Characterization of Fusarium graminearum and Fusarium vorosii originated from Small Cereals Grain in SerbiaABSTRACT
In the present study, the species diversity of Fusarium graminearum species complex was investigated based on morphological, pathogenic, toxicological and genetic characteristics. Fifty-two isolates, derived from small grains from 20 different locations in Serbia, were studied. The phylogenetic analysis of seven selected sequences of three gene regions, translation elongation factor 1-alpha (TEF-1α), histone H3, and β-tubulin, revealed that six isolates were identified as F. graminearum sensu stricto and one as Fusarium vorosii. The TEF-1α and histone H3 genes were found to be sufficiently informative to distinguish the species F. vorosii. These species are of particular concern due to their ability to synthesize mycotoxins that affect both human and animal health. In this study, it was confirmed that all isolates tested belong to the 15ADON chemotype. Since previous investigations have shown that climate change is the leading cause of the appearance of new, potentially more toxic species, future research must pay special attention to changes in the population of this complex. Given that there is little information in the literature about the damage of the F. vorosii species in the production of small grains, this work aimed to examine its aggressiveness and toxicity and thus determine the potential danger of its spreading. Results of the present study showed that the genetic diversity of isolates of F. graminearum species complex (FGSC) in Serbia, as well as their potential for toxin production and aggressiveness, indicate that continuous study of this species is necessary, both in Serbia and the world outside.
Keywords
FGSC; chemotype; mycotoxin; phylogeny
Introduction
Fusarium head blight (FHB) is one of the most widespread and destructive cereal diseases worldwide. Disease on a wheat spike occurs worldwide and causes significant economic losses (Okorski et al., 2022). In addition to substantial economic losses, the FHB is also associated with the contamination of grains by mycotoxins. A large number of mycotoxins has been identified in cereals and they are capable of seriously damaging human and animal health (Omotayo et al., 2019). A major pathogen of FHB is Fusarium graminearum Schwabe, previously identified as a single species, and now a species complex known as the F. graminearum species complex (FGSC). FGSC was demonstrated to be genetically very heterogeneous and is considered to be a complex of at least 15 phylogenetically distinct species, including F. graminearum sensu stricto, F. asiaticum, F. acacia-mearnsii, F. aethiopicum, F. boothii, F. mesoamericanum, F. austroamericanum, F. cortaderiae, F. brasilicum, F. meridionale, F. vorosii, F. gerlachii, F. ussurianum, F. louisianense, and F. nepalense (Sarver et al., 2011). Most FGSC species are native to the southern hemisphere except F. graminearum s.s., which is cosmopolitan (Desjardins and Proctor, 2011). However, F. vorosii occurs mainly in Asia and Europe (Starkey et al., 2007). Potentially due to climate change, migration of FGSC species resulted in their global distribution.
The diversity of FGSC is also reflected in the synthesis of different mycotoxins. F. graminearum s.s., for example, synthesizes a large number of mycotoxins of which the most important ones are trichothecenes of type B, deoxynivalenol (DON), its acetyl ester derivatives (3-acetyldeoxynivalenol - 3ADON, 15-acetyldeoxynivalenol - 15ADON) and nivalenol (NIV) (Lee et al., 2001).
Surveys have shown that F. graminearum s.s. is one Serbia’s most economically important FGSC species on cereals and industrial crops (Lević et al., 2009). Since the F. vorosii species was first identified in Hungary (Starkey et al., 2007), one of the neighboring countries, its emergence in Serbia has been expected. The first occurrence of F. vorosii on wheat in Serbia was recorded in 2021 (Obradović et al., 2022). Due to the potential danger of this species in the production of cereals, as well as the potential impact on the quality of feed and food, the aim of this work was to study its toxigenic and pathogenic characteristics. Therefore, the objectives of this study were (i) to identify FGSC species present on wheat (Triticum aestivum L.) and barley (Hordeum vulgare) in Serbia using in depth conventional morphological and molecular methods, (ii) to determine the toxicological profile of mycotoxins, and (iii) to characterize selected isolates for pathogenicity.
Materials and Methods
Fungal isolates
From the collection of fungi of the Maize Research Institute “Zemun Polje”, Zemun, Serbia, 52 stored isolates, previously identified as F. graminearum based on their morphological characteristics, were used for preliminary screening for molecular diversity. The isolates were originated from asymptomatic samples of wheat (34) and barley (18) grains that were collected from 20 different locations in northern Serbia between 1997 and 2010 (Table 1). They were retrieved from potato dextrose agar (PDA; 200 g potato, 20 g dextrose, 17 g agar, 1 L H2O), and purified according to a single spore isolation procedure (Leslie and Summerell, 2006) and incubated for two weeks in alternating fluorescent light and darkness at intervals of 12 h each to stimulate conidial formation (Burgess et al., 1994).
DNA isolation and molecular identification
For genomic DNA extraction, 52 isolates were cultured on the PDA medium at 25 °C for seven days, and then mycelia were collected using sterile sticks. DNA isolation was performed using a commercial DNeasy Plant Mini Kit (Qiagen), following the manufacturer’s instructions. Molecular identification was performed by the amplification of the TEF-1α gene (O’Donnell et al., 2004; Starkey et al., 2007; Zhang et al., 2012). Polymerase Chain Reaction (PCR) was performed in a reaction mixture with a final volume of 25 μL consisting of 1X PCR Master Mix (Fermentas), 0.4 μM of each primer (ef1/ef2), and 1 μL DNA (approximately 50 ng) under conditions described by Geiser et al. (2004). A reaction mixture containing sterile distilled water (SDW) but no active reagents was used as the negative control. PCR products were separated in a 1 % agarose gel, stained with ethidium bromide, and visualized with an ultraviolet (UV) transilluminator.
All PCR amplicons obtained were sequenced in both directions by a commercial service (Macrogen Inc.) using the same primers used for amplification. The sequences obtained were assembled using Pregap4 and Gap4 (v. 1.5) from the Staden Package and aligned using Clustal X under the MEGA software version 11. After manually verifying the alignment, pairwise distances were calculated in MEGA 11. All isolates were then manually grouped based on sequence identity, and representative sequences from each group were compared to all publicly available sequences using the Basic Local Alignment Search Tool (BLAST) algorithm in the National Center for Biotechnology Information (NCBI) database. The isolates were clustered by constructing the phylogenetic tree of all 52 tested isolates and strains of F. graminearum, F. vorosii, F. boothii and F. asiaticum retrieved from the NCBI GenBank. The phylogenetic tree was constructed by MEGA 11 using the Maximum Parsimony method with search level 1, 1000 Bootstrap Replications, random addition of sequences in ten replicates and the “Gaps/Missing Data Treatment” option set to “Use all sites”.
Morphological characterization of representative isolates
Following the obtained results of the TEF-1α sequence comparison and its BLAST analyses, out of 52 tested isolates, seven were chosen as representative isolates (one isolate from each of the five TEF- groups as well as isolates 1517 and 1339/2) and used for in-depth studies. Five of the seven selected isolates (687/2, 1339/2, 1343, 1348, 1486/2) originated from wheat grain and two (1517 and 1772) from barley grain.
The isolates were subcultured on a PDA for morphological identification and characterization. After five days, macroscopic characteristics such as colony appearance, color, and pigmentation on the PDA were assessed. Microscopic characteristics, macroconidia shape and size were assessed by growing the isolates for 10-14 days on Synthetic Nutrition Agar (SNA; 23 g agar, 1 g KH2PO4, 1 g KNO3, 0.5 g MgSO4 × 7H2O, 0.5 g KCl, 0.2 g glucose, 0.2 g sucrose, 0.6 mL 1 M NaOH, 1 L H2O) and Carnation Leaf Agar (CLA; 20 g agar, 1 L H2O, and sterile carnation leaf fragments) (Fisher et al., 1982; Burgess et al., 1994) in alternating UV light and darkness at intervals of 12 h each. Microscopic characteristics of the isolates were examined in situ using a compound microscope (Karl Zeiss, Axio) equipped with software for recording. For each isolate, 50 randomly selected macroconidia were measured. The results of the macroconidia size were tested for homogeneity and subjected to a one-way analysis of variance (ANOVA). The significance was evaluated using Duncan’s test at p < 0.05. Statistical analyses were conducted using the general procedures of Statistica v.10 (StatSoft Inc.).
Molecular characterization and phylogenetic analysis
Seven selected isolates were further molecularly characterized by the amplification of the β-tubulin and histone H3 genes by using T1/T22 and H3-1a/H3-1b primers, respectively (O’Donnell and Cigelnik, 1997; O’Donnell et al., 1998). The visualization and sequencing of the obtained amplicons were performed as described above. The sequences obtained were assembled, manually edited, compared to all publicly available sequences through the BLAST algorithm in the NCBI database and lastly through phylogenetic analyses they were compared with retrieved sequences of 32 representative strains belonging to 15 different phylogenetic species within the FGSC available in GenBank originating from different parts of the world. The sequences obtained from all three genes of all seven isolates were deposited in the GenBank database, and their assigned accession numbers are shown in Table 2.
– Isolates of Fusarium graminearum species complex (FGSC) identified in this study and retrieved from the GenBank database with their corresponding accession numbers.
In order to summarize the results of all obtained sequences obtained, the concatenated tree of TEF-1α, β-tubulin, and histone H3 genes was constructed using Maximum Parsimony analyses with the MEGA 11 performing a 1000-repeat bootstrap analysis with search level 1 and the “Gaps/Missing Data Treatment” option with a partial deletion and a cutoff value of 95 %. The phylogenetic tree was rooted in an external clade of the species F. pseudograminearum (NRRL 28062) formerly known as F. graminearum group I (Aoki and O’Donnell, 1999). In addition, all sequences of all three tested genes belonging to F. vorosii species were retrieved from NCBI and compared with isolate 1339/2 in the MEGA 11 program.
Molecular detection of trichothecene chemotypes
Two multiplex PCR assays were performed to determine the trichothecene chemotype of same seven representative isolates, targeting the TRI3 and TRI12 genes (Ward et al., 2002) under conditions described by Obradović et al. (2017). Both assays were performed in a reaction mixture with a final volume of 10 μL consisting of 1X PCR Master Mix (Fermentas), 0.2 μM of each primer, and 1 μL DNA (approximately 50 ng). Six microliters of each PCR product were separated in a 1 % agarose gel, stained with ethidium bromide, and visualized with a UV transilluminator. The isolate of F. graminearum (3729), for which previous analyses confirmed the existence of the gene for the creation of 15ADON, was used as a positive control. Distilled water was used as a negative control.
Chemical detection of trichothecene chemotypes
The ability of mycotoxin production of the seven selected isolates was assessed (Logrieco et al., 1995). The five plugs from seven days old colonies of each isolate on PDA were used to inoculate 50 g sterilized maize kernels (45 % moisture) and incubated for three weeks. A qualitative and quantitative determination of trichothecenes (DON, 3ADON, 15ADON, NIV) was performed by liquid chromatography equipped with tandem mass spectrophotometry including a Diode Array Detector (DAD). A ground grain sample (5 g) was extracted with 1 g NaCl mixed with a 25 mL solvent (70 % solution of methanol and distilled water) and filtered through the Whatman paper (nº 1). Filtered samples were passed through the MycoSep 113 Trich and MycoSep 230 Niv column (Romer Labs) followed by filtering through 17 mm, polytetrafluoroethylenemembrane 0.45 µM and using an autosampler (WPS-300SL) injected into Dionex The Ultimate 3000 (Thermo Scientific) liquid chromatographic system equipped with a DAD-3000 detector (Thermo Scientific). The chromatographic separation of 3ADON, 15ADON and NIV was carried out on an analytical column of Acclaim Polar Advantage II, C18 (150 × 4.6 mm, 3 μM) at 25 °C. The injection volume was 10 μL. To separate 3ADON and 15ADON as a mobile phase, a mixture of water: acetonitrile (90:10; mL) at the linear flow of 1 mL min–1 for 15 min was used. Chromatographies were monitored at 221 nm (Mateo et al., 2001). As a mobile phase for NIV chromatographic separation, a water: acetonitrile: methanol (90:5:5; mL) mixture at the linear flow of 0.8 mL min–1 for 15 min was used. Chromatographies were monitored at 218 nm (Yue et al., 2010). The negative control was distilled water, and the positive control was F. graminearum (3729) isolate, for which previous analyses had confirmed the ability to synthesize 15ADON. The analyses were performed twice, and the pooled results of both analyses were presented.
Pathogenicity test
The pathogenicity test was performed on a spike of wheat under field conditions (Mesterházy et al., 1999). The wheat spike was artificially inoculated at the wheat flowering feeks stage with a spore suspension of seven selected isolates (individually) at approximately 1 × 105 conidia mL–1 concentration. Thirty randomly selected plants per isolate were inoculated in three replications (a total 90 of plants per isolate). Each replicate received 30 mL of inoculum, while the control plants were treated with sterile water. After inoculation, wheat spikes were isolated by previously moistened polyvinyl chloride bags, which were removed after 48 h. The aggressiveness of isolates (number of infected spikelets per head) was assessed two weeks after inoculation on a scale of 1-7 (Blandino et al., 2012). Briefly, a scale in which each numerical value corresponds to a percentage interval of surfaces exhibiting visible symptoms of the disease was assessed according to the following score ranges: 1 = 0-5 %, 2 = 5-15 %, 3 = 15-30 %; 4 = 30-50 %, 5 = 50-75 %, 6 = 75-90 %, 7 = 90-100 %. The scores were converted to percentages and each score was replaced with the mid-point of the interval. The infection level results were verified for normality by Colmogorov-Smirnov & Liliefors test, tested for variance homogeneity by Bartlett’s test and subjected to a one-way ANOVA. The means were compared using Duncan’s test at p < 0.05 significance level. Statistical analyses were undertaken following the general procedures of Statistica v.10 (StatSoft Inc.).
Results
Molecular identification of the tested isolates
In all 52 tested isolates, the TEF-1α gene was successfully amplified by producing an amplicon of the expected size. Assembly of obtained sequences produced 663-666 bp long sequences, which, when compared between themselves, were grouped into five groups by their sequence identity (Table 1). Isolates belonging to groups I to IV were 100 % identical with isolates from the same group; inside group V two isolates (798 and 1348) had one T less inside the non-coding region (place 226 in alignment) but still shared 100 % sequence identity with other isolates from group V; isolate 1517 shared a 99.85 % identity with isolates from group I, while isolate 1339/2 shared a 99.09 % identity with isolates from group I and 98.79 % from group IV. Intergroup comparison (excluding isolates 1517 and 1339/2) showed that group III and V, as well as groups I and V, shared the highest sequence identity of 99.85 %, while groups II and IV shared the lowest sequence identity of 99.34 %. Therefore, one isolate from groups I to IV was randomly selected for BLAST analysis, with two isolates from group V (1348 and 2823) and isolates 1517 and 1339/2.
BLAST analysis showed that each representative of groups I to IV, isolates from group V, and isolate 1517, shared a 100 % sequence identity with different isolates of F. graminearum from Poland, China, USA and South Africa. On the other hand, isolate 1339/2 shared a 99.25 % sequence identity with the isolate of F. boothii (KX881786) from Belgium and 98.95 % with F. asiaticum (LC500691) from Japan, as well as with F. boothii (MK896870) from China. Additionally, because of the unclear results from the BLAST analysis of the isolate 1339/2 this sequence was compared through BLAST analysis against each Fusarium species separately, and this analysis showed that it shares 100 % identity with two NRRL strains of F. vorosii: NRRL 45790 (FJ240302) and NRRL 37605 (MW233119).
A Maximum Parsimony phylogenetic tree constructed with all 52 tested isolates and isolates of F. graminearum, F. vorosii, F. boothii and F. asiaticum also suggested the close relationship of the 51 isolates to different isolates of F. graminearum and isolate 1339/2 to F. vorosii (data not shown).
Following these results, isolates 687/2 (group I), 1772 (group II), 1486/2 (group III), 1343 (group IV), 1348 (group V), 1517 and 1339/2 were selected for further in-depth analyses as representatives of the 52 isolates tested.
Morphological characterization of representative isolates
All seven representative isolates formed dense, aerial mycelia on the PDA. The color varied from white, light pink, pink to yellowish brown. The isolates formed 4-6 septate macroconidia with a characteristic foot-shaped basal cell, a conical apex cell, a convex dorsal side, and an almost straight ventral side. Minor statistical differences in the length and width of the macroconidia were observed among the isolates examined. The mean length of the conidia in the isolates was 46.28 μM, while the width was 5.03 μM. Isolate 1343 had the longest macroconidia (53.09 μM), while isolate 1339/2 had the widest conidia (5.77 μM) (Table 3).
Molecular characterization and phylogenetic analysis
In all seven representative isolates β-tubulin and histone H3 genes were successfully amplified by producing an amplicon of the expected size. BLAST analyses of the sequences obtained showed that all isolates except 1339/2 shared a 99.84-100 %/99.77-100 % sequence identity of β-tubulin/histone H3 gene sequences with different isolates of F. graminearum. That isolate 1339/2 shared 100 % sequence identity of both genes with F. vorosii strain NRRL37605 (DQ459643 and DQ459728).
The concatenated Maximum Parsimony tree of the TEF-1α, β-tubulin, and histone H3 sequences of seven Serbian isolates and 32 representative strains from 15 different phylogenetic species within FGSC retrieved from GenBank showed separation into distinct clades. Six isolates (1343, 1348, 1772, 1486/2, 1517, 687/2) were grouped with solid bootstrap support with reference strains of F. graminearum s.s. In contrast, isolate 1339/2 was grouped with reference isolates of F. vorosii (NRRL37605) from Hungary with solid bootstrap support of 99. Based on phylogeny, Serbian F. vorosii was clearly separated from closely related species such as F. graminearum s.s. and other members of the FGSC (Figure 1).
– Maximum Parsimony tree constructed of concatenated sequences of translation elongation factor 1-alpha (TEF-1a), b-tubulin and histone H3 genes of seven Serbian isolates, 32 reference Fusarium graminearum species complex (FGSC) isolates, and outgroup strain Fusarium pseudograminearum (NRRL 28062). The tree was generated in MEGA 11. Bootstrap analyses were performed with 1,000 replicates, and bootstrap values (> 50 %) are shown on the tree. Serbian isolates appear in bold.
Since the concatenated tree confirmed that isolate 1339/2 belongs to the F. vorosii species, in order to compare the Serbian strain to other F. vorosii strains worldwide, the sequences of three tested genes of isolate 1339/2 were compared to all publicly available sequences of F. vorosii isolates. One sequence of β-tubulin, four of histon H3 (two from China and two from Russia) and 16 of TEF-1α gene were available in the NCBI database. According to the histon H3 gene, the Serbian strain is more closely related to strains from Russia (isolated from wheat and barley - FJ240248 and FJ240250) than to strains from China (isolated from maize - OQ124441). According to the sequence of the TEF-1α gene, the Serbian strain shared 100 % sequence identity with strains from Hungary (wheat - MW233119), South Korea (rice, barley and corn - KX266182, KX266183, KX266179 and KX266178) and Russia (wheat and barley - FJ240302 and FJ240304). The comparison of TEF-1α gene sequences showed that, out of the three most informative nucleotide differences, one was inside the coding region leading to the change of amino acid when translated, with a different polarity of side chains.
Molecular detection of trichothecene chemotypes
Two multiplex PCR assays for the TRI3 and TRI12 genes performed to determine trichothecene chemotypes resulted in the amplification of 610 bp and 670 bp long fragments for the TRI3 and TRI12 genes, respectively, in all seven isolates tested, identifying them all as the seven representative isolates of the 15ADON chemotype.
Chemical detection of trichothecene chemotypes
Chemical analyses of seven representative isolates to determine chemotypes showed diversity in the quality and quantity of the production of DON derivatives. The barley isolates produced the highest 15ADON concentration (49.84 mg g–1), while the wheat isolates produced the lowest concentration (9.58 mg g–1). The isolate identified as F. vorosii synthesized only 15ADON at a concentration of 28.71 mg g–1, while 3ADON and NIV were not detected in contrast to the F. graminearum s.s. isolates which synthesized more derivatives. The average production of NIV and 3ADON was lower compared to 15ADON. All isolates tested presented in this paper, regardless of their origin, belonged to the 15ADON chemotype (Table 4).
Pathogenicity
After the artificial inoculation of wheat spikes, the first ear symptoms were observed during grain formation and ripening. The infection at the time of flowering spread to the spikes causing a premature ripening of the grains. The pathogen was reisolated from the inoculated wheat spikes. The appearance of the colony and morphological characteristics of the isolate corresponded completely to the original, which confirmed Koch’s postulates. Symptoms were not observed on the control ears inoculated with SDW. All isolates investigated exhibited pathogenicity on wheat regardless of the origin of the isolates (wheat, barley). The isolates showed statistically significant disease intensity (p < 0.05). It was observed that isolate 1339/2 (F. vorosii) exhibited the lowest degree of virulence on the wheat spike (1.98), while the highest degree of virulence (5.06) was exhibited in isolate 1348 identified as F. graminearum s.s. isolated from wheat (Table 3).
Discussion
Information on the distribution of FGSC species and their chemotypes is crucial because of the increased risk of mycotoxin contamination of cereals. This is also necessary for developing an FHB management strategy and to improving the understanding of the prevalence and importance of the diversity of FGSC species worldwide. Until recently, it was considered that the F. graminearum s.s. species is the only and widely distributed species on cereals in Serbia and that it is the single species causing the Fusarium rot of small grains. The results obtained in this study characterized the previously reported F. vorosii species in the agroecological conditions of Serbia on wheat grain (Obradović et al., 2022). For the first time in Europe, it was identified in the neighboring country of Hungary (Starkey et al., 2007). Considering the geographical proximity and similar climatic and agroecological conditions, the presence of F. vorosii was expected in Serbia, and their distribution has been poorly studied. F. vorosii has been detected in Hungary and Japan, Starkey et al. (2007), while Lee et al. (2016) identified this species in Korea more recently.
The critical biogeographic distribution study of the complex F. graminearum presented by O’Donnell et al. (2004) indicated that F. graminearum s.s. is the most widespread and dominant species in the world which correlates with the situation in Serbia according to our study. In this study, the species F. vorosii was identified in Kikinda, a town in the northern part of Serbia, near the border with Hungary, where it was identified for the first time on a grain of wheat (Starkey et al., 2007). Given the geographical proximity and similar climatic and agroecological conditions, the appearance of this species was to be expected in this region. Literature data indicate that the species F. asiaticum, F. vorosii and F. ussurianum are localized in Asia (O’Donnell et al., 2004). Due to climate changes and international trade, the migration of these species has been recorded; thus, most of the species have now spread further than the originating continent. There is a lack of information on the distribution of FGSC species in Europe. The species F. graminearum s.s. was found to be dominant in France (Boutigny et al., 2014), Germany (Talas et al., 2011), Italy (Somma et al., 2014) and Norway (Aamot et al., 2015). The species F. graminearum s.s. has been observed to be widespread in the areas where wheat and maize are grown in crop rotation (Zhang et al., 2012). In contrast, in the areas where wheat is grown in crop rotation with rice, the species has been identified, in the main, as F. asiaticum (Lee et al., 2010). A precise geographical distribution of FGSC species on wheat and barley was observed in Japan where the species F. graminearum s.s. was isolated in the north. In contrast, the dominant species in the south was F. asiaticum (Suga et al., 2008). Another obvious example of geographical distribution was recorded in Iran, in the western part of the country, where 125 of the total 129 isolates were identified as F. graminearum s.s. originating from wheat. On the other hand, in eastern Iran, the population is much more diverse, with nine FGSC species identified among 26 isolates (Davari et al., 2013). F. vorosii has so far been identified on two continents, but on different crops. The species was identified for the first time in Europe on wheat in 2007 (Starkey et al., 2007), and then in Serbia in 2021 (Obradović et al., 2022), while in Asia, the mentioned species was identified on barley, corn and rice (Lee et al., 2016).
Our study confirmed, as many studies have previously shown, that identification of FGSC species based on morphological characters alone is inconclusive since differences between species are small, if any. The appearance of the macroconidia, which is most commonly used for identification, often overlaps. Considerable variability in colony appearance on PDA was observed among isolates examined in this study, consistent with the results of several previous studies (O’Donnell et al., 2004; Starkey et al., 2007). The colony appearance of F. vorosii (isolate 1339/2) on PDA was no different from the colony appearance of the other isolates tested, as has been previously reported by Starkey et al. (2007). The same conclusion was reached by Lee et al. (2016) who identified F. vorosii on wheat grains in Korea and found no difference in colony appearance among F. graminearum s.s., F. asiaticum, and F. vorosii on PDA. The morphological characteristics of the conidia of the Serbian isolates correspond to the descriptions of the macroconidia of FGSC species (Aoki et al., 2012; Starkey et al., 2007). The average length of the conidia of the Serbian isolate of F. vorosii was 44.84 μM and the width was 5.77 μM, which is similar to the results of Lee et al. (2016). However, the overlapping average width values and small differences in macroconidia length among FGSC species suggest that this feature cannot be used as a taxonomic criterion (Aoki et al., 2012; O’Donnell et al., 2004). It is noted that the macroconidia of F. vorosii are broader compared to other FGSC species, but this is not sufficient for reliable identification (Starkey et al., 2007; Aoki et al., 2012).
All isolates tested were pathogenic on the wheat spike in the field. The F. vorosii isolate showed lower virulence compared to the other Fusarium isolates tested. When examining the pathogenicity of F. vorosii and F. graminearum s.s. isolates on maize, barley, and rice grain, Lee et al. (2016) found wide variability in the pathogenicity of F. vorosii isolates and concluded that pathogenicity was not dependent on the origin of the isolates.
Previous studies have shown that the chemotype 15ADON is predominant in Europe (Obradović et al., 2017) and the chemotype NIV in Asia (Lee et al., 2016). In this study, molecular and chemical analyses confirmed that all seven FGSC tested isolates belong to the 15ADON chemotype. Similar to the majority of previously published F. vorosii isolates, the wheat-derived Serbian isolate 1339/2 belongs to the 15ADON chemotype (Aoki et al., 2012; O’Donnell et al., 2008; Starkey et al., 2007), while Lee et al. (2016) demonstrated the presence of both the 15ADON and NIV chemotypes of F. vorosii isolates. The genes TRI3 and TRI12, used in this study, were also used to differentiate between the 15ADON, 3ADON and NIV producers of F. graminearum by other researchers (Astolfi et al., 2011; Ellis and Munkvold, 2014; Crippin et al., 2019).
The occurrence of F. vorosii on wheat in Europe (Starkey et al., 2007; Obradović et al., 2022), as well as on barley, maize and rice in Asia may indicate that F. vorosii has already adapted to these cereals (Lee et al., 2016). Although the incidence of F. vorosii has been low so far, it can potentially cause FHB on cereals. Genetic diversity of isolates of F. vorosii in Serbia, as well as their potential for toxin production, aggressiveness and adaptability to different climatic conditions, indicates that a continuous study of this species is necessary, both in Serbia and in the world outside.
Acknowledgments
This study was supported by the Ministry of Science, Technological Development and Innovation of the Republic of Serbia (Grant N° 451-03-47/2023-01/200040, 451-03-47/2023-01/200116 and 451-03-47/2023-01/200022) and CREDIT Vibes – Twinning green-editing vibes for FϴϴD (Grant N° 101059942).
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Edited by
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Edited by:
Alice Kazuko Inoue-Nagata
