Fusarium. Whole-genome ANI analysis showed that FvRN1 strain had a sequence similarity of about 0.42% to 0.70% with 15ADON type strain, which may be related to the difference in trichothecene genotypes (Fig. 3). The genomic sequence similarity between the two Fv strains has been shown to be considerably high in 15 FGSC species, particularly in the Asian FGSC clade (Lee et al., 2016b; Yli-Mattila et al., 2009).
Based on the completely assembled whole-genome information of Fv strains, we analyzed the gene clusters involved in secondary metabolite biosynthesis, including mycotoxins, in close FGSC members such as F. asiaticum and F. graminearum in the phylogenetic tree. Genes involved in the biosynthesis of fungal SMs are generally clustered, referred to as SMGCs, and contribute to the biosynthetic process via complex mechanisms (Westphal et al., 2021). Fungi synthesize particular SMs via SMGCs, which provide key advantages for fungal adaptation, such as virulence (e.g., trichothecene, zearalenone) and communication (Macheleidt et al., 2016; Tralamazza et al., 2019). In our previous study, we compared SMGCs found in the genomes of F. asiaticum and F. graminearum using antiSMASH analysis and manual curation based on other publications, and we discovered 12 new SMGCs in F. asiaticum (Jeong et al., 2023). Using the same methodology, we compared the distribution of SMGCs in the genomes of Fv and F. asiaticum, and found that Fv had 76-79 SMGCs, which is slightly less than that of F. asiaticum (Supplementary Table 1). We also discovered a newly identified cluster (C89) present only in Fv and 23 clusters involved in the biosynthesis of known metabolites (Adpressa et al., 2019; Sieber et al., 2014; Westphal et al., 2021).
Among the genes involved in trichothecene biosynthesis, 12 TRI genes, TRI3 to TRI14 gene, are referred to as core genes of TRI gene cluster (Lu et al., 2021; Sieber et al., 2014). Nucleotide polymorphisms of TRI13 and TRI7 among 12 core TRI genes determine the NIV or DON production of F. graminearum, F. asiaticum, and F. culmorum (Lee et al., 2014; Yörük and Albayrak, 2012). In this study, we revealed that chemotype-specific polymorphisms within the 12 core TRI genes also exist in the genome of Fv strains (Fig. 4). In FvW15A1, the front part of the TRI7 gene (probably the promoter), and part of the TRI13 gene were confirmed to be deleted, similar to other 15ADON type strains of FGSC members. This trend was observed in DON-producing strains, implying that the absence or dysfunction of TRI7 and TRI13 genes is a characteristic of the DON chemotype (Jennings et al., 2004; Pasquali et al., 2011). Comprehensive analyses of genetic polymorphisms and expression patterns within the TRI gene cluster of FGSC species, including Fv, are known to be able to elucidate the evolutionary and adaptive development of mycotoxin production and host-specific pathogenicity (Rep and Kistler, 2010; Villafana et al., 2019).
In this study, we compared the mycological and genomic characteristics of Fv strains obtained from FHB-infected crops in Korea with those of F. asiaticum, a representative species of the Asian clade within the FGSC. Based on the high-quality reference genomes of two strains, Fv-NIV and 15ADON type, we report for the first time the genomic characteristics of Fv chemotypes that were previously unknown through comparative analysis of the SMGCs and TRI gene clusters with close FGSC species. Although the Fv strains obtained in this study were less pathogenic than the F. asiaticum strains, they showed very high sporulation ability, which is one of the important factors in fungal infection dynamics, indicating their potential as FHB-causing agents in nature. The reference genome sequence and SMGC analysis results provided in our study will enable comparative studies on the genetic diversity of Fusarium species within FGSC.
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