Primers designed for genetic genotyping of FGSC so far.
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.
- Fusarium graminearum species complex
- Fusarium mycotoxin
- trichothecene genotype
The fungal phytopathogen in
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 diseases through the world. A survey made by the journal
Up to now, more than 70
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 cosmopolitan species. Applying the genealogical concordance phylogenetic species recognition (GCPSR), FGSC was first divided into seven phylogenetic lineages in 2000 . 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
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 (
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 .
Up to now, more than 200 trichothecenes have been identified . 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
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 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 . 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.  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 .
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 .
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
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 . This can be due to the fact that NIV is a virulence factor useful for maize colonization , 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 .
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
The composition of FGSC population appears to be host and location dependent. The results by Zhang et al.  and Shen et al.  indicated that
The distribution of FGSC may correlate with annual temperature. Qu et al.  reported that temperature affected the geographic distribution of
However, more recently the study by Zhang et al.  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.
6. FGSC fitness vary
Phylogenetic analyses of trichothecene gene cluster demonstrated that genotype polymorphism is trans-specific and have been maintained by balancing selection on the ancestral pathogens, and genotype differences may have a significant impact on pathogen fitness .
The FGSC strains with different genotype showed different fitness to the ecological environment, such as the hosts, temperature, rotation, and so on. 3-ADON producer was more aggressive than 15-ADON population in susceptible wheat, and also the 3-ADON isolates exhibit a higher DON production than the 15-ADON isolates. Similar conclusions were made by Zhang et al.  that
Changes in DON chemotypes distribution were reported for FGSC from Canada, USA, and Northern Europe. Recently, Nicolli et al.  assessed a range of fitness-related traits (perithecia formation, mycelial growth, sporulation and germination, pathogenicity, and sensitivity to tebuconazole) with 30 strains representatives of 3ADON-, 15ADON-, and NIV-producers. The pathogenicity assay results indicated that strains with the DON chemotypes were generally more aggressive than the NIV ones .
Phenotypic analyses indicated that
FGSC from wheat-maize rotation regions on wheat spikes and maize stalks in Henan province, China, was determined by Hao et al. , and significant differences were found in the frequencies of
7. Genetic genotype determination of FGSC
Traditionally, chemotyping of FGSC strains has been carried out using gas chromatography/mass spectroscopy. This method can be time-consuming and expensive. The genome sequences of several FGSC strains have been published. The trichothecene core gene cluster nucleotide sequences of many strains representatives 3-ADON, 15-ADON, and NIV genotypes have also been deposited in the GenBank. The availability of this information makes it possible to reveal the structural features and allowed selection of several primer sets used successfully in PCR experiments for the molecular characterization of the various chemotypes. Molecular genetic assays allow for high throughput screening of large numbers of field isolates.
Lee et al.  sequenced the gene cluster for trichothecene biosynthesis from a 15-ADON producer (strain H-11) and a NIV producer (strain 88-1), and sequence polymorphisms within the
Lee et al.  subsequently sequenced the
The PCR assays to
The work by Lee et al. [34, 35] and Brown et al.  indicated that the genes
By comparing the published sequences for
Based on information reported and deposited by Ward et al. , three primer sets were designed to the
Li et al.  found that the intergenic sequences between
Based on the sequences of FGSC described by Lee et al.  and Ward et al., a series of PCR assays have been designed to
The possibility to distinguish by a singleplex PCR 3-ADON, 15-ADON, and NIV genotypes was not yet resolved until very recently. Wang et al.  developed a
Recently Suzuki et al.  reported a multiplex PCR assay for simultaneous identification of the species and trichothecene genotypes for
An alternative method based on
Due to the toxicological differences between DON and NIV, it is important to monitor the population and determine the chemotypes of strains present in any given geographic region. Mycotoxin producing capability of a certain strain could be established both through biochemical and molecular techniques. The biochemical approach involves the incubation and extraction of mycotoxins, the methods being complicated and time consuming. The molecular techniques are based on detection of specific gene by using specific primers. All these molecular methods developed for genotype analysis are based on nucleotide diversity of trichothecene synthesis genes. Chemotype characterization has been extensively used to characterize FGSC for their toxigenic potential . The information about the genetic genotyping methods developed so far, such as targeted gene, primer name, primer sequence, and amplification fragment sizes are summarized in Table 1.
|Target gene||Primers||Sequences (5′ to 3′)||Fragment size (bp)||Chemotypes||References|
|3CON||TGGCAAAGACTGGTTCAC||243||3-ADON||Ward et al. |
|Tri303F||GATGGCCGCAAGTGGA||586||3-ADON||Jennings et al. [40, 41]|
|708||15-ADON||Quarta et al. |
|420||15-ADON (||Suzuki et al. |
|300||DON||Li et al. |
|1100||DON (||Suzuki et al. |
|173–327||15-ADON||Lee et al. |
|458–535||DON||Chandler et al. |
|625||NIV||Quarta et al. |
|342||3-ADON||Zhang et al. |
|334||3-ADON||Wang et al. |
|410||3-ADON||Ward et al. |
|470||DON||Kim et al. |
|234||DON||Waalwijk et al. |
|282||DON||Chandler et al. |
|583||15-ADON||Wang et al. |
More effective and accuracy genetic methods are needed. We are doing genomic sequencing of FGSC strains with different trichothecene genotypes, and we believe some new molecular genetic methods will be developed based on the genomic data.
8. Newly identified trichothecene mycotoxins
In addition to the well characterized fungal mycotoxins, plant-derived mycotoxin metabolites, masked mycotoxins, have emerged as important co-contaminants in cereals [53, 54]. The most commonly detected masked mycotoxin conjugates are β-linked glucose-conjugates of trichothecenes, such as DON-3-glucoside (Figure 6). The possible hydrolysis of masked mycotoxins back to their toxic parents during mammalian digestion raises great concerns. Recently, a new series of type-A trichothecene, NX-toxins (Figure 7), produced by FGSC were characterized . In vitro translation assays indicated that NX-3 can inhibit protein biosynthesis to almost the same extent as DON . Comprehensive work on intestinal hydrolysis, absorption, metabolism, and toxicity of newly characterized mycotoxins need to be determined (Figure 7).
The knowledge about the mycotoxins chemotypes could contribute to a better management of fungal infections and breeding of resistance, in order to obtain grains of better quality. The results will also contribute to improve our understanding of the ecology and epidemiology of FGSC members, which may be of value for improving models for assessing the risk or epidemics and mycotoxin production. Genetic genotyping has been proved to be a useful tool for predicting trichothecene type produced by FGSC, and future work on the more effective tools for genotype determination is needed. The discovery of novel toxic metabolites belonging to trichothecenes, such as NX-toxins is also suggesting that the prevalence, distribution, and genetic diversity of FGSC require continuous monitoring. Further research on the biosynthesis molecular mechanism of trichothecene, especially the novel mycotoxins is needed.
The authors acknowledged the financial support from National Natural Science Foundation of China (31871896, 31602124, and 31401598), Shanghai Agriculture Commission Basic Research Project (Grant number 2014 NO.7-3-7), and Shanghai Academy of Agricultural Sciences (Grant number 210ZJ1812).