**Abstract**

The fungal phytopathogen in *Fusarium* species can cause Fusarium head blight of wheat, barley, oats, and other small cereal grain crops worldwide. Most importantly, these fungi can produce different kinds of mycoxins, and they are harmful to humans and animal health. FAO reported that approximately 25% of the world's grains were contaminated by mycotoxins annually. This chapter will focus on several topics as below: (1) composition of *Fusarium graminearum* species complex; (2) genotype determination of *Fusarium graminearum* species complex strains from different hosts and their population structure changes; (3) genetic approaches to genotype determination in type B-trichothecene producing *Fusaria* fungi; and (4) some newly identified trichothecene mycotoxins, their toxicity, and distribution of the producers.

**Keywords:** *Fusarium graminearum* species complex, trichothecene, Fusarium mycotoxin, trichothecene genotype

### **1. Introduction**

The fungal phytopathogen in *Fusarium graminearum* species complex (FGSC) are the primary etiological agent of Fusarium head blight (FHB) of wheat, barley, oats, and other small cereal grain crops worldwide. Besides, the Gibberella ear rot (GER) caused by FGSC and the related species *F. verticillioides* is one of the most devastating diseases on maize. FHB and GER are economically devastating plant disease that greatly limits grain yield and quality. Warm and humid weather conditions at the flowering stage are conducive to disease development. During the 1990s, economic losses in cereals (wheat and barley) caused by *Fusarium* were estimated at close to US \$3 billion (US \$2.5 billion in wheat and US \$400 million in barley) and US \$520 million (US \$220 million in wheat and US \$300 million in barley) in the United States and Canada, respectively [1]. It was reported that due to the changes in climatic conditions and in agricultural practices, outbreaks of FHB have occurred more frequent and serious in China. From 2008 to 2015, serious yield loss of wheat caused by FHB was occurred in more than 5 million ha each year.

In addition, infested grain is often contaminated with Fusarium toxins which are harmful to human and animal health and pose a serious threat to food or feed safety. FHB and GER are among the most destructive and economically important

**Figure 1.** *Chemical structure of Deoxynivalenol (DON).*

#### **Figure 2.**

*Chemical structure of Zearalenone (ZEN).*

**Figure 3.** *Chemical structure of Fumonisin B1 (FB1).*

diseases through the world. A survey made by the journal *Molecular Plant Pathology* from the international community, and resulted in the generation of a top 10 fungal plant pathogen list with FGSC in fourth place [2].

Up to now, more than 70 *Fusarium* species have been identified within the *Fusaria* genus. FGSC, *F. verticillioides*, *F. culmorum*, *F. oxysporum*, *F. solani*, *F. proliferatum*, *F. poae*, *F. equiseti*, and *F. fujikuroi* are the most commonly isolated species worldwide on wheat, maize and other plants. The most important thing is that, many different kinds of mycotoxins can be produced by these molds, such as deoxynivalenol (DON, **Figure 1**), zearalenone (ZEN, **Figure 2**), and fumonisin B1 (FB1, **Figure 3**) are the most prevalent Fusarium mycotoxins in cereal grains and they are very important in food and feed safety. It is clear now that one mold species may produce many different kinds of mycotoxins, and the same mycotoxin may be produced by several species. For example, FGSC can produce trichothecene and zearalenone, while trichothecene can be produced by FGSC, *F. culmorum*, *F. poae*, and *F. equiseti*. This chapter mainly focused on the FGSC and summarized the genetic methods used for trichothecene genotype determination of the strains.

### **2. Composition and identification of FGSC strains**

Prior to 2000, due to the failure of morphological species recognition to accurately assess species limits for the FGSC, the species complex were considered a single

**67**

**Figure 4.**

*Chemical structure of T-2 toxin.*

Fusarium graminearum *Species Complex and Trichothecene Genotype*

cosmopolitan species. Applying the genealogical concordance phylogenetic species recognition (GCPSR), FGSC was first divided into seven phylogenetic lineages in 2000 [3]. Phylogenetic analyses of multilocus genotyping (MLGT) of DNA sequences from portions of 13 housing keeping genes, combined with GCPSR and molecular marker technologies, it revealed that this morphospecies comprises at least 16 biogeographically structured, phylogenetically distinct species. After that the species designation *Fusarium graminearum* have been *sensu stricto* in some conditions. Up to now, 15 of the 16 species have been formally described, including *F. acaciae-mearnsii*, *F. aethiopicum*, *F. asiaticum*, *F. austroamericanum*, *F. boothii*, *F. brasilicum*, *F. cortaderiae*, *F. gerlachii*, *F. graminearum sensu stricto*, *F. louisianense*, *F. meridionale*, *F. mesoamericanum*, *F. nepalense*, *F. ussurianum*, *F. vorosii*, and one additional species was informally recognized based on genealogical exclusivity and conidial morphology on SNA [4].

Proper species identification is critical to research aimed at improving disease and mycoxins control programs. However, it is difficult to discriminate the FGSC strains accurately by morphological characters. A partial region of the translation elongation factor 1 alpha gene (*TEF-1α*) was widely used for molecular identification of *Fusarium* genus. Some specific databases were created for *Fusarium* DNA sequence alignment analysis. For example, similarity searches of the obtained sequences can be performed with the Pairwise DNA alignments network service of the Fusarium MLST database (http://www.westerdijkinstitute.nl/fusarium/), Basic Local Alignment Search Tool (BLAST) network service of the Fusarium ID database (http://www.fusariumdb.org/index.php), and NCBI nucleotide database.

In addition to yield reduction, the FGSC fungi are also of concern because they can produce different kinds of mycotoxins, e.g. zearalenone (**Figure 2**) and trichothecenes (**Figures 4** and **5**) in infested grains. Mycotoxin contamination can occur in both unprocessed and processed grains, representing a risk for human and animal health. Deleterious health effects caused by different mycotoxins include nephropa-

Up to now, more than 200 trichothecenes have been identified [6]. Due to the chemical structure diverse, trichothecenes are divided into four types, namely type A (have a single bond at carbon atom 8, C-8), e.g. T-2 toxin (**Figure 4**), type B (have a keto at C-8), type C (have an epoxide at C-7, 8), and type D (have a macrocyclic ring between C-4 and C-15). All trichothecenes share a common tricyclic 12, 13-epoxytrichothec-9-ene, and they are derived from the isoprenoid intermediate

Among these mycotoxins, type B trichothecenes (**Figure 5**) are the most common detected in cereal grains and their related products. They are distinguished from type A by the presence of a keto function at C-8, and include deoxynivalenol (DON) and its acetylated forms 3-acetyldeoxynivalenol (3-ADON) and 15-acetyldeoxynivalenol (15-ADON), as well as nivalenol (NIV) and its acetylated form 4-acetylnivalenol (4-ANIV). DON is characterized by the absence of a hydroxyl

farnesyl pyrophosphate via a series of biochemical reactions in *Fusarium*.

*DOI: http://dx.doi.org/10.5772/intechopen.89045*

**3. Mycotoxins produced by FGSC**

thy, infertility, cancer or death [5].

Fusarium graminearum *Species Complex and Trichothecene Genotype DOI: http://dx.doi.org/10.5772/intechopen.89045*

*Mycotoxins and Food Safety*

**Figure 2.**

**Figure 1.**

**Figure 3.**

*Chemical structure of Zearalenone (ZEN).*

*Chemical structure of Deoxynivalenol (DON).*

*Chemical structure of Fumonisin B1 (FB1).*

diseases through the world. A survey made by the journal *Molecular Plant Pathology* from the international community, and resulted in the generation of a top 10 fungal

Up to now, more than 70 *Fusarium* species have been identified within the *Fusaria* genus. FGSC, *F. verticillioides*, *F. culmorum*, *F. oxysporum*, *F. solani*,

*F. proliferatum*, *F. poae*, *F. equiseti*, and *F. fujikuroi* are the most commonly isolated species worldwide on wheat, maize and other plants. The most important thing is that, many different kinds of mycotoxins can be produced by these molds, such as deoxynivalenol (DON, **Figure 1**), zearalenone (ZEN, **Figure 2**), and fumonisin B1 (FB1, **Figure 3**) are the most prevalent Fusarium mycotoxins in cereal grains and they are very important in food and feed safety. It is clear now that one mold species may produce many different kinds of mycotoxins, and the same mycotoxin may be produced by several species. For example, FGSC can produce trichothecene and zearalenone, while trichothecene can be produced by FGSC, *F. culmorum*,

*F. poae*, and *F. equiseti*. This chapter mainly focused on the FGSC and summarized the genetic methods used for trichothecene genotype determination of the strains.

Prior to 2000, due to the failure of morphological species recognition to accurately

assess species limits for the FGSC, the species complex were considered a single

plant pathogen list with FGSC in fourth place [2].

**2. Composition and identification of FGSC strains**

**66**

cosmopolitan species. Applying the genealogical concordance phylogenetic species recognition (GCPSR), FGSC was first divided into seven phylogenetic lineages in 2000 [3]. Phylogenetic analyses of multilocus genotyping (MLGT) of DNA sequences from portions of 13 housing keeping genes, combined with GCPSR and molecular marker technologies, it revealed that this morphospecies comprises at least 16 biogeographically structured, phylogenetically distinct species. After that the species designation *Fusarium graminearum* have been *sensu stricto* in some conditions. Up to now, 15 of the 16 species have been formally described, including *F. acaciae-mearnsii*, *F. aethiopicum*, *F. asiaticum*, *F. austroamericanum*, *F. boothii*, *F. brasilicum*, *F. cortaderiae*, *F. gerlachii*, *F. graminearum sensu stricto*, *F. louisianense*, *F. meridionale*, *F. mesoamericanum*, *F. nepalense*, *F. ussurianum*, *F. vorosii*, and one additional species was informally recognized based on genealogical exclusivity and conidial morphology on SNA [4].

Proper species identification is critical to research aimed at improving disease and mycoxins control programs. However, it is difficult to discriminate the FGSC strains accurately by morphological characters. A partial region of the translation elongation factor 1 alpha gene (*TEF-1α*) was widely used for molecular identification of *Fusarium* genus. Some specific databases were created for *Fusarium* DNA sequence alignment analysis. For example, similarity searches of the obtained sequences can be performed with the Pairwise DNA alignments network service of the Fusarium MLST database (http://www.westerdijkinstitute.nl/fusarium/), Basic Local Alignment Search Tool (BLAST) network service of the Fusarium ID database (http://www.fusariumdb.org/index.php), and NCBI nucleotide database.

## **3. Mycotoxins produced by FGSC**

In addition to yield reduction, the FGSC fungi are also of concern because they can produce different kinds of mycotoxins, e.g. zearalenone (**Figure 2**) and trichothecenes (**Figures 4** and **5**) in infested grains. Mycotoxin contamination can occur in both unprocessed and processed grains, representing a risk for human and animal health. Deleterious health effects caused by different mycotoxins include nephropathy, infertility, cancer or death [5].

Up to now, more than 200 trichothecenes have been identified [6]. Due to the chemical structure diverse, trichothecenes are divided into four types, namely type A (have a single bond at carbon atom 8, C-8), e.g. T-2 toxin (**Figure 4**), type B (have a keto at C-8), type C (have an epoxide at C-7, 8), and type D (have a macrocyclic ring between C-4 and C-15). All trichothecenes share a common tricyclic 12, 13-epoxytrichothec-9-ene, and they are derived from the isoprenoid intermediate farnesyl pyrophosphate via a series of biochemical reactions in *Fusarium*.

Among these mycotoxins, type B trichothecenes (**Figure 5**) are the most common detected in cereal grains and their related products. They are distinguished from type A by the presence of a keto function at C-8, and include deoxynivalenol (DON) and its acetylated forms 3-acetyldeoxynivalenol (3-ADON) and 15-acetyldeoxynivalenol (15-ADON), as well as nivalenol (NIV) and its acetylated form 4-acetylnivalenol (4-ANIV). DON is characterized by the absence of a hydroxyl

**Figure 4.** *Chemical structure of T-2 toxin.*

**Figure 5.**

*Chemical structures of deoxynivalenol (DON), 3-acetyl deoxynivalenol (3-ADON), 15-acetyl deoxynivalenol (15-ADON), nivalenol (NIV), and 4-acetyl nivalenol (4-ANIV).*

function at C-4, whereas NIV is characterized by the presence of a hydroxyl function at C-4. 3-ADON and 15-ADON are the acetylated forms of DON at C-3 and C-15, respectively. Meanwhile, NIV and 4-ANIV can be differed by the absence (NIV) and presence (4-ANIV) of an acetyl function at C-4 (**Figure 5**).

Type B trichothecenes are of the greatest concern in wheat and barley-growing regions worldwide, because they can represent a major threat to food and feed safety. These toxins are potent inhibitors of protein synthesis and are responsible for neurologic, gastrointestinal, immune function and other disorders. Although type B trichothecenes differ only slightly from each other in terms of the pattern and position of acetylation or hydroxylation, these changes can greatly affect the toxicity and the activity of these chemical compounds [7]. For example, DON is associated with feed refusal, vomiting and suppressed immune functions, and NIV is more toxic to humans and domestic animals than is DON. Determination of these trichothecene variations are important because the toxicity of DON and NIV may vary according to the eukaryotic organism affected. Minervini et al. [8] found that NIV was approximately four times more toxic than DON to human cells. Conversely, DON is 10 to 24 times more toxic to plant cells than NIV [9].

Type B trichothecenes are mainly produced by FGSC. Due to the ability of FGSC strains that producing different kinds of trichothecenes, three strain-specific trichothecene genotypes (chemotypes) were identified in FGSC: the 3-ADON genotype produces DON and 3-ADON, the 15-ADON genotype produces DON and 15-ADON, and the NIV genotype produces NIV and its acetylated derivatives 4-ANIV [10].

### **4. Mycotoxins act as virulence on certain hosts**

Evidence is presented to show that trichothecene toxins act as virulence factors on certain hosts. Strains carrying a disrupted trichodiene synthase gene *Tri5* do not produce trichothecenes or their biosynthetic intermediates [11]. Disruption of the trichodiene synthase which catalyzes the first step in trichothecene biosynthesis in a 15-ADON producer GZ3639 exhibited reduced virulence on seedlings and heads of wheat, but wild-type virulence on seedlings of maize. The results indicated that trichothecene production contributes to the virulence of FGSC on wheat [12]. Eudes et al. [13] confirmed earlier findings that trichothecenes are a principal determinant

**69**

Fusarium graminearum *Species Complex and Trichothecene Genotype*

out China, and NIV producers preferentially to maize [17].

**5. Distribution and population changes of FGSC**

north and closer to Canada. In some regions, the

and southern Russia and some parts of Asia.

The species composition and genotype prevalence of FGSC vary widely in different regions, which reflecting the level of risk factors in feed/food safety. Investigations on *Fusarium* species isolated from wheat, barley, and maize crops have been reported in the last two decades. Dynamic changes of species composition and chemotype proportion have been found in different agricultural ecosystems worldwide. Prior to 2000, strains from the United States and Canada were almost exclusively 15-ADON producers, while they have been increasingly replaced by the 3-ADON producers in some major wheat-growing regions, e.g. the frequency of the 3-ADON genotype in western Canada increased more than 14-fold between 1998 and 2004 [18]. Also Schmale et al. [19] analyzed the trichothecene genotypes of *Gibberella zeae* collected from winter wheat fields in the eastern parts of the US. They revealed an increasing gradient in 3-ADON distribution from south to

*F. graminearum* 15-ADON chemotype is being replaced by the 3-ADON chemotype. The epidemiology data indicated that 3-ADON chemotype dominates in northern Europe, while 15-ADON chemotype dominates in North America, central Europe

The composition of FGSC population appears to be host and location dependent. The results by Zhang et al. [20] and Shen et al. [21] indicated that *F. asiaticum* was the predominated in wheat. Among the 97 FGSC assayed from rice (30 strains), maize (33 strains), and wheat (34 strains) by Qiu and Shi [22], 73 strains were identified as *F. asiaticum* and 63 of them were collected from rice or wheat. The remaining 24 strains belonged to *F. graminearum sensu stricto* and 23 of them were isolated from maize, only 1 strain was collected from wheat. Similarly, FGSC strains were isolated from GER samples in South Korea with *F. graminearum sensu stricto* to be the dominant species which account for 75% of the FGSC [23], while *F. asiaticum* was the dominant species (78.5%) on Korean rice and followed by *F. graminearum sensu stricto* [24].

The distribution of FGSC may correlate with annual temperature. Qu et al. [25] reported that temperature affected the geographic distribution of *F. graminearum* 

of *F. graminearum* aggressiveness on wheat cultivars. More recently, Maier et al. [14] investigated the involvement of trichothecenes in the virulence of the pathogen by disrupting the *Tri5* gene encoding the first committed enzyme trichodiene synthase in FGSC strains with 3-ADON, 15-ADON, and NIV genotype, respectively. The results demonstrated that disruption mutants can cause disease symptoms on the inoculated spikelet but the symptoms cannot spread into other spikelets on wheat. And on maize, mutants derived from the NIV genotype strain caused less disease than their progenitor strain, while no significant difference compared to the wild-type strains were observed on barley [14]. Trichothecenes are therefore also potent phytotoxins and act as virulence factors of pathogenic fungi thus facilitate tissue colonization on sensitive host plants, e.g. of DON produced by *F. graminearum* in wheat [15]. Host preference was identified among the FGSC on wheat, maize, barley, and rice in certain regions. Several studies suggested that maize played a significant role for the presence of the NIV genotype for FGSC. NIV producers were found to be more aggressive toward maize compared to DON producers [16]. This can be due to the fact that NIV is a virulence factor useful for maize colonization [14], and therefore the plant probably represents an ecological niche for hosting the NIV genotype strains. A high proportion of NIV producers of FGSC on maize were observed in China by our group based on a collection from 59 districts in 19 provinces through-

*DOI: http://dx.doi.org/10.5772/intechopen.89045*

Fusarium graminearum *Species Complex and Trichothecene Genotype DOI: http://dx.doi.org/10.5772/intechopen.89045*

*Mycotoxins and Food Safety*

function at C-4, whereas NIV is characterized by the presence of a hydroxyl function at C-4. 3-ADON and 15-ADON are the acetylated forms of DON at C-3 and C-15, respectively. Meanwhile, NIV and 4-ANIV can be differed by the absence

*Chemical structures of deoxynivalenol (DON), 3-acetyl deoxynivalenol (3-ADON), 15-acetyl deoxynivalenol* 

Type B trichothecenes are of the greatest concern in wheat and barley-growing regions worldwide, because they can represent a major threat to food and feed safety. These toxins are potent inhibitors of protein synthesis and are responsible for neurologic, gastrointestinal, immune function and other disorders. Although type B trichothecenes differ only slightly from each other in terms of the pattern and position of acetylation or hydroxylation, these changes can greatly affect the toxicity and the activity of these chemical compounds [7]. For example, DON is associated with feed refusal, vomiting and suppressed immune functions, and NIV is more toxic to humans and domestic animals than is DON. Determination of these trichothecene variations are important because the toxicity of DON and NIV may vary according to the eukaryotic organism affected. Minervini et al. [8] found that NIV was approximately four times more toxic than DON to human cells.

(NIV) and presence (4-ANIV) of an acetyl function at C-4 (**Figure 5**).

*(15-ADON), nivalenol (NIV), and 4-acetyl nivalenol (4-ANIV).*

Conversely, DON is 10 to 24 times more toxic to plant cells than NIV [9].

**4. Mycotoxins act as virulence on certain hosts**

Type B trichothecenes are mainly produced by FGSC. Due to the ability of FGSC strains that producing different kinds of trichothecenes, three strain-specific trichothecene genotypes (chemotypes) were identified in FGSC: the 3-ADON genotype produces DON and 3-ADON, the 15-ADON genotype produces DON and 15-ADON, and the NIV genotype produces NIV and its acetylated derivatives

Evidence is presented to show that trichothecene toxins act as virulence factors on certain hosts. Strains carrying a disrupted trichodiene synthase gene *Tri5* do not produce trichothecenes or their biosynthetic intermediates [11]. Disruption of the trichodiene synthase which catalyzes the first step in trichothecene biosynthesis in a 15-ADON producer GZ3639 exhibited reduced virulence on seedlings and heads of wheat, but wild-type virulence on seedlings of maize. The results indicated that trichothecene production contributes to the virulence of FGSC on wheat [12]. Eudes et al. [13] confirmed earlier findings that trichothecenes are a principal determinant

**68**

4-ANIV [10].

**Figure 5.**

of *F. graminearum* aggressiveness on wheat cultivars. More recently, Maier et al. [14] investigated the involvement of trichothecenes in the virulence of the pathogen by disrupting the *Tri5* gene encoding the first committed enzyme trichodiene synthase in FGSC strains with 3-ADON, 15-ADON, and NIV genotype, respectively. The results demonstrated that disruption mutants can cause disease symptoms on the inoculated spikelet but the symptoms cannot spread into other spikelets on wheat. And on maize, mutants derived from the NIV genotype strain caused less disease than their progenitor strain, while no significant difference compared to the wild-type strains were observed on barley [14]. Trichothecenes are therefore also potent phytotoxins and act as virulence factors of pathogenic fungi thus facilitate tissue colonization on sensitive host plants, e.g. of DON produced by *F. graminearum* in wheat [15].

Host preference was identified among the FGSC on wheat, maize, barley, and rice in certain regions. Several studies suggested that maize played a significant role for the presence of the NIV genotype for FGSC. NIV producers were found to be more aggressive toward maize compared to DON producers [16]. This can be due to the fact that NIV is a virulence factor useful for maize colonization [14], and therefore the plant probably represents an ecological niche for hosting the NIV genotype strains. A high proportion of NIV producers of FGSC on maize were observed in China by our group based on a collection from 59 districts in 19 provinces throughout China, and NIV producers preferentially to maize [17].

## **5. Distribution and population changes of FGSC**

The species composition and genotype prevalence of FGSC vary widely in different regions, which reflecting the level of risk factors in feed/food safety. Investigations on *Fusarium* species isolated from wheat, barley, and maize crops have been reported in the last two decades. Dynamic changes of species composition and chemotype proportion have been found in different agricultural ecosystems worldwide. Prior to 2000, strains from the United States and Canada were almost exclusively 15-ADON producers, while they have been increasingly replaced by the 3-ADON producers in some major wheat-growing regions, e.g. the frequency of the 3-ADON genotype in western Canada increased more than 14-fold between 1998 and 2004 [18]. Also Schmale et al. [19] analyzed the trichothecene genotypes of *Gibberella zeae* collected from winter wheat fields in the eastern parts of the US. They revealed an increasing gradient in 3-ADON distribution from south to north and closer to Canada. In some regions, the

*F. graminearum* 15-ADON chemotype is being replaced by the 3-ADON chemotype. The epidemiology data indicated that 3-ADON chemotype dominates in northern Europe, while 15-ADON chemotype dominates in North America, central Europe and southern Russia and some parts of Asia.

The composition of FGSC population appears to be host and location dependent. The results by Zhang et al. [20] and Shen et al. [21] indicated that *F. asiaticum* was the predominated in wheat. Among the 97 FGSC assayed from rice (30 strains), maize (33 strains), and wheat (34 strains) by Qiu and Shi [22], 73 strains were identified as *F. asiaticum* and 63 of them were collected from rice or wheat. The remaining 24 strains belonged to *F. graminearum sensu stricto* and 23 of them were isolated from maize, only 1 strain was collected from wheat. Similarly, FGSC strains were isolated from GER samples in South Korea with *F. graminearum sensu stricto* to be the dominant species which account for 75% of the FGSC [23], while *F. asiaticum* was the dominant species (78.5%) on Korean rice and followed by *F. graminearum sensu stricto* [24].

The distribution of FGSC may correlate with annual temperature. Qu et al. [25] reported that temperature affected the geographic distribution of *F. graminearum* 

*sensu stricto* and *F. asiaticum* on wheat spikes in China. A comprehensive study on FGSC from wheat was conducted by Zhang et al. [20]. They found that the geographic distribution of FGSC associated with the annual average temperature. The cooler temperatures (annual average temperature ≤15°C) appear to favor *F. graminearum sensu stricto*, while the warmer regions (annual average temperature ≥15°C) appear to favor *F. asiaticum*. A hypothesis was made that the distribution of FGSC members are climate dependent [20].

*F. graminearum sensu stricto* with the 15-ADON genotype and *F. asiaticum* with either the NIV or the 3-ADON genotype were the dominant causal agents on wheat, and the two species dominated the northern and southern regions of China, respectively, which is consistent with earlier studies [20, 26, 27].

However, more recently the study by Zhang et al. [28] indicated that temperature may not be the only factor in the distribution of FGSC and that other, yet unknown factors affected their distribution. To explain genotype distribution in different geographic areas, hypotheses based on grain seed shipment, international trade, long-distance spore transportation, and environmental favorable conditions were proposed.
