**1. Introduction**

Plant pathogens have developed sophisticated penetration, infection, and colonization strategies to suppress plant defense mechanisms of susceptible hosts and cause disease. The display of minor symptoms by the plant is of least concern but excessive tissue damage and crop loss result in serious economic losses with adverse implications for society, especially in poor countries. To gain insights into how these plant pathogens develop the sophisticated strategies of attack, platforms such as next-generation sequencing (NGS), and its companion applications like proteomics, are currently being exploited. Pertinent scientific inquiry of pathogenicity and virulence processes of pathogens, comprehensive and integrated investigations remain imperative and must take advantage of such high-tech platforms as NGS to generate multitudes of useful datasets. Among microbes associated with the plant are fungi which encounter plants, penetrate and colonize plant tissue to either cause disease or live within the colonized plant in a symbiotic relationship with the host. Fungi which cause diseases are called fungal pathogens and their adverse e ffects can range from tiny spots on the plant

surface to significant symptom expression and tissue collapse. In causing diseases, fungi employ various strategies. They can penetrate the host using penetration structures and develop a large network of hyphae within the intercellular spaces. Through the hyphae, these fungi draw nutrients from the living plant cells. This mode of attack is called biotrophy. Alternatively, fungal pathogens kill the host tissue and derive nutrients from the dead tissue. This mode of attack is called necrotrophy. In the hemi-biotrophic mode of attack, the fungus successively adopts a necrotrophic lifestyle after biotrophy. The invasion of the plant by the pathogen is usually carried out through the production of factors used for plant tissue manipulation to gain physical access into the tissues as well as draw nutrients. These factors enable the plant to cause disease as well as advance the infection within the interior of the tissue, ultimately worsening the disease condition. The ability of a fungus to cause disease is termed pathogenicity, and the ability to worsen the disease is called virulence. The terms pathogenicity factors and virulence factors are loosely used to refer to any substance a pathogen uses to parasitize the plant. We will also use these concepts jointly and will not attempt to classify the various disease-causing factors as either pathogenicity or virulence factors. As a necrotrophic fungus, the wheat pathogen *Fusarium graminearum* Schwabe (teleomorph *Gibberellae zeae*) produces, among other things, cell wall-degrading enzymes (CWDEs) and toxins. Plant tissue exposed to the CWDEs and the toxins loses firmness, macerates and cell contents leak. As the cell contents provide nourishment to the fungus, it grows and advances into the inner tissues. The disease spreads and eventually, depending on other factors, the a ffected part dies. With its ability to produce toxins, *F. graminearum*, a homothallic (self-fertile) ascomycetous fungus, of the Order: Sordariomycetes and Family: Nectriaceae causes Fusarium head blight (FHB), one of the most economically important diseases of wheat, barley, rice and other grain crops worldwide [1–5]. Also known as scab, this devastating disease was first described in 1884 in England and since then, its prominence has increased worldwide with outbreaks reported in temperate and semitropical areas globally [1,6–9]. Under favorable conditions, FHB can advance from initial infection to the destruction of the entire crop within a few weeks [1]. A combination of factors, global warming, increase in wheat production under irrigation and the concomitant increase in no-till practices is the likely cause of the recent resurgence of FHB. *F. graminearum* is favored by high temperatures and irrigation splash disseminates its propagules. Wheat spikes infected by *F. graminearum* have a bleached appearance and grain infected with this fungus is shriveled with pale grey color and an occasional pinkish discoloration. Over and above these undesirable qualities, ingestion of significant amounts of mycotoxin-contaminated grain may cause vomiting, headache, and dizziness in humans. Animals may lose weight and su ffer anorexia. Serious e ffects of ingestion of large amounts of mycotoxins include leukoencephalomalacia in horses, pulmonary edema in swine, and kidney and liver cancers in mice (mentioned in Proctor et al. [10]), and a plethora of records link mycotoxin consumption to cancer in humans. However, some of these sicknesses are linked to mycotoxins produced by other *Fusarium* species, and not *F. graminearum.* The primary mycotoxin produced by *F. graminearum* is the trichothecene deoxynivalenol (DON) (see Figure 1 for the classification of DON) and other toxins produced include zearalenone, nivalenol (NIV), 4-acetylnivalenol (4-ANIV), and DON derivatives 3- and 15-acetyldeoxynivalenol (3-ADON and 15-ADON) [2,11–14]. The serious e ffects of crop infection by *F. graminearum* require in-depth studies on the pathogen, primarily its pathogenicity and virulence factors which can be comprehensively studied using, among other techniques, whole-genome sequencing, transcriptomics using the convenient, reliable, and large data-generating tools of NGS and proteomics. Recently, a multitude of data has been generated for *F. graminearum* either to understand its genome organization or its gene composition as well as genes involved in plant attack. Although more work is still required, the pace at which this information is generated is too high to allow su fficient time for synthesis, organization, and communication. Meanwhile, with the dynamics of climate change, the relations between the pathogen and the plant require active and rapid utilization of the data which is generated for use to ensure plant health and ultimately good human livelihood. Given this background, it remains important to reflect on the work which has been undertaken in *F. graminearum* research and collate it as a useful resource for various *F. graminearum* workers. Furthermore, the work to understand

the genome of *F. graminearum* and its pathogenicity and virulence factors requires acceleration taking advantage of NGS technologies and its supplementing proteomics applications. This is the purpose of this current review, which aimed to trace progress made in discovering pathogenicity and virulence factors, in general, as well as using NGS, and to some extent, proteomics. We present more than 100 *F. graminearum* factors which either directly perform pathogenicity and virulence functions or are indirectly linked to pathogenicity and virulence. We conclude that a multitude of pathogenicity and virulence factors have been discovered, however, more work needs to be done taking advantage of NGS and its companion applications of proteomics. This review article is organized as follows: We provide a narrative background of pathogenicity and virulence, information on *F. graminearum* and information on NGS. We then discuss in detail the various pathogenicity and virulence factors of *F. graminearum* and elaborate on the pathogenicity and virulence factors discovered using NGS (see Figure 2 for the thought process and article organization) and, to some extent, those discovered using proteomics. We collated information from various recent scientific publications to uniquely reflect the pathogenicity and virulence factors. We conclude that a multitude of pathogenicity and virulence factors have been discovered, however, more work needs to be done taking advantage of NGS and its companion applications of proteomics.

**Figure 1.** The classification of the major toxin produced by *Fusarium graminearum*, DON. The major toxins produced by *Fusarium* species are fumonisins, trichothecenes, and zearalenone. DON is a type B trichothecene which has derivatives 3-ADON and 15-ADON.


**Figure 2.** An illustration of concept development and the organization of the article.

### **2. Next-Generation Sequencing, Its Relevance in Studying Plant Pathogenic Fungi and** *Fusarium graminearum*

Nucleic acid sequencing has evolved from determining a few nucleotides of a nucleic acid fragment to its current form of generating large datasets of millions of reads which cover significant genomes of organisms. The generation of these large genomic and RNA datasets has been exploited by researchers to unravel various complexities of genome organization and function in many organisms. Nucleic acid sequencing is the establishment of the order of nucleotides of a given DNA or RNA fragment [15,16]. Earlier efforts to sequence nucleic acids, such as Sanger sequencing, would determine a stretch of a few nucleotides. These earlier sequencing efforts were slow and costly and the need for cheaper and faster sequencing methods grew. This demand drove the development of NGS. NGS, also termed high-throughput or massively parallel sequencing, is a genre of technologies that allow for thousands to billions of DNA fragments to be sequenced simultaneously and independently [17,18]. Although different platforms are commercially available, the NGS workflow features are generally similar, with four major steps: (i) DNA library preparation, (ii) clonal amplification, (iii) massive parallel sequencing, and (iv) data analysis [19,20]. In just the past five years, a number of instruments have been invented. The NGS platform Illumina NextSeq was invented in 2014, followed by the Ion Torrent S5/S5XL 540 in 2015. These were followed by Pacific Biosciences PacBio Sequel in 2016 [21]. These are among the instruments which have been recently invented and utilized to generate large datasets for organisms. One of the first inventions was the 454 GS FLX (454 Life Sciences, Branford, CT, USA). It utilizes an amplicon-based technology to create libraries from genetic regions selected with pairs of multiple primers [18,22,23]. On the contrary, Ion Torrent utilizes the pH variation sequencing method [18,24], whereas the modern platforms, such as Illumina MiSeq and Gene Reader System, utilize the fluorescence emission sequencing method [24,25]. NGS technologies have impressively accelerated research and are becoming employed routinely to seek solutions in different areas of research, as well as provide insights into pathogenicity and virulence of various plant pathogens [26–34]. Although NGS is more affordable than first-generation sequencing (considering time and money), it is still not within the reach of many laboratories in undeveloped countries [16]. However, sequencing services are offered by many sequencing companies at relatively low prices, and therefore, the need to buy the sequencers is less compelling for many. Despite the many challenges associated with NGS, such as inaccurate sequencing of homopolymer regions on certain NGS platforms which can lead to sequencing errors [16], as well as the lack of expertise in downstream processes such as data analysis, there is still success in the generation of sequences. NGS has been exploited to uncover genome organization and

sequence for various plant pathogenic fungi. Plant pathogenic fungi were identified as important in some of the ambitious earlier projects in the area of characterization of fungi, such as the Fungal Genome Initiative [35,36], the Fungal Genomics Program [37,38] and its extension, the 1000 Fungal Genomes (1KFG) Project [39]. By extension, the plant pathogenic fungus *F. graminearum* has benefited tremendously and still does so from the various sequencing initiatives. The genome of *F. graminearum* has been sequenced completely and published for the benefit of *Fusarium* workers worldwide. This is in addition to the numerous gene sequences available in various databases. The complete genome and sequence resources have provided useful information on the biology, pathogenicity, and virulence of *F. graminearum*. A multitude of genes coding for pathogenicity and virulence factors of *F. graminearum* has been uncovered and the valuable information analyzed by researchers to deduce the much-needed insight into this devastating plant pathogen. We trace the important pathogenicity and virulence factors discovered and focus our attention on those which were discovered using NGS, and to some extent those which were discovered using proteomics. Within the context of this review article, pathogenicity factors and virulence factors are any substances produced by the fungus to gain access into the plant, manipulate it for its benefit, thus causing disease, as well as advance pathogenicity, thus causing the severity of the disease. Genes encoding these pathogenicity and virulence factors are themselves called pathogenicity and virulence factors. The terms pathogenicity and virulence are used jointly in this article. There does not seem to be clarity on the distinction between pathogenicity factors and virulence factors, and therefore, many publications seem to utilize these terms loosely. There is grea<sup>t</sup> justification to generate genome sequence information on *F. graminearum*. In order to o ffset the serious pathogenic problems caused by *F. graminearum* and to develop insights into the virulence and antagonistic defense mechanisms in host plants, it appears imperative to undertake the identification of the pathogenicity and virulence factors which make up the attack arsenal of *F. graminearum*. For this reason, the MIPS Fusarium graminearum Genome Database (FGDB) with an updated set with an estimated 14,000 genes and downstream analysis in a live gene validation process was established to provide a comprehensive genomic and molecular analysis [40]. Because *Fusarium* species, including *F. graminearum*, are among the most important phytopathogenic and toxigenic fungi, it is essential to understand the molecular underpinnings of their pathogenicity and virulence. Based on the comparative genomic analysis conducted on three phenotypically diverse species that include *F. graminearum*, it was revealed that among others, this particular homothallic fungal species, *F. graminearum*, has shown a relatively narrow host range that includes important crops like wheat (mentioned in Ma et al. [41]). Before discussing pathogenicity and virulence factors of *F. graminearum*, we look at pathogenicity and virulence factors of plant pathogens generally.

### **3. Pathogenicity and Virulence Factors of Plant Pathogens**

Pathogenicity is the ability of an organism to cause disease, virulence, on the other hand, refers to the magnitude of the disease caused. There are three modes of attack by fungi. One mode requires 'cooperation' from the plant and living plant tissues. The fungus forms structures for penetration and a network of hyphae intertwined within the colonized plant tissues. Nutrients in the plants are extracted by this mass of hyphal structures formed in the intercellular spaces of the plant. This mode is called biotrophy (Figure 3). Other fungi release a barrage of degradative enzymes and toxins to lyse plant tissues and in so doing feed on the leaking cell contents and degraded tissue. This is called necrotrophy (Figure 4). Hemi-biotrophy is a hybrid mode between biotrophy and necrotrophy. Biotrophs produce proteins which circumvent the defense responses of the plant. These secreted proteins may have a counteracting e ffect on the plant's defense proteins. This protein interaction in a match-match situation plays out as gene-for-gene interaction. This was found to be the case with flax rust of wheat caused by *Melampsora lini* and wheat rusts caused by the Puccinias. This gene-for-gene interaction is evidence of co-evolution between the plant and its pathogen. The mechanism of attack is less systematic in necrotrophs in which toxin production can be unspecific. Necrotrophs flood the plant with CWDEs and toxins which "chew up" the tissue. The hard physical defense structures of the plant, such as

pectin, lignin, and glucans, crumble and the cells collapse. Pathogenicity and virulence-associated with plant pathogens are made possible by the secretion of pathogenicity and virulence factors which are molecules that help the pathogen colonize the plant [42,43]. For the purpose of this review, genes which are involved in the production of pathogenicity and virulence factors produced are themselves called pathogenicity and virulence factors.

**Figure 3.** An illustration of biotrophy between a plant pathogenic fungus and a plant. The grey circle represents a fungal spore. Dotted lines represent the germ tube which has developed to form a network of hyphae, and the green rectangles represent plant cells. Finally, symptoms are expressed.

**Figure 4.** An illustration of necrotrophy between a plant pathogenic fungus and a plant. The grey circle represents a fungal spore, the orange arrows represent secreted pathogenicity and virulence factors. The green rectangles represent plant cells, the plant cells become deformed, ripped, and cell contents leak. Finally, symptoms are expressed.

Decades of research have disclosed that plant pathogens have used a notable assortment of proteins and toxins as virulence factors [44]. Several categories of pathogenicity genes, namely (i) gene signals, (ii) genes generating or detoxifying toxins, (iii) metabolic enzymes, (iv) genes involved in the generation of particular infection structures, and (v) CWDEs, can be identified (mentioned in Duyvesteijn et al. [45]). Vadlapudi and Naidu [46] have identified several fungal processes and molecules which contribute to fungal pathogenicity or virulence which have the ability to harm the plants, and these include cell-wall-degrading proteins and toxins. Reception and transduction of signals in the early phase of plant infection play a vital role in triggering development and morphogenesis mechanisms involved in the penetration of the plant host. Several plant pathogens produce toxins that can harm the tissues of plants. Toxins may be either non-specific or host selective. The link between host selective toxin (HST) secretion and pathogenicity in plant pathogens and between sensitivity to toxins and susceptibility to disease in plants provides convincing proof that HSTs can be accountable for host-selective infection and disease growth [47]. Non-specific toxins harm phylogenetically unrelated plant cells, while HSTs are both pathogenicity determinants on various hosts and their genetic varieties. *Loculoascomycetes* produce the most recognized fungal HSTs. Trichothecenes are also compounds that are toxic to plants, and they are believed to be pathogenicity and/or virulence factors. The protein region of the oomycete *Phytophthora* family of the *phytotoxin*-like scr74 gene, the C-terminal of *Phytophthora* RXLR effector paralogues and a single amino acid polymorphism in the *Phytophthora* EPIC1 effector were related to the capacity to specialize in a fresh host [48–50]. Stukenbrock and McDonald [51] also reported polymorphism data showing the spread of two codons in the host-specific necrotrophic effector ToxA produced by *Pyrenophora tritici-repentis* and *Stagonospora nodorum*. Various fungi are models for studying plant-directed toxins. These include *Alternaria alternata*, *Sclerotinia sclerotiorum*, *Botrytis cineria,* and *Fusarium*. Certain plant species can also be used as models to study the effect of toxins on plants as it was demonstrated with trichothecenes on the unicellular plant *Chlamydomonas reinhardtii* [52]. The production of CWDEs (e.g., cellulases, hemicellulases, xylanases, and pectinases) is one of the processes used by fungi to invade plants. CWDEs degrade the complex polymers, cellulose, hemicellulose, xylan, and pectin to gain access to the contents of the plant cell. The enzymatic action of the CWDEs crumbles the cell wall, its integrity is weakened and eventually it collapses. Coupled with toxin production, the production of CWDEs is part of the infection process of *F. graminearum* when it attacks wheat spikelets. These compounds have been reported to help the infection spread to other tissues [53]. Several pathogenicity factors identified in *Fusarium* are also part of preserved complexes or pathways, such as mitogen-activated protein kinases. However, specific genes of *Fusarium* involved in host-pathogen interactions such as HST, elicitors, or Avr genes are mainly undefined, apart from the effectors secreted in xylem. For the purposes of this review article, attention is focused on pathogenicity and virulence factors which are produced by *F. graminearum* when it infects its various hosts which are primarily grain crops.

### *3.1. Pathogenicity and Virulence Factors of Fusarium graminearum*

*F. graminearum* causes FHB, a devastating disease of wheat and other crops which include maize (*Zea mays* L.) and barley (*Hordeum vulgare* L.) [1,54] (mentioned in Desjardins et al. [54]). The rate at which this fungus attacks and infects its host plant is dependent on different mechanisms involving the secretion of extracellular enzymes and mycotoxins. Wanjiru and colleagues [53] show that the pathogenicity of *F. graminearum* is dependent on the extracellular enzymes secreted. The fungus enters the host via the epidermal cell walls with the help of infection-hyphae, leading to the reduction of the host plant's cellulose, pectin, and xylan. Upon gaining access into the plant, *F. graminearum* produces various depolymerase enzymes such as CWDEs that macerate plant tissue. Jenczmionka and Schäfer [55] showed that the production of CWDEs such as pectinases, amylases, cellulases, xylanases, proteases, and lipases by *F. graminearum* enhances its penetration and proliferation in the attacked plant. The production of CWDEs is systematic, regulated, and programmed by the pathogen during attack. To prove regulation and co-ordination, Jenczmionka and Schäfer [55] showed that the *F. graminearum* Gpmk1 MAP kinase regulates the ability of the fungus to induce extracellular endoglucanase, xylanolytic as well as proteolytic activities.

With the disruption of the cell wall, *F. graminearum* uses the polysaccharides in the plant cell wall as a source of nutrients when advancing infection into the deeper underlying tissue [56]. These CWDEs have also been reported to elicit primary immune responses in plants [57–61]. According to Kikot and colleagues [62], various studies have shown a link between the presence of pectic enzymes, disease symptoms, and virulence, indicating CWDEs as decisive factors in the process of phytopathogenic fungi. Plant defense requires the pathogen to engage in a more sophisticated attack mode to overcome this defense. The production of CWDEs alone does not suffice, and therefore, other pathogenicity and virulence factors must come into play. Over and above CWDEs, *F. graminearum* attacks host plants through the release of mycotoxins which include primarily the trichothecene DON, also known as vomitoxin [63]. Sella and colleagues [64] reported that *F. graminearum* secretes lipase, DON, OS-2 (a stress-activated kinase) and Gpmk1 MAP kinases as essential pathogenicity and virulent factors necessary for the development of full disease symptoms in soybean seedlings, and OS-2 is involved in overcoming the resistance possessed by the soybean phytoalexin. Voigt and colleagues [65] observed that the lipase secreted by *F. graminearum* encoded by the FGL1 gene is a virulent factor that enhances the fungal pathogenicity against wheat and maize. Through functional annotation by gene deletion, Zhang and colleagues [66] also discovered the involvement of *FgNoxR* in conidiation, germination, sexual development and pathogenicity of *F. graminearum*. In another study, Jia and colleagues [67], through gene deletion and mutant analysis, identified a putative secondary metabolite biosynthesis gene cluster *fg3\_54* responsible for cell-to-cell penetration. The invasiveness of the *fg3\_54-*deleted *F. graminearum* strains is restored by fusaoctaxin A (an octapeptide), leading to the conclusion that fusaoctaxin A is a virulence factor necessary for cell-to-cell invasion of wheat by *F. graminearum*. Gardiner and colleagues [68] point out that DON is associated with fungal virulence in wheat and barley and plays a crucial role in the spread of FHB. Moreover, DON elicits various defense responses in wheat which include hydrogen peroxide production, programmed cell death [69]. Trichothecenes are secondary metabolites secreted by many fungi belonging to the genera *Fusarium*, *Myrothecium*, *Stachybotrys,* and *Trichoderma*. Ueno [70] classifies trichothecenes into four types based on the unique characteristics of their chemical structures. *Fusarium* spp. secrete mostly type A and type B trichothecenes and not type C and type D [71]. These two types are characterized by the presence of a carbonyl group attached to C-8 of the sequiterpenoid backbone of trichothecenes. The presence of additional 7, 8 epoxides di fferentiates type C from the other types. Type D contains a macrocyclic ring that connects C-4 and C-15 of the sequiterpenoid backbone. Not a matter of discussion in the current review is the formation of toxin glycosides, also called masked mycotoxins, which are formed by cereal crops which are infected with mycotoxins. Toxin glycosides may be converted to toxins during digestion by the consumers of grain. Of grea<sup>t</sup> concern is the fact that toxin glycosides may escape detection by routine laboratory procedures (taken from McCormick et al. [72]. *F. graminearum* secretes type B trichothecenes DON, 15-ADON and 3-ADON or NIV. Lanoseth and Elen [73] reported the presence of DON, 3-ADON and NIV in infected oats. In *F. graminearum*-infected barley, wheat and corn, DON is the most common toxin produced by the fungus [74]. Ueno [70] indicates that the mechanism of action of trichothecenes involves binding the toxin to the ribosomes and inhibiting the synthesis of proteins.

A wide variety of proteins plays essential roles in the synthesis of trichothecenes, and most are encoded by genes within a trichothecene biosynthetic cluster (*Tri* cluster) [75,76]. The *Tri* cluster encodes 13 proteins that are responsible for the secretion of toxins in *F. graminearum*. Jenczmionka and colleagues [77] reported the role of MAP kinase Gpmk1/MAP1 in the production of *F. graminearum* conidia, which implies that the removal of the enzyme could result in a reduction in conidia production and eventually disease spread and pathogenicity. Urban and colleagues [78] observed that in their study the mutants of <sup>Δ</sup>gpmk1/map<sup>1</sup> did not cause any disease to wheat, although they were able to produce DON in the infected plant. It was further discovered that Gpmk1 was involved in the regulation of CWDEs (endo-1,4-b-glucanase and other proteolytic, xylanolytic, and lipolytic enzymes) expression [55]. Ochiai and colleagues [79] reported the activities of a protein kinase cascade in trichothecene biosynthesis. According to the authors, the protein kinases are actively involved in histidine kinase signal transduction. Alteration of the genes leads to the reduction in the expression of *Tri6*, *Tri4* and NIV production in rice cultures. Perithecia, ascospore, conidia formation and spore germination are impacted by the activities of the serine/threonine-protein kinase gene (*GzSNF1*) [80]. *GzSNF1* is also involved in the expression of *F. graminearum* genes which encode for endo-1,4-ß-xylanase 1 precursor (GzXYL1), an endo-1,4-ß-xylanase 2 precursor (GzXYL2) and an extracellular ß-xylosidase (GzXLP) which are involved in depolymerization and plant cell wall degradation. Lee and colleagues [80] also report a reduction in the virulence of <sup>Δ</sup>gzsnf1 mutants on barley, proving the importance of the GzSNF1 gene in pathogenicity and virulence. The docking and fusion of secretory vesicles are required for vegetative and sexual growth in fungi. In the process of docking and fusion, v-SNARE proteins are fused with cargo proteins into vesicles. SNARE proteins are soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptors.

Hong [81] reports that the fusion of the apposing membranes which belong to the transport intermediate together with the target compartment is sped up by the interaction between the membrane-integrated t-SNARE protein and v-SNARE protein. In *F. graminearum*, syntaxin-like t-SNARE proteins are encoded by *GzSYN1* and *GzSYN2* and the deletion of these genes results in decreased virulence of the fungus on barley [82]. Syntaxin a is a multidomain protein with a globular amino terminal domain, a SNARE domain, and a carboxyl terminal transmembrane domain. In Δgzsyn1 mutants, perithecia were found to be unevenly distributed despite retaining reproductive ability with a decrease in the radial hyphal growth in culture. The Δgzsyn2 mutants are sterile females that could act like males in outcrosses with Δmat1-2 strains, resulting in the normal formation of perithecia. Radial hyphal growth of Δgzsyn2 mutants looks like the wild-type strains although the mycelia of both Δgzsyn1 and Δgzsyn2 mutants are generally thinner compared to those of wild-type strains [82].

Secreted lipase in *F. graminearum* is encoded by Fgl1 genes and the alteration of these genes in the fungus can reduce the virulence of the fungus in wheat with symptoms not spreading beyond the spikelets adjacent to inoculated spikelets [65]. Voigt and colleagues [65] further reported that in their study the maize plant infected with Δfgl1 mutants showed slight symptoms and the disease severity estimation was lower in the cobs infected with the mutant compared to the wild-type. The ability of fungi to colonize plants and initiate disease formation is dependent on the transmembrane transport proteins. These protein transporters can also exhibit virulence activities via the provision of protection from poisonous secondary metabolites secreted by host plants or even the fungus itself [83]. Many studies have indicated the roles of multiple ATP-binding cassette transporters or major facilitator superfamily (MFS) proteins in virulence activities of the fungi such as *Botrytis cinerea*, *Cochliobolus carbonum*, *Cercospora kikuchii*, Fusaria, *Magnaporthe grisea*, *Mycosphaerella graminicola* and *Penicillium digitatum* [83–92]. The same applies to *F. graminearum*, ATP-Binding Cassette Transporter Gene *FgABCC9* was found to be involved in the fungus's pathogenicity towards wheat [93]. *F. graminearum* attacks its host plant through the secretion of mycotoxins characterized as virulence factors. These secreted virulence factors and the level of secretion depend on the presence of the relevant genes that facilitate their secretion. Presently, trichothecene mycotoxins have been identified as the major virulence factors associated with *F. graminearum* [94,95]. The trichothecene gene cluster has diverse allelic genes with some of the genes found outside of the main cluster [95]. The allelic variations in *fgTri8* are responsible for producing the DON derivatives [96]. Furthermore, allelic di fferences in *fgTri1* are linked to the formation of the alternate trichothecene NX-2, whereas *fgTri13* and *fgTri7* are responsible for the trichothecene NIV [97,98]. The genes associated with the trichothecene production are not the only genes discovered to be responsible for the pathogenicity and virulence of *F. graminearum*. *FGSG\_04694*, a gene-encoding polyketide synthase (PKS2) responsible for mycelial growth and fungi virulence, was found in *F. graminearum* [99]. Ga ffoor and colleagues [100] confirm the function of PKS2 as they observed decreased mycelial growth and virulence in a mutant PKS2 found in *F. graminearum*. PKS2 is an accessory gene with irregular patterns of conservation that is also found in *F. graminearum* responsible for secondary metabolism and virulence [101,102]. The genes within these accessory gene clusters include terpene synthase-encoding genes *FGSG\_08181*, *FGSG\_08182*, which encode a putative transcription factor and three putative cytochrome P450 genes *FGSG\_17088*, *FGSG\_08183* and *FGSG\_08187* [101,102]. Harris and colleagues [103] observed the expression of these accessory genes in cereals when infected by the fungus, which implies that they are involved in the virulence of the fungus.

*ZIF1* that is conserved in filamentous ascomycetes encodes a bZIP (basic leucine zipper) transcription factor. The absence of *FgZIF1* affects the virulence and reproduction ability of *F. graminearum*. Expression of *FgZIF1* in mozif1 of *M. oryzae* mutants shows clearly that this transcription factor is functionally conserved in *F. graminearum* and *M. oryzae* [104]. Topoisomerase I encoded by *TOP1* is another enzyme involved in the pathogenicity and conidia formation of fungi. The removal of *TOP1* in *F. graminearum* was found to reduce disease expression in infected spikelets and abolition of spore formation [105]. *FGSG\_10057* encodes a Zn(II)2Cys6-type transcription factor in *F. graminearum* that enhances radial growth and virulence activity of the fungus in wheat [106]. Most of these factors that enhance virulence are proteins and these proteins need to be transported to the areas in the host plant where disease symptoms are expressed. Most recently, it was found that *F. graminearum FgCWM1* encodes a cell wall mannoprotein which plays a role in pathogenicity. *FgCWM1* mutants exhibit reduced pathogenicity in wheat [107]. Another recent discovery is that of a SNARE gene *FgSec22* which was found to be required for vegetative growth, pathogenesis and DON biosynthesis in *F. graminearum* [108]. Similarly, an *F. graminearum* mitochondrial gene mitochondrial gene *FgEch1* was found to be important for conidiation, DON production, and plant infection [109]. Another recently discovered pathogenicity and virulence factor is FgPEX4, which is important for development, cell wall integrity and pathogenicity [110]. Elucidation and complete delineation of the pathogenicity and virulence factors of *F. graminearum* must still be done by increasing e fforts in whole-genome sequencing of strains from various parts of the world and by conducting in planta gene expression studies to assess genes which are di fferently expressed during the infection of a host plant by *F. graminearum*. Nowadays these can be conveniently done using NGS. The pathogenicity and virulence factors of *F. graminearum* can be conveniently summed under CWDEs, toxins, toxin biosynthesis genes, and other pathogenicity and virulence determinants. In this section, an elaborate explanation of the di fferent pathogenicity and virulence factors was provided. Below we provide a convenient list of these pathogenicity and virulence factors under di fferent topics, namely, CWDEs, toxins, toxin biosynthesis genes and other pathogenicity and virulence determinants (Table 1). The pathogenicity and virulence factors are therefore summarized under their respective headings. In trying to categorize pathogenicity and virulence factors there may be some ambiguities which may lead to unclear categorizations.


**Table 1.** List of pathogenicity and virulence factors of *Fusarium graminearum*.

189
