Strategies for Controlling the Sporulation in Fusarium spp.

Fusarium species are the most destructive phytopathogenic and toxin-producing fungi, causing serious diseases in almost all economically important plants. Sporulation is an essential part of the life cycle of Fusarium. Fusarium most frequently produces three different types of asexual spores, i.e., macroconidia, chlamydospores, and microconidia. It also produces meiotic spores, but fewer than 20% of Fusaria have a known sexual cycle. Therefore, the asexual spores of the Fusarium species play an important role in their propagation and infection. This review places special emphasis on current developments in artificial anti-sporulation techniques as well as features of Fusarium’s asexual sporulation regulation, such as temperature, light, pH, host tissue, and nutrients. This description of sporulation regulation aspects and artificial anti-sporulation strategies will help to shed light on the ways to effectively control Fusarium diseases by inhibiting the production of spores, which eventually improves the production of food plants.


Introduction
Fusarium species are the most important phytopathogenic and toxic fungi distributed worldwide, and their spores act as infective propagules that initiate infection [1,2]. Fusaria are soil-born filamentous fungi, belonging to the class Ascomycetes and family Hypocreaceae. The genus Fusarium, which was characterized for the first time by Link in 1809, consists of hundreds of species, many of which are found in the soil, and some of them are associated with plants [3,4]. This fungus is found in tropical, subtropical, and also in temperate regions [5]. Fusarium produces white-, pink-, red-, purple-, salmon-, or grey-colored colonies with velvet to cottony surfaces. The capacity of this fungus to grow on a variety of substrates and its highly effective spore dispersal ability account for its extensive dissemination [6,7]. Therefore, understanding the strategies for the regulation and inhibition of the sporulation of Fusarium species is important for controlling their propagation and infection.
Some Fusaria are harmful to agricultural products, animals, and humans, because many of them are phytopathogenic and produce mycotoxins on plants that can adversely affect humans and animals if they enter the food chain [8][9][10]. Fusarium produces a variety of noxious secondary metabolites, such as fumonisins, zearalenone, and trichothecenes, that infect agricultural commodities and create a risk for human health and consumption [11]. The genome of Fusarium verticillioides, Fusarium graminearum, and Fusarium oxysporum f.sp. lycopersici contains about 46 secondary metabolite biosynthesis gene clusters that encode these mycotoxins [12]. There are more than 145 distinct Fusarium species, of which about one-seventh produce toxins [13]. As plant pathogens, Fusarium species result in significant economic damages and harvest losses [14].
Fusarium is one of the most economically destructive plant pathogens, causing major diseases in nearly all economically important plants and resulting in billion dollars of losses in the field of agriculture worldwide [15]. It is also capable of infecting crops in the moderate climate zones of the world. Fusarium produces mycotoxins such as Trichothecenes that can act as a source of infection in plant diseases [16][17][18][19][20][21]. As Fusarium is a soil-born

Sporulation in Fusarium
Spores and fruiting bodies are the two most important morphological characters used by mycologists to categorize fungi into genera and to differentiate closely related species [28]. In fungi, asexual propagules are produced throughout the life cycle, typically requiring less investment for each propagule than for sexual spores, and dispersal is their sole function [29]. Fusarium species generate sexual spores and three different types of asexual spores. Less than 20% of Fusarium species, however, reproduce sexually, and not all Fusarium species produce all forms of spores [30]. As long as a food source is present, asexual spores are continuously produced in Fusarium and other fungi.

Sexual Spores
Some Fusarium species generate sexual spores ( Figure 1). The role of sexual development in Fusarium spp. Is diverse. In F. graminearum, ascospore is an important primary inoculum that causes head blight disease of wheat and barley [31]. Furthermore, for disease production, sexual development was shown to be essential in F. graminearum, as it undergoes both sexual and asexual stages. In Fusarim solani f.sp. pisi, ascospores are not essential as propagules for dispersal [32]. As we know, the majority of Fusarium species produce fruiting structures in a culture (laboratory), but in the field, sexual development is rare. This is why out of the 12 species with Gibberella teleomorphs, fruiting is common only in G. zeae, whereas G. fujikuroi occasionally produce fruiting bodies in the field. The remaining species (G. baccata, G. ircinate, G. coronicola, G. avenaceae, G. moniliformis, G. nygamai, G. pulicaris, G. intermedia, G. subglutinans, and G. thapsina) have rarely or never produced fruiting structures in nature [33]. However, the majority of these species develop perithecia in the laboratory on natural or artificial substrates [3].

Asexual Spores
Fusarium species can produce three different forms of asexual spores (mitotic), including macroconidia, chlamydospores, and microconidia ( Figure 1). These asexual spores are the most efficient means of reproduction and dispersal, and they also act as the main source of plant infection. These contagious propagules are crucial components of the disease cycle. They are also important for survival and protection in harsh environmental conditions [3,34,35]. Furthermore, chlamydospores play a vital role in the survival of Fusarium wilt diseases and thus cause more severe disease symptoms [36,37]. Chlamydospores are thick-walled cells that arise from mycelial hyphae, and conidia are produced in sporodochia, which are clusters of conidia-producing cells in a slimy mass [29]. Likewise, macroconidia are fusiform to sickle-shaped, multi-celled by transverse septa, with a foot-shaped basal cell. Microconidia can be globose, oval, and reni-form to fusiform, and they are often single-celled, though they can also be three-to five-celled. Only a few species generate microconidia in chains, but most do so in solitary or slimy heads [38]. Although Fusarium produces both sexual and asexual spores, asexual reproduction is more common. In this review, we focus on asexual reproduction.

Genetic Pathway Responsible for Spore Formation
With the advancement in molecular techniques, several genes in Fusarium that are involved in sporulation have been characterized. For example, in F. graminearum, several genes were identified and expressed that are reported to be involved in spore formation processes [39]. Similarly, in Fusarium and Aspergillus, mycotoxin production and sporulation are both regulated by G protein-signaling (RGS) pathways. Further, it was revealed that a number of genes were identified that are involved in the process of sporulation, altering several signal transduction pathway steps [40][41][42]. Furthermore, several regulators are also involved with asexual reproduction in F. graminearum. For instance, several genes required for conidiation are regulated by transcriptional factor AbaA, suggesting that AbaA is essential for asexual sporulation [43]. Meanwhile, in F. graminearum, WetA is required for conidiogenesis and maturation of the conidia [44].
Moreover, FgFlbA (RGS proteins) is required for conidiation in Fusarium, as it induces conidiation in F. graminearum [45]. For F. graminearum to produce asexual spores, a number of other proteins are also required. However, the appropriate expression of HEX1, which encodes the hexagonal peroxisome protein, is essential for controlling conidiogenesis [46]. Similarly, the autophagy-related lipase Atg15 is also essential for morphogenesis and conidia formation [47]. In addition, Mes1 (methyl salicylate esterase), a homologue of MeSA, is necessary for conidiogenesis in F. graminearum [48]. The deletion of velvet genes veA and velB showed increased conidial production [49-51]. Additional proteins involved in conidiation include Mid1 (mating-induced death), HDF1 (histone deacetylase), CATs (carnitine acetyltransferases; CAT1 and CAT2), Acl (ATP citrate lyase), and Top1 (topoisomerase I) [52-56]. In a similar way, the actin binding protein and Fimbrin are also key factors in the conidiation process, as they increase the production of conidia in F. graminearum [57].

Growth Conditions and Environmental Factors Affecting Sporulation
Sporulation is mostly induced or stimulated by endogenous and environmental factors [40,58]. Environmental conditions that trigger sporulation include nutrient depletion, osmotic stress, oxidative stress, carbon and nitrogen status, calcium signaling, pH, aerial stimuli, desiccation, changes in CO 2 partial pressure, secondary metabolites produced by competing organisms, and light. Similarly, endogenous factors such as conidiogenone, sporogen PF-1, and volatile organic compounds also stimulate conidiation [59-61]. However, various fungal species have diverse responses to these stimuli.

Temperature
Temperature is an essential component that affects the growth and sporulation of Fusarium as well as the host's susceptibility to diseases ( Figure 2) [62 -64]. Generally, Fusarium species can be grown in several temperature ranges [64][65][66][67]. However, the optimum temperature for growth and sporulation of Fusarium is 25-30 • C [68]. The optimal temperature for maximum growth and sporulation of F. oxysporum f.sp. ricini was shown to be 27 ± 2 • C on potato dextrose agar media [69].
J. Fungi 2023, 9, x FOR PEER REVIEW 5 of 17 temperature for maximum growth and sporulation of F. oxysporum f.sp. ricini was shown to be 27 ± 2 °C on potato dextrose agar media [69].

Light
Light is considered to be one of the most important factors for spore formation ( Figure  2). However, in some species of Basidiomycetes, Myxomycetes, Ascomycetes, and Zygomycetes, near-UV light irradiation successfully induced sporulation [70][71][72][73]. Excessive irradiation can inhibit sporulation. However, a wavelength ranging from 350-500 nm proved to be effective in enhancing sporulation [74][75][76]. For efficient sporulation, 12 h light and 12 h darkness is the best method available [77,78]. Under 12 h light and 12 h dark conditions, F. solani developed concentric sporulation ring patterns, but this pattern was lost when the fungus was exposed to continuous light or darkness [79]. Numerous other fungi, including Fusarium, were stimulated to sporulate by near-UV light, either on their own or in combination with cool white light [78,80]. Light also stimulated the production of metabolites in Fusarium, and in several other species, it also promoted the production of conidia and sexual fruiting bodies [81]. Several light-sensing proteins, such as White Collar-1 and the Vivid protein, and a few transcription factors, such as BLR-1 (blue-light regulator) and BLR-2, have been found to be involved in fungal sporulation [82][83][84][85][86][87][88][89]. Some Fusarium species also conidiate in the dark, rather than under continuous illumination, such as Fusarium fujikuroi [90].

pH
Fusarium mostly need various pH values for growth and sporulation ( Figure 2) [91,92]. An acidic pH is most suitable for the growth and sporulation of F. oxysporum and F. solani [93]. G. fujikuroi and F. oxysporum were shown to grow and sporulate at 5-5.5 pH [94,95]. The best pH for the growth and sporulation of F. oxysporum was proved to be 5.5 to 7 [91,92,96].

Light
Light is considered to be one of the most important factors for spore formation ( Figure 2). However, in some species of Basidiomycetes, Myxomycetes, Ascomycetes, and Zygomycetes, near-UV light irradiation successfully induced sporulation [70][71][72][73]. Excessive irradiation can inhibit sporulation. However, a wavelength ranging from 350-500 nm proved to be effective in enhancing sporulation [74][75][76]. For efficient sporulation, 12 h light and 12 h darkness is the best method available [77,78]. Under 12 h light and 12 h dark conditions, F. solani developed concentric sporulation ring patterns, but this pattern was lost when the fungus was exposed to continuous light or darkness [79]. Numerous other fungi, including Fusarium, were stimulated to sporulate by near-UV light, either on their own or in combination with cool white light [78,80]. Light also stimulated the production of metabolites in Fusarium, and in several other species, it also promoted the production of conidia and sexual fruiting bodies [81]. Several light-sensing proteins, such as White Collar-1 and the Vivid protein, and a few transcription factors, such as BLR-1 (blue-light regulator) and BLR-2, have been found to be involved in fungal sporulation [82][83][84][85][86][87][88][89]. Some Fusarium species also conidiate in the dark, rather than under continuous illumination, such as Fusarium fujikuroi [90].

PH
Fusarium mostly need various pH values for growth and sporulation ( Figure 2) [91,92]. An acidic pH is most suitable for the growth and sporulation of F. oxysporum and F. solani [93]. G. fujikuroi and F. oxysporum were shown to grow and sporulate at 5-5.5 pH [94,95]. The best pH for the growth and sporulation of F. oxysporum was proved to be 5.5 to 7 [91,92,96].

Host Tissue
In pathogenic fungi, host tissues may also be used to stimulate sporulation ( Figure 2). Banana petioles were shown to increase the sporulation of endophytic fungi isolated from wild banana (Musa acuminata) leaves [97]. The leaves of Rhododendron pulchrum cv. Ohmurasaki were autoclaved and used to enhance the sporulation of Guignardia endophyllicola [98]. Similarly, the leaves of Dianthus caryophyllus were also reported to be effective for conidiation in Fusarium and Pestalotiopsis species [99][100][101]. Though some plant tissues were also observed to be effective in inducing sporulation, such as in some Botryosphaeriaceae spp., pycnidia were stimulated using autoclaved pine needle [70]. Autoclaved corn hulls promoted macroconidia and mycelial growth of F. graminearum. Wheat bran and carnation leaves induced mycelial growth and macroconidia in F. graminearum and F. proliferatum [102].
Biotin also plays important role in the sporulation process. Due to its presence in plant tissue, it might change the formation of the cell walls and oleic acid, altering the expression of the genes related to sporulation [103][104][105][106][107]. Similarly, in mulberry leaves, biotin enhanced the sporulation of Colletotrichum dematium [108].

Nutrition
Some nutritional factors such as microelements, carbon, and nitrogen sources also influence sporulation (Figure 2) [109]. Therefore, several fungi need a particular amount of carbon and nitrogen for sporulation [110,111]. Moreover, sporulation is induced with reduced mycelial growth, and it is inhibited under factors that promote rapid mycelial growth [70]. Hence, food shortage or low nutrient media enhance sporulation [112,113]. Synthetic nutrient-poor agar medium, water agar media, and half-or 1 4 -strength potato dextrose agar (PDA) are some low-nutrient media that induce sporulation [114]. The polysaccharides starch and inulin were shown to induce sporulation in F. oxysporum.
For fungal isolation and culture, PDA is the most commonly used medium. Similarly, potato sucrose agar, Czapek yeast autolysate agar, yeast extract-phosphate medium, cornmeal agar, malt dextrose agar, V8 vegetable juice agar, potato carrot agar, and malt extract agar are also widely used mediums [115]. These media promoted the growth of many endophytic and pathogenic fungi, but they were not very effective in enhancing the sporulation of sterile isolates [116,117]. Furthermore PDA, MEA, and oatmeal agar were shown to be the best mediums for the induction of sporulation in Fusarium [93,118,119]. Similarly, MB and PDB media also promoted sporulation in Fusarium [93].

Artificial Control of Sporulation in Fusarium
Fusarium diseases are a major interruption to food production and are very difficult to control [120]. Farmers still use synthetic fungicides to control Fusarium disease. There are several other reasons to completely stop or minimize the use of synthetic chemicals, aside from their negative impact on the environment.

Biological Control Agents
Nowadays, botanical fungicides are used instead of synthetic fungicides for safety considerations. The botanical fungicides are developed from the extracts of higher plants, and these plant extracts contain antifungal and anti-microbial compounds that act as an antisporulation agent to control fungal diseases ( Figure 2). In Indonesia, 37,000 plant species have been identified, but only 1% of them have been used as botanical fungicides [121]. Several tropical plant extracts possess antifungal activities that control plant pathogens [122][123][124][125]. Four species of plants, namely, Eugenia aromatica, Piper bettle, Alpinia galanga, and Sphaeranthus indicus, have been used as antifungal agents to control F. oxysporum f.sp. vanilae [126]. Similarly, the extracts of 14 tropical plants inhibited the growth of F. oxysporum f.sp. capsici, which causes Fusarium wilt in paprika [125]. Plant extracts of garlic, ginger, onion, neem, vinca, Indian pennywort, wild sage, marigold, and goat weed showed a complete inhibition of sporulation against Fusarium moniliforme [127]. Pea seed extract was used to inhibit the sporulation of Fusarium oxysporum f.sp. pisi race2 [128]. Chinese gall was found to be effective in inhibiting the sporulation of Fusarium graminearum, and tillecur and white mustard seed flour were found to be best in inhibiting conidia in in vitro conditions [129]. Higher plants also produced secondary metabolites such as phenolic acid, cafeic acid, chlorogenic acid, and scopoletin, which are toxic to pathogens [130]. Aloe vera and clove plant extracts significantly inhibited the growth and spore formation of F. oxysporum f.sp. lycopersici [131]. Clove contains eugenol, and Aloe vera contains phenolic compounds as an antifungal agent [132,133].

Essential Oils
Essential oil is also used as an antifungal agent against pathogenic fungi and is one of the most promising natural products for fungal inhibition. The main components of essential oil are carvacrol, thymol, and terpenes/terpenoids, which act as antifungal agents. The cell wall, cytoplasm, and mitochondria are the main targets for antifungal agents [153,154]. The antifungal agents can deactivate the fungus by disrupting the cell membrane and inhibiting the cell wall formation, the action of mitochondrial dehydrogenases, and efflux pumps. Because of their low molecular weight and high lipophilic nature, terpenes are capable of damaging the cell wall and cell membrane of fungi and also inhibiting its sporulation [154]. Similarly, the essential oil of Litsea cubeba contains citral, which acts as an antifungal agent against F. moniliforme and F. solani, affecting their cell wall and membrane, and it also inhibits DNA, RNA, and protein biosynthesis [155,156]. Garlic oil was shown to inhibit the mycelial growth and sporulation in F. oxysporum, which causes wilting in chili [137]. Other researchers have also used garlic against many diseases and reported that garlic contains a sulphur-containing antibiotic that is toxic to plant pathogens [157][158][159][160]. Garlic also contains allicin, which is the main antifungal compound [161,162]. Mint oil and clove oil reduced spore formation and the growth of F. oxysporum f.sp. lycopersici [131]. Rosmarinus officinalis essential oil reduced the sporulation of F. verticillioides [163].

Mycovirus
Mycoviruses have also been used as natural enemies for the management of pathogenic fungi (Figure 2) [164][165][166][167]. These can trigger targets and in some cases suppress RNA silencing, which is the antiviral response of the fungus. Viruses defend themselves from the antiviral response of the fungus by suppressing RNA silencing. Mycoviruses regulate gene expression of the host fungus and also downregulate genes involved in virulence and growth. Wu et al. (2017) used the Sclerotinia sclerotiorum 4 (SsMYR4) infection to downregulate the critical cellular activities and singling pathways of the host [168]. Moreover, the F. graminearum virus China 9 (FgV-ch9) and the F. graminearum viruses FgV1 and FgV2 induced hypovirulence in pathogenic fungi such as F. graminearum [169,170]. Thus, the relation of F. graminearum isolate china 9 with dsRNA mycovirus (Fgv-ch9) showed a significant reduction in conidiation [171]. In 2018, Lemus-Minor et al. used F. oxysporum f.sp. dianthi virus 1 (FodV1) to induce hypovirulence in F. oxysporum. This resulted in reduced mycelial growth, conidiation, and virulence on carnation plants, suggesting it functions as a biocontrol agent for Fusarium wilt of carnation [171].

Rhizospheric Bacteria
Some rhizospheric bacterial species are employed as biological control agents, shielding plants from soil-borne diseases and promoting plant growth (Figure 2). Streptomyces albospinus CT205 and Bacillus sp. str. SV101 and SV104 have been used as biocontrol agents to inhibit Fusarium wilt [172,173]. In 2014, Zhao et al. used Bacillus subtilis SG6, which inhibits the growth and sporulation of Fusarium graminearum, to break down the cell wall of F. graminearum by producing chitinase [174]. Paenibacillus polymyxa NSY50 inhibited the growth of F. oxysporum in the rhizosphere of cucumber and thus protected the plant from pathogen invasion [175].

Chemical Supplements
Some chemical supplements are also used to control pathogenic fungi ( Figure 2). Chitosan is known to inhibit spore formation and to act as antifungal agent. The fungal cell membrane is the primary target of chitosan [176]. The interaction of negatively charged phospholipid of the fungal cell membrane and positively charged chitosan increases membrane permeability that results in the leakage of cellular contents, which ultimately results in cell death [177]. They also function as chelating agents and bind to trace elements, hence rendering the vital nutrients inaccessible for the normal growth of fungi. Chitosan also punctures the fungal cell wall and binds to its DNA to inhibit the synthesis of mRNA [178,179]. Its inhibitory effect was proved with soil-borne phytopathogenic fungi, including Fusarium wilt pathogens [180][181][182]. It also inhibited the growth and sporulation of F. solani and F. oxysporum f.sp. cubense race 4 (FocR4) [183,184]. Nano chitin whisker also significantly inhibited the mycelial growth and conidiation of Fusarium species [185].
Potassium phosphonate inhibited the production of microconidia in F. oxysporum [186]. Similarly, pregnenolone inhibited sporulation in Fusarium graminearum. Pregnenolone might be targeted to the transcriptional factors required for sporulation [187]. Sulfamethoxazole and the indole alkaloid gramine are two natural compounds that decreased disease symptoms caused by F. graminearum in Arabidopsis and wheat [188].
Methyl jasmonate is a signaling molecule that modulates plant defense responses. It stimulates phenolic acids, flavonoids, and phytoalexins responsible for the plant's defense against pathogens. [189][190][191][192]. Methyl jasmonate induced a defensin-like protein in Pganax notoginsen (PnDEFL1), which showed resistance to F. solani in transgenic tobacco [193]. Methyl jasmonate had an inhibitory effect on the sporulation and mycelial growth of F. solani. Radial growth and sporulation were significantly inhibited in F. oxysporum and F. solani by using different concentrations of salicylic acid [184,194]. Coumarin also inhibited the sporulation of Fusarium oxysporum f.sp. niveum by suppressing activities of pathogenesis-related enzymes [195].

Transgenic Approaches to Control of Sporulation in Fusarium
For transgenic approaches to sporulation control in Fusarium, RNA interference (RNAi) is frequently used as a tool to regulate gene expression and provide protection against viruses and pathogens [196][197][198][199]. It was first reported in 1990 by Napoli and Jorgensen [200]. RNAi is activated in the presence of double-stranded RNA (dsRNA) in the host plant and degrades the double-stranded RNA molecule into single-stranded RNA molecules, hence causing silence or knockdown of the targeted gene of the pathogen. This artificial manipulation of gene silencing is used in both transgenic and non-transgenic plants and can be used to control Fusarium growth and sporulation by silencing the genes responsible for conidiation ( Figure 3). Generally, there are two ways to perform RNA interference.
against viruses and pathogens [196][197][198][199]. It was first reported in 1990 by Napoli and Jorgensen [200]. RNAi is activated in the presence of double-stranded RNA (dsRNA) in the host plant and degrades the double-stranded RNA molecule into single-stranded RNA molecules, hence causing silence or knockdown of the targeted gene of the pathogen. This artificial manipulation of gene silencing is used in both transgenic and nontransgenic plants and can be used to control Fusarium growth and sporulation by silencing the genes responsible for conidiation ( Figure 3). Generally, there are two ways to perform RNA interference.

Host-Delivered RNAi or Host-Induced Gene Silencing
Host-delivered RNAi (HD-RNAi) uses the host plant as a delivery system and silences the targeted gene of the pathogen [196,201]. In this approach, the siRNA or dsRNA is transformed to the host plant, thus targeting the gene of the pathogen. When this transgenic plant becomes infected, and the pathogen starts feeding from the host, the small interfering RNA (siRNA) and dsRNA molecules from the plant are transferred to the pathogen cells, hence activating an RNAi response in the pathogen and silencing the targeted gene of the pathogen [202]. This strategy was used on various Fusarium species. It was tested in tobacco against F. verticillioides. When the pathogen started feeding off transgenic tobacco plants, GUS-RNAi expressed and significantly silenced the GUS gene in the pathogen [203]. This technology was also used against F. graminearum in Arabidopsis and barley and significantly silenced three fungal cytochrome P450 lanosterol C-14 α-demethylase (CYP51) genes and also increased resistance against pathogens [204]. The silencing of the Cmk1 gene (Colletotrichum lagenarium MAP kinase) in C. lagenarium showed a reduction in conidiation [205]. Similarly, Fmk1, Hog1, and Pbs2 are mitogen-activated protein kinase genes responsible for fungal growth, development, sporulation, and virulence, and so the silencing of these genes in F. oxysporum showed reduced growth, sporulation, and pathogenicity [206]. However, silencing of the Hog1 gene in F. graminearum showed significantly reduced conidiation [207]. Furthermore, the silencing of FOW2 and chsV (class V chitin synthase) in F. oxysporum and F. solani showed reduced mycelial growth and sporulation, which confirmed their involvement in pathogenicity [208]. In F. oxysporum f.sp cubense, the SGE1 gene (Six gene expression 1) is involved in pathogenicity and virulence. Therefore, silencing of this gene showed reduced sporulation and pathogenicity [209]. Moreover, the ODC gene (ornithine decarboxylase) in F. oxysporum is important for fungal growth and causes Fusarium wilt in tomato. Hence, the silencing of this gene showed resistance to Fusarium wilt in tomato [210].

Host-Delivered RNAi or Host-Induced Gene Silencing
Host-delivered RNAi (HD-RNAi) uses the host plant as a delivery system and silences the targeted gene of the pathogen [196,201]. In this approach, the siRNA or dsRNA is transformed to the host plant, thus targeting the gene of the pathogen. When this transgenic plant becomes infected, and the pathogen starts feeding from the host, the small interfering RNA (siRNA) and dsRNA molecules from the plant are transferred to the pathogen cells, hence activating an RNAi response in the pathogen and silencing the targeted gene of the pathogen [202]. This strategy was used on various Fusarium species. It was tested in tobacco against F. verticillioides. When the pathogen started feeding off transgenic tobacco plants, GUS-RNAi expressed and significantly silenced the GUS gene in the pathogen [203]. This technology was also used against F. graminearum in Arabidopsis and barley and significantly silenced three fungal cytochrome P450 lanosterol C-14 α-demethylase (CYP51) genes and also increased resistance against pathogens [204]. The silencing of the Cmk1 gene (Colletotrichum lagenarium MAP kinase) in C. lagenarium showed a reduction in conidiation [205]. Similarly, Fmk1, Hog1, and Pbs2 are mitogen-activated protein kinase genes responsible for fungal growth, development, sporulation, and virulence, and so the silencing of these genes in F. oxysporum showed reduced growth, sporulation, and pathogenicity [206]. However, silencing of the Hog1 gene in F. graminearum showed significantly reduced conidiation [207]. Furthermore, the silencing of FOW2 and chsV (class V chitin synthase) in F. oxysporum and F. solani showed reduced mycelial growth and sporulation, which confirmed their involvement in pathogenicity [208]. In F. oxysporum f.sp cubense, the SGE1 gene (Six gene expression 1) is involved in pathogenicity and virulence. Therefore, silencing of this gene showed reduced sporulation and pathogenicity [209]. Moreover, the ODC gene (ornithine decarboxylase) in F. oxysporum is important for fungal growth and causes Fusarium wilt in tomato. Hence, the silencing of this gene showed resistance to Fusarium wilt in tomato [210].

Spray-Induced Gene Silencing
The exogenous application of dsRNA and siRNA is another very promising approach to gene silencing [211,212]. The siRNA and dsRNA target the essential pathogen gene on the plant surface. They can also be sprayed on a wounded surface of the plant, and then this siRNA or dsRNA is taken up by the plants and transferred through the vascular system of fungi. This is an environmentally friendly strategy and is easily accepted by the public and biosafety authorities, and it is optimized faster than HIGS [213]. Koch et al. used this method on Fusarium and sprayed barley leaves with CYP3-dsRNA to check the growth of F. graminearum, and they found that the growth and conidiation of F. graminearum was inhibited by CYP3-dsRNA [214]. Myo5 dsRNA was sprayed on a wounded surface of the plant and silenced the Myo5 gene in the fungus. Myo5 has five segments, Myo5-3, Myo5-4, Myo5-5, Myo5-7, and Myo5-8, and all of these were significantly silenced by dsRNAs. As a result, both the sexual and asexual reproduction of F. asiaticum were significantly reduced. Meanwhile, Myo5-8 significantly reduced the growth of F. asiaticum, F. tricinctum, F. graminearum, and F. oxysporum f.sp. lycopersici [215].

Future Perspectives in Sporulation Control in Fusarium
Fusarium is one of the most harmful plant pathogens that causes wilt diseases of crops. Fusarium spores are easily spread in the field, causing invasive and disseminated infections. Fusarium sporulation is mostly induced or stimulated by endogenous and environmental factors. Several strategies have been developed to control the production of spores. In particular, various biocontrol agents and chemicals were used to control Fusarium sporulation, but most of these experiments were performed under in vitro conditions, so they should be validated under field conditions. We expect that more efficient biocontrol agents and chemicals will be identified from further field experiments. The management of Fusarium diseases by gene silencing was also considered to be a powerful method to control the sporulation of Fusarium, and more studies should be carried out in the future to characterize and identify the genes that are involved in sporulation. Currently, two genes in the ergosterol synthetic pathway that are relevant to the sporulation of Fusarium were identified by our team (unpublished). The control of the sporulation of Fusarium and then the control of the spread of wilt diseases will eventually become a new approach to increase crop yield and quality.