Eco-Friendly Biocontrol Strategies of Alternaria Phytopathogen Fungus: A Focus on Gene-Editing Techniques

: Agricultural food production is greatly affected by postharvest diseases worldwide, such as the diseases caused by Alternaria species, which are very common in several crops. The management of fungal infections around the world largely relies on fungicides. In this context, the control of diseases such as early blight caused by Alternaria solani in potatoes and Alternaria linariae in tomatoes has mainly consisted of the application of fungicides, with negative impacts on the environment and human health. Recently, the application of ‘omics’ and gene editing through the CRISPR/Cas9 system and RNAi technologies demonstrated their effectiveness as emerging greener alternatives for controlling phytopathogenic fungi. Additionally, coatings based on essential oils and microbial antagonists suggest alternative strategies for controlling phytopathogenic fungi that are respectful of the environment. This review presents an exhaustive literature review focused on using greener alternatives to the traditional management of postharvest diseases associated with Alternaria species, such as inhibiting pathogenicity from their phytopathogenic genes using gene editing based on CRISPR/Cas9 and RNAi technologies. The review also presents coatings based on essential oils and microbial antagonists as greener strategies for Alternaria control. Biological processes of maximum efﬁciency can replace chemical methods for controlling phytopathogenic fungi, preserving healthy conditions in agricultural lands and ecosystems. This is possible with the rise of ‘omic’ technologies, the CRISPR/Cas9 tool, and RNAi technology. Greener control methods of Alternaria fungi can increase agricultural production, improving the economy and global health. genetic modiﬁcation in plants. Gene suppression by target RNA silencing plays a critical role in studying phytopathogenic fungal control C.C.-L.; D.C.C.-H., E.F.-L., Y.P.-R. and C.D.G.-T.; visualization, C.D.G.-T. and C.C.-L.; supervision, C.D.G.-T., E.F.-L. and C.C.-L.; funding acquisition, C.D.G.-T. and E.F.-L. All authors


Introduction
The control of phytopathogenic fungi in agricultural production is of global interest since they cause significant economic losses to producers [1]. About six million fungi are known; however, about 200 species have some pathogenicity associated with diseases affecting biodiversity, ecology, agriculture, and food security worldwide [2]. Some estimates of losses caused by phytopathogenic fungi in crops are 21.5% for wheat, 30% for rice, 22.5% for corn, 17.2% for potatoes, and 21.4% for soybeans, especially in the postharvest production stage [3]. Within these fungi, Alternaria species are ubiquitous phytopathogenic fungi that cause deadly and common diseases in crops, including Alternaria leaf spot and Alternaria brown spot, characterized by necrotic spots on plants' vegetative tissue [4].
which have desirable biological activities and a high control potential in phytopathogenic fungi [20]. The use of microbial biocontrol agents in the control of postharvest diseases is also promising for the management of black spot disease caused by A. alternata, as reported in Ziziphus jujuba, through the antagonistic yeast Debaryomyces nepalensis [21].
This comprehensive review focuses on controlling the pathogenicity of causal species such as Alternaria alternata, Alternaria tenuissima, Alternaria burnsii, and Alternaria brassicicola through gene editing studies and using green alternatives such as essential oils and antagonist microbials. These alternatives have emerged with a potential for improving plant resistance, also contributing to controlling diseases caused by ingesting mycotoxins from contaminated food, and increasing the fertility of arable agricultural spaces, ultimately allowing the preservation of ecosystems.

Alternaria spp.
Alternaria is a genus of the phylum Ascomycota (family of Pleosporaceae, order of Pleosporales). It is one of the most ubiquitous fungi, with the characteristic of inhabiting any environment, given its adaptability to different substrates and minerals to grow [22]. Alternaria species are infectious fungi with a broad host diversity in crops. Resistance to multiple environmental conditions and genetic diversity give Alternaria species a great adaptation capacity in different areas of the world [23].
The description of the Alternaria genus dates to 1816, with identification of the first type of Alternaria tenuis. Since then, about a thousand Alternaria species have been reported [24].

Alternaria Infection
Diseases caused by Alternaria species, such as Alternaria leaf spot and Alternaria brown spot, are prevalent in agricultural production [38]. These diseases present necrotic spots on the leaves and vegetative tissues, as shown in Figure 1.

Alternaria Infection
Diseases caused by Alternaria species, such as Alternaria leaf spot and Alternari brown spot, are prevalent in agricultural production [38]. These diseases present necroti spots on the leaves and vegetative tissues, as shown in Figure 1. Alternaria species are endophytic, saprophytic, and pathogenic, related to diseases in preharvest and postharvest production [39]. The sporulation of these fungi facilitates propagation by the breeze and through vectors such as insects, which contract the spores of phytopathogenic fungi and spread them to healthy plants [40]. Advanced Alternaria infections in crops of economic importance generate putrefaction in postharvest production through cell wall degradation, metabolism of fatty acids, sugars, and the increase in secondary metabolites in their hosts, which are discarded in an advanced disease stage [41]. Infection with Alternaria species occurs through contact with the fungus spores. The penetration into the vegetative tissue usually occurs through plant injury or the routes of plant supplies, directly through the epidermis or via stomata. The Alternaria spores remain inactive on the vegetative tissue until they find some path to enter the host or recognize nutrients and minerals. Subsequently, a germination process starts through the secretion of enzymes and mycotoxins. In contrast, the host plants of these pathogens produce new substances (generally oligosaccharides) in response to the infection process, which slows down the spread of the infection. Oligosaccharides induce defense enzymes in plants, significantly improving resistance to diseases transmitted by the soil or pathogens [42].
The environments most prone to damage by Alternaria on plants are humid zones with a mild acid to neutral pH. The infection process usually begins in fruits at an advanced stage of maturity, where the defense system progressively weakens and tissues soften, while nutrient and pH conditions are ideal [43].
The pathogenicity and virulence depend on specific genes producing host-specific toxins (HSTs) and non-host-specific toxins (NHSTs). These genes are included in conditionally dispensable chromosomes (CDCs), presumably acquired by horizontal transfer from other pathogens [44,45]. Horizontal gene transfers in pathogenic species increase the range of existing pathogens [46]. In the case of Alternaria species, the pathogenicity is described by genes encoding both HSTs and NHSTs [47]. Meena et al. (2019) reported the molecular characterization of the toxins encoded by Alternaria species, including HSTs and NHSTs. Both HSTs and NHSTs are distributed in four families of toxins: the first belongs to epoxy-decatrienoic acid (EDA)-AK-AF and ACT; the second belongs to cyclic depsipeptide (cyclic tetrapeptide) AM toxin; the third family is the amino pento/polyketide (sphininganine analogs) AAL toxins; and finally, those belonging to polyketides-ACR toxins, which cause degeneration in the membranes of important cell organelles, such as chloroplasts, mitochondria, plasma membrane, Golgi complex, among others [48], as shown in Figure 2.  The production of more than 70 toxins in Alternaria species is known and associated with pathogenicity. Several of them are harmful to human beings and animals [50] Among them, alternariol (AOH), alternariol monomethyl ether (AME), tenuazonic acid (TA), altenuene (ALT), and tentoxin (TEN) are the most widely spread in foods and plants fully characterized by high-performance liquid chromatography coupled to mass spec trometry (HPLC-MS) [51]. The use of spectrometry (UPLC-MS/MS) to identify Alternaria associated mycotoxins was reported in 260 medicinal and edible herbs showing symp toms of Alternaria-associated diseases. AME was the most prevalent mycotoxin with 37.5%, followed by AOH with 22.5%, ALT with 17.5%, TA with 10.83%, alterotoxin with 7.5%, and altenusin with 4.17% [52].
Although the identification of Alternaria species is carried out by molecular methods the production of secondary metabolites has also been used for classification and identi The mechanisms associated with host-specific toxin production linked to the pathogenicity of A. alternata and A. arborescens have been reported by polymerase chain reaction (PCR) assays targeting three genes (AMT1, AMT2, and AMT14), which were identified in a conditionally expendable chromosome involved in the production of host-specific toxins [49].
The production of more than 70 toxins in Alternaria species is known and associated with pathogenicity. Several of them are harmful to human beings and animals [50]. Among them, alternariol (AOH), alternariol monomethyl ether (AME), tenuazonic acid (TA), altenuene (ALT), and tentoxin (TEN) are the most widely spread in foods and plants, fully characterized by high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS) [51]. The use of spectrometry (UPLC-MS/MS) to identify Alternariaassociated mycotoxins was reported in 260 medicinal and edible herbs showing symptoms of Alternaria-associated diseases. AME was the most prevalent mycotoxin with 37.5%, followed by AOH with 22.5%, ALT with 17.5%, TA with 10.83%, alterotoxin with 7.5%, and altenusin with 4.17% [52].
Although the identification of Alternaria species is carried out by molecular methods, the production of secondary metabolites has also been used for classification and identification. In this context, Saleem et al. (2022) reported the molecular characterization of A. tenuissima, A. mimicula, A. infectoria, A. alternata, and A. arborescens in tomatoes and the ability of these species to produce AOH AME, TA, and ALT, using HPLC. Additionally, they used PCR to amplify the ITS region and sequenced the pksJ and pksH genes associated with toxin production [53].
Previous research has suggested molecular variability between species, which presents different levels of virulence. Garganese et al. (2016) recorded a significant correlation between the pathogenicity of Alternaria and the expression of the ACTT1, pksJ, and pksH genes, associated with the synthesis of AOH, AME, TeA, and AlT. The expression of ACTT1 depends specifically on the geographical zones of crops [54]. The study of mycotoxins secreted by Alternaria species is of interest since they allow the rapid identification of diseases in crops associated with single species of phytopathogenic fungi. Interspecies molecular variability presents a big problem for both agricultural producers and consumers. Still, it has expanded the field of study through the metabolic pathways, allowing the development of genetic assemblies and the prediction of genes through omics technologies [55].

Phytopathogenic Genes in Alternaria spp.
Alternaria species (like most crop pathogens) possess chromosomes known as expendable coding chromosomes (ECCs), which contain specific genes encoding HSTs. Gai et al. (2021) studied the HSTs produced by the ACT toxin-encoding gene cluster and reported the genome assembly of A. alternata strain Z7 at the chromosomal level. The genome has 34.24 Mb, 12067 protein-coding genes, 34 ribosomal RNAs (rRNAs), 107 transfer RNAs (tRNAs), and two ECCs, which offer information on the chromosomes directly linked to the pathogenicity of the organism. This is valuable because it allows an understanding of the pathogenicity mechanism of A. alternata [56].
Despite the little information available to study genes that encode toxins from Alternaria species, data mining offers possibilities to build an alternative to control pathogens through proteomic and transcriptomic analysis [57]. Using these technologies, whole genome sequencing was reported for A. solani (altNL03003) [58], the agent causing early blight disease in potato and tomato (genomic DNA sequencing ID: 4190556). Additionally, the genomes of other Altermaroa species have been sequenced and studied in depth [59].
The codominant ASC-1 gene has been identified (also known as ASC, ASC-1, and LASS 2) using comparative genomics, which confers resistance to stem canker caused by the necrotrophic fungus A. alternata f sp. lycopersicis that affects Solanum lycopersicum [60]. The ASC-1 gene is homologous to genes belonging to most eukaryotes (from yeast to humans) and has a conserved function. For example, a yeast strain with deletions in the LAG1 and LAC1 genes (homologous to the ASC gene) complemented the ASC-1 gene [61]. It is known that the different toxins produced by Alternaria species vary in the site of action during infection. Still, all of them converge in the death cell of their hosts [62,63]. Figure 3 shows the homolog of the ASC-1 gene (also reported in homologs of some strains of Alternaria and yeasts) located on chromosome three of S. lycopersicum, which can reveal details about the infection and resistance mechanisms by plants against attacks by phytopathogenic fungi [64]. ternaria and yeasts) located on chromosome three of S. lycopersicum, which can reveal details about the infection and resistance mechanisms by plants against attacks by phytopathogenic fungi [64].
The study of gene expression levels after 18 h of inoculation with A. solani in the breeding lines BD12-65 and BD1235 showed that the ASC-1 gene is involved in the resistance response in the mentioned lines. Studying the metabolic genes, also known as biosynthetic genes, associated with essential metabolic pathways for a species is also fascinating. These biosynthetic genes allow an understanding of the metabolite synthesis or the production of mycotoxins [66]. Figure  4 shows (in purple) the central biosynthetic genes of A. alternata, positioned in their genome (with sizes up to 6066 nt) and other genes (in gray) whose precise functions are unknown. The study of gene expression levels after 18 h of inoculation with A. solani in the breeding lines BD12-65 and BD1235 showed that the ASC-1 gene is involved in the resistance response in the mentioned lines.
Studying the metabolic genes, also known as biosynthetic genes, associated with essential metabolic pathways for a species is also fascinating. These biosynthetic genes allow an understanding of the metabolite synthesis or the production of mycotoxins [66]. Figure 4 shows (in purple) the central biosynthetic genes of A. alternata, positioned in their genome (with sizes up to 6066 nt) and other genes (in gray) whose precise functions are unknown.  The elucidation of genes of interest in phytopathogenic fungi through in silico or in vitro studies, such as genes encoding mycotoxins, as in the case of A. atra (a saprophytic fungus producing leaf blight on the potato) and A. arborescens (producing stem blight) that affects tomato crops and is a producer of host-specific toxins, is of great interest to combat losses associated with diseases transmitted by phytopathogenic fungi since they offer the possibility of applying the gene-editing techniques such as CRISPR/Cas9 or RNAi [67,68].
In this context, Wenderoth et al. (2019) reported the genes encoding AOH and its derivative AME in A. alternata by CRISPR/Cas9-mediated gene knockout [69]. In addition, information on genes associated with synthesizing secondary metabolites was reported. The associated genes consist of a group of 15 kb, including a pksI (polyketide synthase gene), omtI (an O-methyltransferase), and moxI (a FAD-dependent monooxygenase), sdrI (short-chain dehydrogenase), doxI (extradiol dioxygenase response gene), aohR (transcription factor gene). The inhibition of the transcription factor gene aohR generated a significant reduction in AOH levels, consequently decreasing the virulence in citrus, apple, and tomato crops [69].
Liu et al. (2020) reported the regulation of four genes, PksJ, PksF, PksI, and PksA, which are polyketide synthase type and associated with the synthesis of secondary metabolites [70]. The study suggests that the inhibition of PksJ, PksF, PksI, and PksA eliminates the biosynthesis of AOH, AME, and TEA in A. alternata. The elucidation of genes of interest in phytopathogenic fungi through in silico or in vitro studies, such as genes encoding mycotoxins, as in the case of A. atra (a saprophytic fungus producing leaf blight on the potato) and A. arborescens (producing stem blight) that affects tomato crops and is a producer of host-specific toxins, is of great interest to combat losses associated with diseases transmitted by phytopathogenic fungi since they offer the possibility of applying the gene-editing techniques such as CRISPR/Cas9 or RNAi [67,68].
In this context, Wenderoth et al. (2019) reported the genes encoding AOH and its derivative AME in A. alternata by CRISPR/Cas9-mediated gene knockout [69]. In addition, information on genes associated with synthesizing secondary metabolites was reported. The associated genes consist of a group of 15 kb, including a pksI (polyketide synthase gene), omtI (an O-methyltransferase), and moxI (a FAD-dependent monooxygenase), sdrI (shortchain dehydrogenase), doxI (extradiol dioxygenase response gene), aohR (transcription factor gene). The inhibition of the transcription factor gene aohR generated a significant reduction in AOH levels, consequently decreasing the virulence in citrus, apple, and tomato crops [69]. Liu et al. (2020) reported the regulation of four genes, PksJ, PksF, PksI, and PksA, which are polyketide synthase type and associated with the synthesis of secondary metabolites [70]. The study suggests that the inhibition of PksJ, PksF, PksI, and PksA eliminates the biosynthesis of AOH, AME, and TEA in A. alternata.

Chemical Control Strategies
The use of fungicides in agricultural fields is a widespread practice in the world. The excessive use of pesticides and the commercialization of food contaminated with fungicides poisons more than four million people yearly, with 350,000 lethally poisoned people. Additionally, the residues of fungicides in commercialized foods generate more than 20,000 cases of cancer per year [71]. Pereira et al. (2021) reported the countries where chemical compounds are used as the primary treatment to control pathogens, their incidence in the environment, and the adverse effects on non-target species [72].
Additionally, the excessive use of these chemical techniques to control pathogens harms arable fields, causing a reduction in soil fertility.

Essential Oils and Biopolymers Used to Control Alternaria Fungi
Biofungicides based on essential oils and biopolymers to control phytopathogenic fungi have been used as greener alternatives for managing crop pathogens [78]. Essential oils are of particular interest as an alternative for managing diseases transmitted by phytopathogenic fungi, given their efficiency, biodegradability, and non-toxicity in human health and the environment [79]. In recent years, several studies have demonstrated the efficacy of essential oils. For example, Feng et al. (2007) reported the inhibitory effects of essential oils from thyme (Thymus vulgaris), eucalyptus (Eucalyptus globulus Labill), cassia (Cassia didymobotrya), sage (Salvia officinalis), and nutmeg (Myristica Fragans Houtt) against A. alternata, the agent causing necrotic spots [80]. Zaker et al. (2010) demonstrated the antifungal capacity of eucalyptus (Eucalyptus globulus), peppermint (Mentha piperita), Lavandula (Lavandula angustifolia), and alcoholic extracts of datura against A. alternata [81]. Additionally, Huang et al. (2019) reported a solid antifungal capacity of Artemisia lavandulaefolia DC and Artemisia scoparia essential oils, while Artemisia annua oil presented a moderated activity against A. solani [82]. In the same way, the extract of Syzygium aromaticus, Betle piper, Zingiber cassumunar, and Coscinium fenestratum had inhibitory activity against A. brassicicola [83]. The controlled release of essential oils has consisted of the nanoencapsulation of essential oils with antifungal activity [84]. Antifungal lipid nanoemulsions incorporating essential oils have effectively controlled tomato leaf spots caused by A. alternata. In addition, lipid nanoemulsions encapsulating garlic (Allium sativum), chilli (Capsicum annuum L. var annuum), and cinnamon (Cinnamomum zeylanicum) essential oils exhibit high antifungal activity in vivo against A. alternata [85].
It is well known that the chemical structure of essential oil components is fundamental for disease control. For example, the essential oil of basil (Ocimum basilicum) is rich in compounds with antifungal activities (28.7% linalool and 38.2% methyl chavicol) [86], which has an inhibitory effect on conidia germination and growth of A. alternata. However, despite the chemical composition, several external factors limit the inhibitory efficiency of essential oils, such as humidity, light sensitivity, high temperatures, volatility, hydrophobicity, and Agriculture 2022, 12, 1722 9 of 23 high oxygen content environments. These limitations are compensated by encapsulation or coatings with substances such as polymers that protect them from these unfavorable external effects [87]. The development of films or coatings used to preserve food consists of systems in which various biopolymers are combined to achieve beneficial properties. Usually, these coatings are made from proteins, lipids, and carbohydrates [88].
On the other hand, carbohydrate-based biopolymers such as alginate and chitosan are very attractive in protecting crops against fungi due to their antifungal properties and biodegradability [89]. Using chitosan (a derivative of deacetylated chitin found in the outer layer of crustaceans such as shrimp, crab, lobster, and shellfish) as a coating in crop protection against phytopathogenic fungi is well studied. It is considered an ideal resource for sustainable agriculture thanks to its biodegradability, non-toxicity, and biocompatibility properties [90]. Table 2 reports examples of free and encapsulated essential oils used to control Alternaria species in several hosts. -Cherry tomato/in vitro and in vivo Laurus nobilis L.

Antagonistic Microorganisms to Control Alternaria
Research based on antagonist microorganisms (such as antagonistic rhizobacteria) in the control of phytopathogenic fungi is of interest to inhibit fungal growth and enhance the healthy development of plants. More environmentally friendly alternatives use biological fungal control methods by applying antagonistic bacterial and fungal genera of Gliocladium, Bacillus, Coniothyrium, Paecilomices, Phlebiopsis, Pseudomonas, and Rhizobium, among others [115], which are based on mycoparasitism or direct parasitism through bacteria. Bacterial-based parasitism has been studied in previous publications, which report that these associations between antagonistic microorganisms and phytopathogenic fungi are based on the adherence of antagonistic microorganisms to both the conidia and vegetative hyphae of the fungus, which aggressively compete for nutrients [116]. These bacteria have been identified as species belonging to the Bacillus genus, such as B. cereus, B. mycide, and B. subtilis, which have shown high efficiency in controlling Alternaria helianthi. In this context, Kurniawan et al. (2018) evaluated the control of mycelial growth of A. alternata through the use of antagonistic Pseudomonas and Bacillus bacteria, with a 27% inhibition of mycelial growth and 50% inhibition of postharvest rot in blueberry (Vaccinium corymbosum) development [117]. Additionally, antagonistic fungi to control phytopathogenic fungi are an alternative whose importance is increasing given the high percentages of inhibition of many pathogens. Komhorm et al. (2021) evaluated the antagonistic activity of Talaromyces flavus, Talaromyces trachyspermus, Neosartorya fischeri, Eupenicillium sp., and Gongronella butleri against black spot disease in Brassica oleracea var. saberlica caused by A. brassicicola [118]. Table 3 summarizes biological control agents such as fungi and bacteria.
The use of plant growth promoting rhizobacteria (PGPR), in addition to suppressing plant diseases, improves their growth, showing a 13% reduction in the severity of illness transmitted by A. solani and 84.3% protection in samples treated with Achromobacter xylosoxidans, Lysinibacillus fusiformis, and Bacillus subtilis [119]. Additionally, isolates of Pseudomonas aeruginosa and Enterobacter roggenkampii exhibited inhibitory activity against A. alternata. In addition, postharvest treatment of tomato fruits using yeasts such as Meyerozyma guilliermondii showed a 57% reduction in lesions, while using Pseudomonas aeruginosa exhibited 50%, 60% with E. roggenkampii, and 50% with M. guilliermondii [120]. Sharma et al. (2018) studied the antagonistic activity of rhizobacterial isolates against A. brassicae blight in Brassica juncea L, revealing high disease inhibition [121].

Molecular-Based Methods for Controlling Alternaria
Biosynthetic genes are interesting for developing alternative pathogenicity control methods for Alternaria and other phytopathogenic fungi. The alternative techniques intend to replace the use of chemical processes through genome editing strategies based on gene knockout, CRISPR/Cas9, and RNAi techniques [14]. Lu et al. (2019) reported the silencing of the AbSte7 gene, involved in specific aspects of the pathogenicity of A. brassicicola, related to the improvement in the adaptation capacity and inhibition of the host's immune response [11]. The AbSte7 gene's disruption allowed a considerable reduction in the enzymes involved in the host cell wall degradation during infection, suggesting a significant decrease in pathogenicity, opening possibilities for nonconventional biological methods' application in controlling phytopathogenic fungi for agro-industrial production.

Control of Alternaria Pathogenicity Based on CRISPR/Cas9
The activation of biosynthetic genes is of particular interest for controlling the pathogenicity of various fungi. Due to the complexity of the selective activation of genes of interest, few successful cases have been reported, which poses a challenge to studying the biosynthetic potential of genes of interest. However, some activation methodologies consisting of global regulators have been successful [129,130].
Methodological techniques based on CRISPR/Cas9 have significantly impacted biomedical research by correcting errors at the genome level through the activation or deactivation of specific genes. Applications of CRISPR/Cas9 have been reported in genome editing in filamentous fungi [131]. However, few studies are based on Alternaria, despite the wellstudied Aspergillus nidulans [132,133]. CRISPR/Cas9 consists of two essential molecules: a guide RNA complementary to a gene of interest and an endonuclease that breaks specific bonds in the double helix chain, known as Cas proteins (or proteins associated with CRISPR) [134], as shown in Figure 5. for non-conventional biological methods' application in controlling phytopathogenic fungi for agro-industrial production.

Control of Alternaria Pathogenicity Based on CRISPR/Cas9
The activation of biosynthetic genes is of particular interest for controlling the pathogenicity of various fungi. Due to the complexity of the selective activation of genes of interest, few successful cases have been reported, which poses a challenge to studying the biosynthetic potential of genes of interest. However, some activation methodologies consisting of global regulators have been successful [129,130].
Methodological techniques based on CRISPR/Cas9 have significantly impacted biomedical research by correcting errors at the genome level through the activation or deactivation of specific genes. Applications of CRISPR/Cas9 have been reported in genome editing in filamentous fungi [131]. However, few studies are based on Alternaria, despite the well-studied Aspergillus nidulans [132,133]. CRISPR/Cas9 consists of two essential molecules: a guide RNA complementary to a gene of interest and an endonuclease that breaks specific bonds in the double helix chain, known as Cas proteins (or proteins associated with CRISPR) [134], as shown in Figure 5. The biosynthetic potential in phytopathogenic fungi is mainly unknown, which is why the number of reports of plasmids used in CRISPR/Cas9 is increasing, especially in studying fungal genes linked to the pathogenicity of filamentous fungi. In this regard, Roux et al. (2020) reported the activation of biosynthetic gene clusters in filamentous fungi by applying CRISPR/Cas9 to obtain the highest biosynthetic potential encoded in fungal genomes. The technique is based on a CRISPR/dLbCas12a-VPR system targeting the micA gene linked to non-ribosomal peptides similar to synthetases (similar to NRPS), allowing more significant production of microperfuranone and the discovery of dehydrocroperfuranone, demonstrating the utility of the dLbCas112aD156R-VPR variant for CRISPR/Cas9 under culture conditions [135]. Previously, Nødvig et al. (2015) developed a methodology based on CRISPR/Cas9 for use in filamentous fungi based on gRNAguided mutagenesis. The system consists of four CRISPR/Cas9 vectors equipped with fungal markers that facilitate selecting a wide range of fungi [132]. New CRISPR/Cas9 vari- The biosynthetic potential in phytopathogenic fungi is mainly unknown, which is why the number of reports of plasmids used in CRISPR/Cas9 is increasing, especially in studying fungal genes linked to the pathogenicity of filamentous fungi. In this regard, Roux et al. (2020) reported the activation of biosynthetic gene clusters in filamentous fungi by applying CRISPR/Cas9 to obtain the highest biosynthetic potential encoded in fungal genomes. The technique is based on a CRISPR/dLbCas12a-VPR system targeting the micA gene linked to non-ribosomal peptides similar to synthetases (similar to NRPS), allowing more significant production of microperfuranone and the discovery of dehydrocroperfuranone, demonstrating the utility of the dLbCas112aD156R-VPR variant for CRISPR/Cas9 under culture conditions [135]. Previously, Nødvig et al. (2015) developed a methodology based on CRISPR/Cas9 for use in filamentous fungi based on gRNA-guided mutagenesis. The system consists of four CRISPR/Cas9 vectors equipped with fungal markers that facilitate selecting a wide range of fungi [132]. New CRISPR/Cas9 variants that are increasingly efficient in editing fungal genomes have been reported, such as the ribonucleoprotein-based CRISPR/Cas9 variant and methodologies based on the in vivo expression of Cas9 [136]. The establishment of genome editing mediated by the CRISPR/Cas9 system in economically important filamentous fungi is becoming increasingly relevant and exciting in industrial biotechnology and agriculture [137,138].
CRISPR-based gene editing systems such as the CRISPR interference system (CRISPRi) and CRISPR activation strategy (CRISPRa) are used for gene editing. These two systems, CRISPRi and CRISPRa, are used in gene editing strategies in phytopathogenic fungi [139]. Both methods and their variants have allowed a greater understanding of the genes of phytopathogenic species and their regulations through the manipulation of their genomes, also used in novel gene editing methodologies such as excision DNA, base editing, and epigenetic modifications [140,141].
CRISPR/Cas9 transcriptional activity is a powerful system for overexpression of target genes [142], whose efficiency is based on the enzymatic fusion or the transcriptional activation domain with dCas9; dCas9 is a variant of CRISPR/Cas9, consisting of an alteration in the domain of the Cas9 enzymes that decreases the efficiency in DNA breaks. dCas9-mediated transcriptional regulation occurs at the elongation or transcriptional points, reducing the amount of mRNA through competitive inhibition. Interference efficiencies of up to 90% have been reported in eukaryotic cells [143]. The complex formed with dCas9 facilitates the assembly of the RNA transcriptional machinery in a specific target guided by gRNA. This complex potentially increases the expression of genes associated with the biosynthesis of secondary metabolites [144].
The production of gene-associated host-specific metabolites is still largely unknown. However, developing new methodologies based on CRISPR/Cas9 may help elucidate the compounds produced by various genes and the subcellular localization of proteins in phytopathogenic fungi that regulate the transport of nutrients across multiple target fungi to inhibit their growth [145,146]. The CRISPR/Cas9 system has enabled targeted mutagenesis efficiently in plants to improve resistance to fungal diseases [147]. In this context, two different strategies have been used to induce disease resistance in plants with CRISPR/Cas9: (i) the pathogen-gene method, which encompasses engineering a sgRNA into the plant chromosome that directs Cas9 to target a specific pathogen gene, thus hampering pathogenesis, (ii) the plant-gene method which uses a sgRNA that targets endogenous plant genes involved in pathogen interactions and modifies them to either improve the host immune response, or to interfere with the host-recognition pathway of the pathogen [148].
CRISPR/Cas9-induced mutations of disease susceptibility (S) genes have generated resistance to pathogens, including fungi, oomycetes, bacteria, and viruses [149]. However, genome editing-mediated plant resistance to Alternaria species remains relatively unexploited. In a recent review, Dort et al. (2020) gave an overview of CRISPR/Cas systems and their adaptation to the CRISPR/Cas9 technology in plant pathology [147].

RNAi in the Control of Alternaria
Additionally, methodological strategies have been developed based on RNA interference (RNAi), which positively influenced the plants' defense mechanisms against various phytopathogenic fungi [15]. Genetic control methods are promising as emerging biocontrol alternatives through molecular studies, leading to genetic information of fungal species using 'omic' technologies [150]. RNAi is a biological gene silencing mechanism involving the degradation of specific sequences of mRNA mediated by double-stranded RNA, applied in functional gene research and genetic modification in plants. Gene suppression by target RNA silencing plays a critical role in studying phytopathogenic fungal control and host-induced gene silencing through RNAi-generated signals in the plant [151]. Additionally, a spray gene silencing methodology has been reported that protects plants from diseases transmitted by phytopathogenic fungi through direct spraying of dsRNA-targeted resistance genes onto plant tissue, which is an excellent alternative to the use of synthetic or rudimentary fungicides [152].
The mechanism of host-induced gene silencing through RNAi-generated signals in the host plant requires dsRNA expression of specific sequences in the host plant and silencing of target genes [153]. The dsRNA expression implies the transformation of the host plant through the construction of a hairpin dsRNA containing the gene of the target pathogen. The degradation of the target pathogen mRNA occurs through the generation of small RNAi homologous to the target RNA, which protects plants from diseases transmitted by phytopathogenic fungi [154,155].
These induced resistance techniques are well studied and successfully applied to control various necrotrophic fungi and oomycetes through the expression of endogenous fungal genes to stimulate increased resistance to diseases transmitted by phytopathogenic fungi [156].
The techniques of resistance induced in host plants vary according to the variations in the RNAi pathways. RNAi pathways differ in the RNA sources and the mechanisms by which gene silencing is achieved. However, they all follow similar processing steps and are triggered by a dsRNA molecule. The processing of dsRNA generates RNA fragments of 21-25 bp (sRNA) through Dicer enzyme processing. Then, the fragment is incorporated into a protein complex known as the RNA-induced silencing complex (RISC) ( Figure 6) [157,158]. The mechanism of host-induced gene silencing through RNAi-generated signals in the host plant requires dsRNA expression of specific sequences in the host plant and silencing of target genes [153]. The dsRNA expression implies the transformation of the host plant through the construction of a hairpin dsRNA containing the gene of the target pathogen. The degradation of the target pathogen mRNA occurs through the generation of small RNAi homologous to the target RNA, which protects plants from diseases transmitted by phytopathogenic fungi [154,155].
These induced resistance techniques are well studied and successfully applied to control various necrotrophic fungi and oomycetes through the expression of endogenous fungal genes to stimulate increased resistance to diseases transmitted by phytopathogenic fungi [156].
The techniques of resistance induced in host plants vary according to the variations in the RNAi pathways. RNAi pathways differ in the RNA sources and the mechanisms by which gene silencing is achieved. However, they all follow similar processing steps and are triggered by a dsRNA molecule. The processing of dsRNA generates RNA fragments of 21-25 bp (sRNA) through Dicer enzyme processing. Then, the fragment is incorporated into a protein complex known as the RNA-induced silencing complex (RISC) (Figure 6) [157,158]. In recent years, genetically improved cultivars with high levels of disease resistance through the use of RNAi have been one of the alternative methods to control diseases transmitted by phytopathogenic fungi. For example, Thakur et al. (2020) reported Ac-CHS7 gene silencing associated with Alternaria leaf spot disease in Cyamopsis tetragonoloba [160]. Silencing the posttranscriptional gene AcCHS7 associated with mycelial growth and conidiation of Alternaria cyamopsidis is an efficient approach to controlling Alternaria. In the same way, Zhai et al. (2018) used RNAi to silence the tomato PR5 gene for resistance to A. alternata [161]. Zhang et al. (2021) identified two new proteins (MdPR10-2 and MdPR10-1) associated with the pathogenesis of A. alternata f. sp. mali. The two proteins interact with the resistance protein MdRNL2, consisting of a nucleotide-binding domain and leucine-rich repeats (CC-NB-LRRs) interacting with the protein MdRNL6. The two proteins form an MdRNL6-MdRNL2 protein complex which grants resistance to A. alternata f. sp. mali attack in Malus x domestica [162]. The production of MdPR10-2 and MdPR10-1 has been re- Figure 6. Graphical summary of cellular RNAi silencing in the mRNA target. The cell generates RNAi (sRNA) from the degradation of dsRNA. A guide chain of RNAi is incorporated into the RISC that subsequently binds to an mRNA complementary sequence, leading to degradation [159].
In recent years, genetically improved cultivars with high levels of disease resistance through the use of RNAi have been one of the alternative methods to control diseases transmitted by phytopathogenic fungi. For example, Thakur et al. (2020) reported AcCHS7 gene silencing associated with Alternaria leaf spot disease in Cyamopsis tetragonoloba [160]. Silencing the posttranscriptional gene AcCHS7 associated with mycelial growth and conidiation of Alternaria cyamopsidis is an efficient approach to controlling Alternaria. In the same way, Zhai et al. (2018) used RNAi to silence the tomato PR5 gene for resistance to A. alternata [161]. Zhang et al. (2021) identified two new proteins (MdPR10-2 and MdPR10-1) associated with the pathogenesis of A. alternata f. sp. mali. The two proteins interact with the resistance protein MdRNL2, consisting of a nucleotide-binding domain and leucine-rich repeats (CC-NB-LRRs) interacting with the protein MdRNL6. The two proteins form an MdRNL6-MdRNL2 protein complex which grants resistance to A. alternata f. sp. mali attack in Malus x domestica [162]. The production of MdPR10-2 and MdPR10-1 has been reported to significantly enhance the induced resistance in Malus domestica against leaf spot disease of four strains of A. alternata f. sp. mali: BXSB5, ALT1, BXSB7, and GBYB2. It has been suggested that this resistance occurs through the resistance induced in Malus domestica against A. alternata infection through the suppression of aldose-6-phosphate reductase (A6PR), generated by the regulation of 56 nucleotide-binding genes [163]. The overexpression of the resistance protein encoded by the MdNLR16 gene in Malus domestica significantly improves the control of A. alternata disease mediated by the overexpression of the HRIP1 gene. Transcriptional regulation of MdNLR16 occurs through binding MdWRKY79 to the MdNLR16 promoter in response to sorbitol. Thus, sorbitol regulates resistance to A. alternata infection by expressing the MdNLR16 gene. Given the urgent need to increase synergy in sustainable management strategies against crop losses caused by phytopathogenic fungi, RNAi mechanisms play a fundamental role in increasing host resistance [164]. Phytopathogenic gene control strategies through RNAi mechanisms are interesting approaches since these are conserved mechanisms in all kingdoms, considered eco-friendly fungi control alternatives.

Control of Alternaria Phytopathogenicity through Regulation of Transcriptional Factors
The study of transcription factors in phytopathogenic fungi is key to regulating their pathogenicity. Transcription factors are proteins that bind to specific DNA regulatory sequences, which regulate the flow of information from DNA to messenger RNA (mRNA) [165]. Transcription factors as regulatory elements in phytopathogenic fungi play an essential role in their pathogenicity. Their ubiquity has been reported in many phytopathogenic species through fungal genome annotation [166]. Families of transcription factors in species of phytopathogenic fungi are classified according to their DNA-binding domains, which are involved in multiple stages related to infection [167].
Transcription factors consist of specific regulatory proteins that act as intermediaries in the expression of many genes associated with critical biosynthetic processes [168]. Transcription factors in fungi use cofactors such as Zn 2+ , as in the case of the Zn 2+ Cys6 transcription family, which is specific in families of phytopathogenic fungi, mainly in ascomycetes [169]. In this context, Chen et al. (2021) reported six transcription factors involved in the pathogenicity of the A. alternata mandarin pathotype (AaAreA, AaAreB, AaLreB, AaLreA, AaNsdD, and AaSreA). The study revealed a significant decrease in pathogenicity after suppressing the transcription factors AaAreA, AaAreB, AaLreB, AaLreA, AaNsdD, and AaSreA, which significantly reduced virulence. Comparative transcriptome analysis between mutants with deletions in these transcription factors revealed alterations in the expression of genes linked to oxidoreductase activity, secondary metabolism, and amino acid metabolism [170]. In addition, Gai et al. (2022) identified genes involved in transcription factors and critical metabolic pathways by analyzing the A. alternata transcriptome, enabling the analysis of the regulatory and biological roles of genes involved in the transcriptional factor bZIP by mutagenesis [171].
Additionally, Chung et al. (2020) reported the critical aspects of the transcriptional regulator SreA in Alternaria alternata strains [12]. SreA is linked to fungus growth and ACR toxin production, which generate necrotic spots in the vegetative tissue. Mutant strains lacking the transcriptional regulator SreA showed defects or severe alterations in the development and increased susceptibility in the fungal cell wall. Recently, Yang et al. (2022) identified and characterized the putative calcineurin-responsive transcription factor AaCrz1 in A. alternata and suggested that it might regulate the early stage of A. alternata colonization [172]. Fungal pathogenicity is known to evolve as a function of transcription factors, which act as fundamental regulators of gene expression and are associated with the coordinated regulation of molecular virulence [173]. The evolution of transcription factors in fungal pathogenicity is a practical phylogenetic resource to enhance virulence investigations on key pathogen lineages.

Conclusions
In this review, we presented recent literature about eco-friendly methods for controlling the pathogenicity of Alternaria spp. We reviewed the available literature on essential oil and biopolymers methods, antagonist microorganisms, with particular emphasis on molecular-based methods such as CRISPR/Cas9, RNAi, and regulation of transcriptional factors available for Alternaria species. Despite the scarce literature, there is a growing interest in applying greener alternatives to control the diseases caused by Alternaria species, such as coatings of essential oils and microbial antagonists, which are safer and do not present environmental or safety concerns. In recent years, more Alternaria species have been identified based on molecular studies and the genome assembly according to the mycotoxins in infected hosts. The knowledge and understanding of the infection processes with the mycotoxins produced are clues for developing gene-editing strategies such as RNAi and CRISPR/Cas. Additionally, the information related to transcription factors associated with the mycotoxin genes is the clue for the editing strategy, as some studies have revealed. However, despite all the literature reviewed on gene-editing techniques for Alternaria species, the information is still scarce. Therefore, future research collecting genetic information on phytopathogenic species must expand the strategy. The development of new CRISPR/Cas9 systems and RNAi allows the expansion of knowledge about the biosynthetic potential of genes of interest in phytopathogenic species. In this way, it will be possible to improve the synergy of biological control alternatives through their phytopathogenic genes, substituting chemical methods to control phytopathogenic species, generating a sustainable alternative to preserve health and reduce the global incidence.