Interaction between Cocoa and Witches’ Broom Disease: Comparison
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Invaluable methods and resources have been explored to understand the molecular biology of M. perniciosa and fungi-host interactions, it is still important to determine how the biotrophic phase is maintained in M. perniciosa, and at the molecular level, to ascertain how their hosts contribute to the end of this phase of WBD. Comprehending the transition from biotrophic to necrotrophic phase is crucial for control of the disease, as well as for the development of resistant hosts.

  • Theobroma
  • witches’ broom
  • molecular interaction

1. Introduction

“All you need is love. But a little chocolate now and then doesn’t hurt”, is a famous saying by the American cartoonist Charles Schulz. In the field of science, the production of chocolate and cupulate, the main products made from plant species of the genus Theobroma, are threatened by witches’ broom disease (WBD) caused by Moniliophthora perniciosa. Despite this scenario, the Zion Market Research Report [1] indicates that the international chocolate trade was worth of US$ 136.01 billion in 2020 and is forecast to generate revenue of around US$ 192.12 billion in 2028.
Despite this optimistic international outlook, the devastating fungus, M. perniciosa reduced the production of the main raw material, the seeds, by approximately 190,000 tons between 2017 and 2020 [2]. In addition, ICCO [2] warns that the fall in world cocoa production resulting from less favorable weather conditions and increased presence of diseases that affect cocoa trees, such as WBD, will cause a 6.8% decline in real revenue in 2022 (after considering high inflation).
WBD was reported for the first time in Suriname in 1985. Soon thereafter, its presence was also reported in the Amazon region. However, in Brazil, more precisely in the south of the state of Bahia, it was only described in 1989 [3,4][3][4]. The geographic distribution of the disease is shaped by the physiology of the causal agent under different climate conditions [5].
WBD is a tropical disease resulting from the interaction between evergreen trees and the fungal pathogen M. perniciosa. The fungus is hemibiotrophic, with a very peculiar life cycle, because the majority of plant diseases caused by this class of fungi have two infection phases: (i) asymptomatic biotrophic phase (transient); and (ii) necrotrophic phase. This does not apply to M. perniciosa. The biotrophic phase is symptomatic and long-lasting, while less abundant hyphae remain in the apoplast, appropriating the nutrients available there, manipulating the host’s metabolism to generate greater availability of nutrients. This phase lasts an average of 60 days in the living tissues of the hosts. Then, at around 90 days of the molecular battle, the infected tissue becomes necrotic. The fungus undergoes dikaryotization and produces clamp connections, characterizing the second phase of the life cycle: the necrotrophic phase. At this stage, the fungus invades the intracellular space and undergoes primary homothallism as part of its reproductive process [6,7,8,9,10,11][6][7][8][9][10][11].
After the elucidation of the life cycle of M. perniciosa, studies were performed to understand its action mechanism. The infection and development of the fungus were orchestrated in favorable environmental conditions and the nutritional status of the host, resulting in physiological, histological and metabolic changes [12,13,14,15,16][12][13][14][15][16].
The molecular action mechanisms of the fungus have only recently become known, when the genomic sequences of M. perniciosa [10] and its main host, Theobroma cacao [17,18][17][18], were published, along with availability of the dual transcriptome M. perniciosa and Theobroma cacao [11]. These developments have brought the possibility of more detailed molecular studies of these organisms and their mechanisms of action.
Since then, several other studies have been published analyzing how biotrophy is maintained for so long in M. perniciosa, what the molecular mechanisms involved in the necrotrophic phase are, and the participation of hosts in signaling the end of these fungal phases [19,20,21,22,23][19][20][21][22][23].
In summary, the genome and dual transcriptome of M. perniciosa have already been identified, as well as its mitochondrial genome, its genetic variability, and polymorphic regions of the chromosomes [10,11,24,25,26][10][11][24][25][26]. Biochemical modifications of hosts when infected by M. perniciosa have also been investigated [27]. Cultivation protocols have been established, such as the artificial cultivation in cookies that allow the production of basidiocarps [28], followed by molecular studies of the fungus’ necrotrophic phase based on this type of cultivation [23,29][23][29]. With technological advances, improvements in the cultivation and manipulation techniques of the fungus have been achieved [30,31][30][31]. Identification of fungal genes through EST libraries has also occurred [32]. The challenge of in vitro production of the fungus in the biotrophic phase has been overcome [9]. Fungal protein profiles have been traced and key proteins related to pathogenicity and necrosis have been identified [21,22,23,33,34][21][22][23][33][34].

2. Brazil Leads in Production of Knowledge about M. perniciosa

The main host of the fungus M. perniciosa is Theobroma cacao, in terms of economic value. This host is an arboreal, perennial plant that prefers a tropical climate. Upon infecting the host, the fungus causes witches’ broom disease (WBD). WBD was first reported in Suriname in 1895. At that time Brazil was the second-largest producer of cocoa beans in the world. Although there were records of the endemicity of this disease in the Amazon region since the 19th century, including the northern region of Brazil, it was only in 1989 that the disease was detected in cocoa plantations in southern Bahia state. Since then, Brazil’s production has declined to seventh position in the world [61][35]. Since 1959, studies have been conducted seeking to describe and understand WBD. Brazilian researchers have focused on the search for varieties resistant to the disease as well as on the understanding of the interaction between M. perniciosa and its hosts, producing knowledge at the level of functional genomics, including the proteome. The results indicate that the studies between 1959 and 2006 were based on strategies of classical genetic improvement to obtain resistant cultivars. With the results of these studies, Brazilian cocoa farming began showing signs of recovery, although this was not enough, because the new cultivars at that time had a narrow genetic base, i.e., they descended from a single source of resistance, the Scavina-6 variety [4]. In addition, the pathogen has high genetic variability between generations and outgrows host resistance. With technological advances, studies applying sequencing technologies to investigate the fungus and its interaction with hosts revolutionized the understanding of their molecular biology [10[10][11][17][18][25][26],11,17,18,25,26], explaining the significant increase in publications from 2007 onwards. The sources of the publications on the molecular biology of M. perniciosa and its interaction with hosts allow inferring the types of journals that researchers seek to publish their studies. According to Barrios et al. [62][36], the set of journals is organized in descending order in relation to the production of studies on a given theme, from this, one can identify a group of journals that deal basically with a theme, as done in the present study. The production of scientific knowledge about the molecular biology of M. perniciosa in Brazilian research institutions is impressive. Most of these institutions collaborate with each other, which strengthens this field of study in the country and helps forge partnerships among researchers. Thus, the range of information generated from these different studies is expanded, offering opportunities for a better understanding of the pathosystem, with emphasis on Theobroma cacao and M. perniciosa.

3. The Peculiar Battle of a Hemibiotrophic Fungus and Its Hosts

Moniliophthora perniciosa is a hemibiotrophic basidiomycete and the causal agent of WBD [63][37]. Plant diseases caused by fungi with hemibiotrophic life cycle present a brief asymptomatic biotrophic stage, followed by necrosis of the plant, as exemplified by the fungi Magnaporthe oryzae and Colletotrichum graminicola [64,65][38][39]. The life cycle of M. perniciosa is characterized by a symptomatic biotrophic phase of long duration (lasting more than 60 days in the living tissues of the hosts). Initially, favorable environmental conditions, such as optimal humidity, temperature, and wind strength, facilitate the dispersal of unicellular basidiospores, which adhere to rapidly growing meristematic tissues. The germination time of basidiospores is different between resistant and susceptible genotypes, being shorter (2 h) in the former, and longer in the latter (4 h). In this initial phase of the disease and fungal life cycle, an enzymatic arsenal and mechanical traction are aroused by the fungus to achieve successful penetration and morph into the biotrophic phase [50][40]. In the biotrophic phase of M. perniciosa, still with uninucleate hyphae, the fungus invades the plant tissue and grows in the intercellular region of the host, because unlike other hemibiotrophic phytopathogens, M. perniciosa does not yet have structures for nutrient absorption, such as invasive hyphae or haustoria. In turn, the hosts present hypertrophic and hyperplastic anomalous branches and parthenocarpic fruit formation, morphological changes that Mondego et al. described as characteristic of this phase of the disease. In this case, M. perniciosa depends on the nutrients available in the apoplast of the plant tissue, spending a period of 30 to 60 days in this region [10]. The apoplast is the first cellular space that the fungus appropriates. Little is known about the molecular tools that the host makes available, and the fungus appropriates to overcome this barrier. Regarding the apoplast-fungus fluid interface, only Barau et al. investigated the dynamics of carbon in the apoplast fluid in order to start the end of the biotrophic phase. They observed that glucose and fructose levels were higher in the apoplastic fluid of infected plants in the first 25 days of WBD development, and this increase in glucose and fructose was correlated with a decrease in sucrose levels, suggesting increased activity of invertases [19]. Other authors pointed out that this increase in sugars in the host may represent a plant response to infection [27]. Furthermore, it is believed that M. perniciosa manipulates the host’s metabolism to increase the availability of nutrients from the apoplast, ensuring the maintenance of its life cycle [11]. The systematized data from the studies indicated that M. perniciosa can grow and develop in the biotrophic phase, employing different plant carbon sources, mainly sucrose, fructose, glucose, and glycerol, the latter produced as a by-product of lipid degradation [11]. Another carbon source found to be important is methanol. The studies reported that methanol can be an optional carbon source during the biotrophic phase of M. perniciosa. According to these studies, carbon obtained from hosts regulates the developmental transitions of WBD [9,11,13,19,54][9][11][13][19][41]. Other hemibiotrophic fungi, for example, those of the genus Colletotrichum, also use glycerol as an energy source for nutrient transfer from infected plants [66][42]. On the other hand, the host uses nonspecific mechanisms, such as an increase in alkaloids, phenolic compounds and tannins, to try to eliminate the fungus, but without success, since the disease is not avoided, although these compounds can inhibit the germination of basidiospores and cause alteration in the germ tube morphology of the biotrophic phase of M. perniciosa [27]. To understand the specificity of the modulation of these secondary metabolites in the host—M. perniciosa interaction Scarpari et al. and Gesteira et al. warn about the need for additional biochemical studies, as well as detailed analysis of gene expression of these metabolic routes, to contribute to the understanding of the transition mechanisms of the fungus from the biotrophic to the necrotrophic phase and relate them to the biochemical changes that occur in the broom stage green [27]. In the literature, few studies address the biochemical interaction of M. perniciosa and its hosts. Only 30% of the works with this strategy are systematized here, confirming the existence of a gap in the understanding of secondary metabolites and whether this response comprises a specific mechanism or a plant response to any biological stressor. Meinhardt et al. performed in vitro studies of M. perniciosa and observed that after 60–90 days the fungus develops connecting clamps and dicariotized hyphae to invade the intracellular space, and that the rapid change to binucleated mycelia with connecting clamps is characteristic of the necrotrophic phase of this fungus [9]. This change from uninucleate to binucleated mycelia occurs without previous reproduction among compatible individuals, since M. perniciosa is a primary homothallic fungus [10]. Calcium oxalate crystals have been shown to play a role as a virulence factor in the fungus, affecting the success of M. perniciosa infection in the host. On the one hand, the crystals that appear in the necrotrophic mycelium are formed from calcium ions removed from the pectin of the host cells, facilitating their degradation [51,54][41][43]. On the other hand, according to Ceita et al., intracellular growth is accompanied by the development of calcium oxalate crystals, which were found in susceptible uninfected plants, and increased in number after infection with M. perniciosa [12]. In resistant plants, these crystals were not observed, so the authors proposed a new role for calcium oxalate of signaling susceptibility to WBD. Furthermore, Dias et al. observed that the resistant genotype accumulated fewer calcium oxalate crystals and these dissolved in the early stages of infection, giving way to the accumulation of H2O2 [56][44]. Most studies have focused on understanding the behavior and interaction of the fungus with its hosts in the biotrophic phase. Few have examined the necrotrophic phase, perhaps because of the goal of mitigating the symptoms of the disease while still in the biotrophic phase, and because of the data indicating that the green broom phase is a point of compromise in disease progression [11]. However, researchers who have investigated the necrotrophic phase admit that the arrival at the dry broom stage is a slow and adaptive process to the hostile environment, where the host expresses its arsenal of defense. According to Pungartnik et al., a gradual exhaustion of the biochemical mechanisms of the fungus occurs, while necrotic action is induced, and rapid intracellular growth takes place [14]. At that stage, M. perniciosa already has dikaryotic hyphae penetrating the host, generating necrosis of plant cell tissues, i.e., parallel to the death of the infected plant tissue, which occurs after 90 days of infection. M. perniciosa completes its cycle in an increasingly inhospitable environment, with alternating wet and dry periods. The fungus produces basidiomes that release basidiospores, restarting the WBD cycle [14,67,68,69][14][45][46][47]. Sena et al. studied the development of M. perniciosa in resistant and susceptible genotypes of Theobroma cacao. They observed the same pattern of fungal invasion in both genotypes, but at a slower rate in the resistant genotype than in the susceptible one. In the resistant genotype, the beginning of green broom was evident only as of week 5, while in the susceptible genotype this condition was observed from week 3 after inoculation. In addition, the resistant genotype produced smaller brooms, less hypertrophied cortex and phloem, shorter and thinner intercellular hyphal segments, and overall lesser pathogen proliferation [50][40]. Barau et al. reported a curious feature of WBD in Theobroma cacao: when its tissues are necrotized and dead, they remain attached to the plant for a long time [19]. It is believed that this behavior is advantageous to the fungus because it has first access to the biological resources contained in the dead tissues and neutralizes any competition with other microorganisms. Moreover, remaining in dead tissues increases the probability of spore dispersion, including to nearby living tissues, guaranteeing its life cycle in an efficient and peculiar way. This intriguing characteristic of M. perniciosa in dead cocoa tissues was also reported by Purdy and Schmidt [67][45], Scarpari et al. [27] and Meinhardt et al. [9]. Among the research strategies used in the studies reviewed here, analysis of molecular markers was the most-used technique, more so in the hosts than the fungus M. perniciosa. Among the different classes of molecular markers, there were five leading ones (as cited in the results section), only pertaining to two hosts of the fungus, Theobroma cacao L. and Theobroma grandiflorum. In general, these studies aimed to characterize molecular markers to identify genetic variability and resistance genes in the hosts. These studies were published between 2005 and 2011, when the genome of the pathosystem (fungus x host) had not yet been published. Indeed, the molecular markers for Theobroma grandiflorum were only described in 2016 [70][48]. This shows the wide interest in this technique, so SSR or SSR-EST markers are well characterized for some cacao genotypes [71,72,73,74,75,76,77,78] (Supplementary Table S2)[49][50][51][52][53][54][55][56]. When the overall goal of identifying molecular markers in hosts is to develop resistant or high-yielding varieties, reseauthorchers claim that this can be accelerated using marker-assisted selection (MAS), which, associated with the analysis of quantitative trait loci (QTL) that control resistance genes, can be used to select varieties of interest [79,80,81][57][58][59]. However, these associated methods lack refinement, since they rely on intensive genotyping with SNP molecular markers, either via next-generation sequencing (NGS) or by microarrays. However, they are more robust [80,82,83][58][60][61]. Santana et al. analyzed isolates of M. perniciosa using RFLP [4]. However, studies such as these this are scarce, even when prioritizing other classes of markers, unlike host studies. For M. perniciosa, SSR molecular markers were first characterized by Gramacho et al. [84][62] and Silva et al. [85][63] in different isolates. The RAPD molecular markers were also investigated by Andebrhan and Furtek [86][64]. However, considering the co-evolution of M. perniciosa and hosts, it is necessary to investigate the construction of a map of molecular markers for the pathogen, perhaps using the class of SNPs, since they identify single-base polymorphisms.

4. Structural Genomics of the Causal Agent of Witches’ Broom

Descriptions of the genome sequences of the causal agent of WBD, M. perniciosa, published in recent years [10[10][25][26],25,26], along with the genome of one of its main hosts of socioeconomic importance, Theobroma cacao [17[17][18][65],18,87], and the dual transcriptome (M. perniciosa and Theobroma cacao—Atlas Transcriptome) revealed by Teixeira et al. [11], opened the way for application of structural genomics to obtain more detailed molecular information about this pathosystem. Another important development is genetic manipulation through techniques based on CRISPR/cas9 and epigenetic systems, to shed light on problematic organisms such as M. perniciosa. To complete its life cycle, M. perniciosa wages a molecular battle with its host, activating or repressing genes crucial for its success. Genes associated with pectinolytic metabolism are positively regulated in the initial phase of the disease [11], confirming the colonization of M. perniciosa in the host’s mid-layer, rich in pectin, as well as the degradation of this polysaccharide in the initial green stage. In entirely necrotrophic fungi, the pectin degradation activity is more common and well characterized, as is the case of the fungus Botrytis cinerea [88][66]. This reiterates how peculiar the biology of M. perniciosa is. In the process of degradation and demethylation of pectin, methanol is released, used as an energy source, and enters a cascade to form carbon dioxide. In this process, one of the byproducts is glyceraldehyde 3-phosphate, which can be a precursor for the synthesis of oxaloacetate. Oxaloacetate removes calcium ions from the pectin structure, forming calcium oxalate and allowing further enzymatic degradation of pectin by plant cell wall degradation enzymes [54,89][41][67]. This event is particularly evident in susceptible hosts. Previously, studies predicted the formation of CaOX crystals only in the early stage of the disease [12], but its production has also been observed in necrotrophic mycelia of M. perniciosa [51,56][43][44]. The fungus initially grows in the intercellular space of the plant cell, invading the apoplast. In this region, during the proliferative phase of the disease, Barau et al. found a significant increase in glucose and fructose levels in parallel with a reduction in sucrose levels, suggesting upregulation of genes related to cell wall invertases, as well as the accumulation of fungal and plant enzymes for the maintenance of apoplastic hexose from sucrose breakdown [19]. According to these findings, infection initially leads to the accumulation of hexose, followed by consumption of the soluble carbohydrates in the apoplastic fluid. Teixeira et al. reported that M. perniciosa seems to manipulate the metabolism of the host to increase the availability of nutrients in the apoplasts, triggering the formation of monokaryotic hyphae, which favors the availability of more nutrients to the fungus [11]. This characteristic supports the upregulation of fungal genes, identified to encode oligopeptide and monosaccharide transporters, proteases and asparaginase in the initial phase of the disease (green broom). This process degrades apoplastic proteins and enables use of the final peptides [11]. Host manipulation for nutrient acquisition has also been observed in other phytopathogens, such as Colletotrichum gloeosporioides f. sp. malva, which uses glycerol for nutritional transfer [66][42]. Still at the green broom stage, genes encoding antioxidant enzymes such as superoxide dismutase and catalase are upregulated in M. perniciosa according to the studies reviewed here. These enzymes detoxify ROS, such as superoxide anions (O2−), hydroxyl radicals (OH) and hydrogen peroxide (H2O2) and are produced by plants to prevent pathogen invasion and increase stress tolerance [90][68]. In contrast, hosts also activate the gene arsenal of their antioxidant system to develop metabolites and enzymes, such as peroxidases, that prevent entry into the fungus’ intracellular space [91][69]. In the Transcriptome Atlas of Teixeira et al. [11], the pathogenesis related (PR) genes of M. perniciosa are among the most highly expressed genes during interaction with the host Theobroma cacao, with emphasis on MpPR-1, which acts to detoxify lipid toxins produced by the host, thus protecting the fungus. This protein belongs to the class of cysteine-rich secretory proteins, including antigen 5 and pathogenesis-related 1 (CAP), which according to Darwiche et al. are implicated in fungal virulence and immunosuppression [92][70]. Another class of microbial PR whose genes are over-represented in the green broom stage are the protein cerato-platanins (CPs). CPs prevents the plant’s fungal recognition receptors from detecting it, impairing host defense, and favoring success of the biotrophic stage [11]. Also, when studying biotrophic mycelium, Thomazella et al. identified increased expression of the Mp-aox gene accompanied by high mitochondrial alternative oxidase (AOX) enzyme activity in M. perniciosa during the infection process [15]. This suggests the important role of this enzyme during the biotrophic phase. AOX protects the fungus against the harmful effects of mitochondrial respiratory chain byproducts, among them nitric oxide (NO), and further regulates the transition to the neurotrophic phase of WBD. Researchers have hypothesized that although hosts, in response to fungal attack, produce high concentrations of H2O2, triggering programmed cell death (PCD) of infected tissue cells as a defense mechanism, this actually favors the fungus since it makes more nutrients available by stimulating the phase change (biotrophic–necrotrophic) of WBD [12]. A few studies have identified gene expression of M. perniciosa in the necrotrophic phase. Examples are Lanver et al. and Basse et al., who studied the phytopathogen Ustilago maydis, a biotrophic fungus with a dimorphic lifestyle [93,94][71][72]. In its biotrophic phase, it is not pathogenic, unlike M. perniciosa. This indicates the need to understand the action of the fungus as early as possible in its biotrophic phase, to prevent development of the necrotrophic phase. Key genes related to the necrotic phase of the disease are well described as to their expression in the infection process. Examples are the genes that code for the necrosis and ethylene inducing proteins (NEPs). These proteins act in the extracellular medium and were first identified in the fungus Fusarium oxysporum, which also has the ability to induce necrosis in its hosts [33,95,96,97][33][73][74][75]. The onset of necrosis is parallel to the death of infected host tissues [69][47]. Overall, infection by M. perniciosa causes a general derangement in host metabolism and culminates in the expression of key genes in this molecular battle. The information generated by different molecular studies sheds light on the regulatory networks that control fungal pathogenicity, as well as host defense, by identifying the changes in gene expression regulation that occur in this battle. The results directly impact agricultural production and food security. Large-scale gene expression analyses of plant-pathogen interactions are of great relevance to unveil the molecular basis of a specific disease. However, when it comes to M. perniciosa and its hosts, studies focused on genomic editing using CRISPR/cas9 systems as well as the action of epigenetic mechanisms on gene regulation have not yet been published. Some studies have already described CRISPR-Cas9 genome editing technology for fungi of major socioeconomic importance, such as Magnaporthe oryzae [98][76], Neurospora crassa [99][77], Trichoderma reesei [100][78] and Ustilago maydis [101][79]. In the case of Ustilago maydis, for example, the authors aimed to efficiently disrupt functionally redundant target genes in the corn phytopathogen [101][79]. This same pathway has been paved for some of the main hosts of these phytopathogens. An example is Oryza sativa, which is affected by Brusone’s disease in rice, caused by the fungus Magnaporthe oryzae. The study of [102][80] revealed that when the nlr gene, which confers resistance to the disease by rice, was knocked out via CRISPR-Cas9, resistance in transgenic plants was partially reduced. Unlike rice, for Theobroma cacao, the main host of M. perniciosa, there are no studies of the CRISPR-Cas9 system conferring resistance, since there is no established method. The regulation of gene expression under the effects of epigenetic mechanisms is another field not yet explored involving the M. perniciosa pathosystem and its hosts. The understanding and manipulation of resistance responses of plants with great agronomic importance affected by biotic and abiotic factors from an epigenetic standpoint is already well described for some species [41,103,104,105][81][82][83][84]. In this sense, there are no reports of Theobroma cacao, the main host of M. perniciosa, so there are no methods that aim to identify epigenetic mechanisms associated with resistance to the fungus. There is hence a need to clarify the action of epigenetic mechanisms in the regulation of genes that confer pathogenicity in order to understand their action in the different stages of WBD, and, if possible, to manipulate them. In a study of Ustilago maydis, the authors hypothesized that epigenetic control through histone acetylation would act to control the transcription of genes related to pathogenesis, virulence and growth of the fungus [106][85]. Differences in some molecular responses between resistant and susceptible genotypes of Theobroma cacao have been reported. Almost all of them are related to the antioxidative system of the plant, such as higher amounts of H2O2, oxalic acid and/or ascorbic acid, induction of the genes for oxalate oxidase (G-OXO), germinal-type oxalate oxidase (Glp), and dehydroascorbate reductase (Dhar) [12[12][44],56], as well as higher activity of the APX enzyme [57,91][69][86] in resistant genotypes in the interval from 0 h to three days after infection with M. perniciosa. Another interesting situation, discussed previously, is that resistant genotypes produce much smaller amounts of calcium oxalate crystals in the basal state and these are dissolved during the initial phase of infection, leaving in their place H2O2 [56][44]. This information indicates that the key feature of the resistant genotypes is their detoxification mechanism from the first hours of infection.

5. The Hidden Biotechnological Potential of M. perniciosa Pathosystem Proteins

The publication of the genome of M. perniciosa and its main host, T. cacao, has allowed the integration of proteomics studies, helping resolve the puzzle about WBD. Unlike the genome, the proteome reflects biological processes triggered under different physiological conditions or pathological states [107][87]. Proteins expressed during phytopathogenic interactions have been successfully identified through proteomic analyses [108][88]. Furthermore, biochemical and structural characterization of proteins, whether inferred from the genome or identified in the proteome, contributes to unravel the molecular mechanisms of the battle between M. perniciosa and its hosts. In this regard, 19 M. perniciosa proteins of interest have been characterized in vitro: two NEPs [33], four CPs [109[89][90],110], 11 pathogenesis-related PR-1 proteins [92][70], one Acyl-CoA binding protein [111][91] and one inactive chitinase [55][92]. One of the biggest mysteries surrounding WBD is the transition from the biotrophic to the necrotrophic phase, because it is still unclear which factor induces this change. Initially, the hypotheses pointed to action peculiar to M. perniciosa, so the first proteins to be characterized in vitro were those found in the genome of M. perniciosa, similar to proteins from other fungi that had already been shown to cause necrosis in hosts [33]. The proteins MpNEP1, MpNEP2 and MpCP1 induce necrosis in tobacco and cocoa leaves. Although the genes for these three proteins are expressed in the biotrophic mycelium, infected plants show no visible symptoms for many weeks. This suggests that the proteins need a minimum concentration to cause symptoms in tissues [33]. Furthermore, MpNEPs induce ethylene production in the plant long before necrosis symptoms are visible. Although the effects of MpCP1 and MpNEP2 are different, when subjecting leaves to treatment with both proteins, a necrosis effect similar to that found in naturally infected plants occurred [110][90]. Furthermore, MpNEP2 induced detachment of cell membranes and affected ATP synthesis in tobacco and has also been found to be related to an increase in NO synthesis [112][93]. Thanks to the publication of the M. perniciosa genome, it has been possible to find orthologous gene families that had already been studied in other fungi. One of these families was the cerato-platanins, of which 12 genes were identified. These showed different expression profiles during infection [109][89]. MpCP1, MpCP2, MpCP3 and MpCP5 proteins bind to chitin fragments according to in vitro characterization. MpCP5 blocks the perception of chitin monomers by the plant, while MpCP1, MpCP2, and MpCP3 facilitate the process of hyphal growth, fruiting body formation, and substrate adhesion. In addition, aggregates of MpCP2 were able to promote cellulose fragmentation as well as contribute to pollen tube formation [109][89]. Interestingly, in the genome of M. perniciosa, orthologous genes from other fungi, and also from plants have been identified, such as PR-1 (pathogenesis-related 1) family proteins, involved in fungal virulence and immunosuppression. From this family, 11 members have been identified, revealing that sterol and fatty acid binding is important in WBD progression [92][70]. Finally, a MpChi (inactive chitinase) has been shown to function as a putative pathogenicity factor, preventing chitin-triggered immunity by sequestering immunogenic chitin fragments [55][92]. Hundreds of candidate effector proteins have been identified in silico in the M. perniciosa genome [22]. However, it is still necessary to characterize them in vitro and in vivo for a better understanding of the fungal strategy and the WBD process, as well as to identify targets for control of the disease. Efforts to understand the molecular mechanisms during the development of M. perniciosa through proteomics have been carried out in the last decade. Protein profiling of basidiospores has revealed that most of the identified proteins are related to energetic and oxido-reduction processes, in addition to identifying proteins important to hyphal development and branching [113][94]. In turn, the protein profile of M. perniciosa spores in the germination phase revealed proteins associated with fungal filaments, such as septin and kinesin, in addition to positive regulation of oxidative stress-related proteins, such as SOD and catalase, which possibly help in the detoxification of free radicals within the primary hyphae. Virulence factors, such as polyketide synthase and FapR, were also present in the basidiospores and primary hyphae [21]. Two proteomic studies of the necrotrophic phase of the mycelium were identified. One compared the S and C biotypes [20] and the other compared six developmental stages of the fungus according to mycelium color and development (white, yellow, pink, dark pink; primordium and basidiocarp) [23]. In the first study, proteins involved in virulence, pathogenicity and stress response were identified abundantly in all cultures [20]. In the second one, proteins related to IAA metabolism, cell proliferation and cytoskeleton activity were identified most abundantly in the white mycelium phase, suggesting they are required for mycelial development. In the dark pink phase, proteins were identified related to vesicular transport processes mediated by small GTPase signaling, cell wall biogenesis, stress response and response to nutrient deficiency, which were also associated with the primordium phase [23]. Proteomic studies of M. perniciosa subjected to treatments with leaf washes and with a PR-10 of Theobroma cacao were found. Basidiospores of M. perniciosa germinated in the presence of leaf washes from contrasting genotypes of Theobroma cacao showed a distinct protein profile [114][95]. The authors highlighted the reduced ATP synthase of the germinated basidiospores in the leaf wash of the susceptible genotype, suggesting a shift from aerobic to fermentative metabolism. On the other hand, M. perniciosa hyphae treated with TcPR-10 had positive regulation of proteins related to stress response, detoxification, autophagy, and maintenance of fungal homeostasis [34]. A protein–protein interaction network was constructed from the M. perniciosa proteins reported in some of the papers. The centrality values indicated that among the most important proteins for the regulation of the network are glyceraldehyde-3-phosphate dehydrogenase (phosphorylating) and pyruvate kinase, both of which are in the cluster related to organic acid metabolism and are specifically involved in glycolysis, indicating that obtaining energy by this pathway is a key process for the development of the fungus and the disease. It has already been shown that an increase in the amount of glucose in the infected plant occurs in the initial weeks [19[19][27],27], so it is an important energy source for the fungus during the progression of WBD. Cluster 2 had the second most proteins, related to transport, localization, and the cell cycle. This cluster included proteins involved in the formation of structures during cell division, consistent with the development of the fungus, considering the change from monokaryotic to dikaryotic form in the transition from green broom to dry broom [14]. Interestingly, the protein with the most connections within this cluster, an AAA domain-containing protein (E2L7U8), has an unknown function. Another interesting biological process in the PPI network was the metabolism of phospholipids, formed by proteins that are directly related to phosphatidylserine decarboxylase. This enzyme is involved in the synthesis of phosphatidylethanolamine, a mitochondrial phospholipid whose depletion in yeast causes respiratory dysfunction, defects in the assembly of mitochondrial protein complexes and loss of mitochondrial DNA [115,116][96][97]. Therefore, it can be studied as a molecular target to control the development of M. perniciosa. With regard to the hosts, 10 Theobroma cacao proteins of biotechnological interest have been characterized in vitro: four cystatins [117][98], one TcPR-10 [118][99], one β-1,3-1,4-Glucanase (TcGlu2) [119][100], one PR-4b [120][101], and one osmotin [121][102]. All of them have been shown to have a fungicidal effect on M. perniciosa. TcCYSPR04 [122][103] was also characterized in vitro but showed no significant fungicidal effect. Another protein from Theobroma cacao, the Kunitz-type trypsin inhibitor, was tested in vitro for its biotechnological potential and efficiency, but only as a potential larvicide against Helicoverpa armigera [123][104]. The phylloplane is considered the first battleground for cocoa against M. perniciosa, and the resistant genotype CCN51 has an index of short glandular trichomes two times higher than the susceptible genotype, Catongo [124][105]. Water-soluble compounds from the phylloplane of Theobroma cacao have demonstrated an inhibitory effect on germination of spores of the fungus [114][95]. The proteomics of the phylloplane of the cocoa genotype resistant to WBD revealed proteins related to defense, synthesis of defense metabolites and the metabolism of nucleic acids, demonstrating that proteins and water-soluble compounds secreted to the phylloplane of cocoa participate in the defense against pathogens. In this regard, the expression of a phylloplanin, identified in the genome of Theobroma cacao (TcPHYLL), was demonstrated in leaf trichomes of transformed tobacco plants [125][106]. Recently, the protein profile of contrasting genotypes for resistance to WBD inoculated with M. perniciosa and its controls was studied. This revealed that pathogenesis-related proteins (PRs), proteins related to the regulation of oxidative stress and trypsin inhibitors, and a strong detoxification mechanism are involved in WBD resistance [126][107].

References

  1. Zion Market Research Chocolate Market—Global Industry Analysis 2022. Available online: https://www.zionmarketresearch.com/report/chocolate-market (accessed on 13 November 2022).
  2. ICCO International Cocoa Organization (ICCO). Available online: https://www.icco.org/ (accessed on 30 January 2022).
  3. Pereira, J.L.; Ram, A.; Figueiredo, J.M.; Almeida, L.C.C. Primeira Ocorrência de Vassoura-de-Bruxa Na Principal Região Produtora de Cacau Do Brasil. Agrotrópica 1989, 1, 79–81.
  4. Santana, M.F.; de Araújo, E.F.; de Souza, J.T.; Mizubuti, E.S.G.; de Queiroz, M.V. Development of Molecular Markers Based on Retrotransposons for the Analysis of Genetic Variability in Moniliophthora perniciosa. Eur. J. Plant Pathol. 2012, 134, 497–507.
  5. Auhing Arcos, J.A.; Cedeño Moreira, Á.V.; Saucedo Aguiar, S.; Vera Benites, L.F.; Macías Holguín, C.J.; Canchignia Martínez, H.F. Biodiversity of Ecotypes and Aggressivenss Ranges of Moniliophthora Perniciosa, in Theobroma cacao L. National of the Ecuadorian Coast. Sci. Agropecu. 2021, 12, 599–609.
  6. Delgado, J.C.; Cook, A.A. Nuclear Condition of the Basidia, Basidiospores, and Mycelium of Marasmius perniciosus. Can. J. Bot. 1976, 54, 66–72.
  7. Griffith, G.W.; Hedger, J.N. Dual Culture Of Crinipellis perniciosa and Potato callus. Eur. J. Plant Pathol. 1994, 100, 371–379.
  8. Pereira, J.L.; de Almeida, L.C.C.; Santos, S.M. Witches’ Broom Disease of Cocoa in Bahia: Attempts at Eradication and Containment. Crop Prot. 1996, 15, 743–752.
  9. Meinhardt, L.W.; Bellato, C.d.M.; Rincones, J.; Azevedo, R.A.; Cascardo, J.C.M.; Pereira, G.A.G. In Vitro Production of Biotrophic-Like Cultures of Crinipellis Perniciosa, the Causal Agent of Witches’ Broom Disease of Theobroma cacao. Curr. Microbiol. 2006, 52, 191–196.
  10. Mondego, J.M.; Carazzolle, M.F.; Costa, G.G.; Formighieri, E.F.; Parizzi, L.P.; Rincones, J.; Cotomacci, C.; Carraro, D.M.; Cunha, A.F.; Carrer, H.; et al. A Genome Survey of Moniliophthora perniciosa Gives New Insights into Witches’ Broom Disease of Cacao. BMC Genom. 2008, 9, 548.
  11. Teixeira, P.J.P.L.; Thomazella, D.P.d.T.; Reis, O.; do Prado, P.F.V.; do Rio, M.C.S.; Fiorin, G.L.; José, J.; Costa, G.G.L.; Negri, V.A.; Mondego, J.M.C.; et al. High-Resolution Transcript Profiling of the Atypical Biotrophic Interaction between Theobroma Cacao and the Fungal Pathogen Moniliophthora perniciosa. Plant Cell 2014, 26, 4245–4269.
  12. de Oliveira Ceita, G.; Macêdo, J.N.A.; Santos, T.B.; Alemanno, L.; da Silva Gesteira, A.; Micheli, F.; Mariano, A.C.; Gramacho, K.P.; da Costa Silva, D.; Meinhardt, L.; et al. Involvement of Calcium Oxalate Degradation during Programmed Cell Death in Theobroma Cacao Tissues Triggered by the Hemibiotrophic Fungus Moniliophthora perniciosa. Plant Sci. 2007, 173, 106–117.
  13. Alvim, F.C.; Mattos, E.M.; Pirovani, C.P.; Gramacho, K.; Pungartnik, C.; Brendel, M.; Cascardo, J.C.M.; Vincentz, M. Carbon Source-Induced Changes in the Physiology of the Cacao Pathogen Moniliophthora perniciosa (Basidiomycetes) Affect Mycelial Morphology and Secretion of Necrosis-Inducing Proteins. Genet. Mol. Res. 2009, 8, 1035–1050.
  14. Pungartnik, C.; Melo, S.C.O.; Basso, T.S.; Macena, W.G.; Cascardo, J.C.M.; Brendel, M. Reactive Oxygen Species and Autophagy Play a Role in Survival and Differentiation of the Phytopathogen Moniliophthora perniciosa. Fungal Genet. Biol. 2009, 46, 461–472.
  15. Thomazella, D.P.T.; Teixeira, P.J.P.L.; Oliveira, H.C.; Saviani, E.E.; Rincones, J.; Toni, I.M.; Reis, O.; Garcia, O.; Meinhardt, L.W.; Salgado, I.; et al. The Hemibiotrophic Cacao Pathogen Moniliophthora perniciosa Depends on a Mitochondrial Alternative Oxidase for Biotrophic Development. New Phytol. 2012, 194, 1025–1034.
  16. Argôlo Santos Carvalho, H.; de Andrade Silva, E.M.; Carvalho Santos, S.; Micheli, F. Polygalacturonases from Moniliophthora perniciosa Are Regulated by Fermentable Carbon Sources and Possible Post-Translational Modifications. Fungal Genet. Biol. 2013, 60, 110–121.
  17. Argout, X.; Salse, J.; Aury, J.-M.; Guiltinan, M.J.; Droc, G.; Gouzy, J.; Allegre, M.; Chaparro, C.; Legavre, T.; Maximova, S.N.; et al. The Genome of Theobroma cacao. Nat. Genet. 2011, 43, 101–108.
  18. Motamayor, J.C.; Mockaitis, K.; Schmutz, J.; Haiminen, N.; III, D.L.; Cornejo, O.; Findley, S.D.; Zheng, P.; Utro, F.; Royaert, S.; et al. The Genome Sequence of the Most Widely Cultivated Cacao Type and Its Use to Identify Candidate Genes Regulating Pod Color. Genome Biol. 2013, 14, r53.
  19. Barau, J.; Grandis, A.; Carvalho, V.M.d.A.; Teixeira, G.S.; Zaparoli, G.H.A.; do Rio, M.C.S.; Rincones, J.; Buckeridge, M.S.; Pereira, G.A.G. Apoplastic and Intracellular Plant Sugars Regulate Developmental Transitions in Witches’ Broom Disease of Cacao. J. Exp. Bot. 2015, 66, 1325–1337.
  20. Pierre, S.; Griffith, G.W.; Morphew, R.M.; Mur, L.A.J.; Scott, I.M. Saprotrophic Proteomes of Biotypes of the Witches’ Broom Pathogen Moniliophthora perniciosa. Fungal Biol. 2017, 121, 743–753.
  21. Mares, J.H.; Gramacho, K.P.; Santos, E.C.; Da Silva Santiago, A.; Santana, J.O.; De Sousa, A.O.; Alvim, F.C.; Pirovani, C.P. Proteomic Analysis during of Spore Germination of Moniliophthora perniciosa, the Causal Agent of Witches’ Broom Disease in Cacao. BMC Microbiol. 2017, 17, 176.
  22. Barbosa, C.S.; da Fonseca, R.R.; Batista, T.M.; Barreto, M.A.; Argolo, C.S.; de Carvalho, M.R.; do Amaral, D.O.J.; Silva, E.M.d.A.; Arévalo-Gardini, E.; Hidalgo, K.S.; et al. Genome Sequence and Effectorome of Moniliophthora perniciosa and Moniliophthora Roreri Subpopulations. BMC Genom. 2018, 19, 509.
  23. Gomes, D.S.; de Andrade Silva, E.M.; de Andrade Rosa, E.C.; Silva Gualberto, N.G.; de Jesus Souza, M.Á.; Santos, G.; Pirovani, C.P.; Micheli, F. Identification of a Key Protein Set Involved in Moniliophthora perniciosa Necrotrophic Mycelium and Basidiocarp Development. Fungal Genet. Biol. 2021, 157, 103635.
  24. Rincones, J.; Meinhardt, L.W.; Vidal, B.C.; Pereira, G.A.G. Electrophoretic Karyotype Analysis of Crinipellis Perniciosa, the Causal Agent of Witches’ Broom Disease of Theobroma cacao. Mycol. Res. 2003, 107, 452–458.
  25. Rincones, J.; MazzottiI, G.D.; Griffith, G.W.; Pomela, A.; Figueira, A.; Leal, G.A.; Queiroz, M.V.; Pereira, J.F.; Azevedo, R.A.; Pereira, G.A.G.; et al. Genetic Variability and Chromosome-Length Polymorphisms of the Witches’ Broom Pathogen Crinipellis perniciosa from Various Plant Hosts in South America. Mycol. Res. 2006, 110, 821–832.
  26. Formighieri, E.F.; Tiburcio, R.A.; Armas, E.D.; Medrano, F.J.; Shimo, H.; Carels, N.; Góes-Neto, A.; Cotomacci, C.; Carazzolle, M.F.; Sardinha-Pinto, N.; et al. The Mitochondrial Genome of the Phytopathogenic Basidiomycete Moniliophthora perniciosa Is 109kb in Size and Contains a Stable Integrated Plasmid. Mycol. Res. 2008, 112, 1136–1152.
  27. Scarpari, L.M. Biochemical Changes during the Development of Witches’ Broom: The Most Important Disease of Cocoa in Brazil Caused by Crinipellis perniciosa. J. Exp. Bot. 2005, 56, 865–877.
  28. Niella, G.R.; Castro, H.A.; Silva, L.H.C.P.; Carvalho, J.A. Aperfeiçoamento Da Metodologia de Produção Artificial de Basidiocarpos de Crinipellis perniciosa. Fitopatol. Bras. Brasília 1999, 24, 523–527.
  29. Gomes, D.S.; Lopes, M.A.; Menezes, S.P.; Ribeiro, L.F.; Dias, C.V.; Andrade, B.S.; de Jesus, R.M.; Pires, A.B.L.; Goes-Neto, A.; Micheli, F. Mycelial Development Preceding Basidioma Formation in Moniliophthora perniciosa Is Associated to Chitin, Sugar and Nutrient Metabolism Alterations Involving Autophagy. Fungal Genet. Biol. 2016, 86, 33–46.
  30. Lima, J.O.; dos Santos, J.K.; Pereira, J.F.; de Resende, M.L.V.; de Araújo, E.F.; de Queiroz, M.V. Development of a Transformation System for Crinipellis perniciosa, the Causal Agent of Witches’ Broom in Cocoa Plants. Curr. Genet. 2003, 42, 236–240.
  31. Filho, D.F.; Pungartnik, C.; Cascardo, J.C.M.; Brendel, M. Broken Hyphae of the Basidiomycete Crinipellis perniciosa Allow Quantitative Assay of Toxicity. Curr. Microbiol. 2006, 52, 407–412.
  32. Gesteira, A.S.; Micheli, F.; Carels, N.; Da Silva, A.C.; Gramacho, K.P.; Schuster, I.; Macêdo, J.N.; Pereira, G.A.G.; Cascardo, J.C.M. Comparative Analysis of Expressed Genes from Cacao Meristems Infected by Moniliophthora perniciosa. Ann. Bot. 2007, 100, 129–140.
  33. Garcia, O.; Macedo, J.A.N.; Tibúrcio, R.; Zaparoli, G.; Rincones, J.; Bittencourt, L.M.C.; Ceita, G.O.; Micheli, F.; Gesteira, A.; Mariano, A.C.; et al. Characterization of Necrosis and Ethylene-Inducing Proteins (NEP) in the Basidiomycete Moniliophthora perniciosa, the Causal Agent of Witches’ Broom in Theobroma Cacao. Mycol. Res. 2007, 111, 443–455.
  34. Silva, F.A.C.; Pirovani, C.P.; Menezes, S.; Pungartnik, C.; Santiago, A.S.; Costa, M.G.C.; Micheli, F.; Gesteira, A.S. Proteomic Response of Moniliophthora perniciosa Exposed to Pathogenesis-Related Protein-10 from Theobroma Cacao. Genet. Mol. Res. 2013, 12, 4855–4868.
  35. FAO. (Food and Agriculture Organization) Food and Agriculture Organization of the United Nations. 2020. Available online: https://www.fao.org/statistics/en/ (accessed on 30 January 2022).
  36. Barrios, M.; Borrego, A.; Vilaginés, A.; Ollé, C.; Somoza, M. A Bibliometric Study of Psychological Research on Tourism. Scientometrics 2008, 77, 453–467.
  37. Aime, M.C.; Phillips-Mora, W. The Causal Agents of Witches’ Broom and Frosty Pod Rot of Cacao (Chocolate, Theobroma cacao) Form a New Lineage of Marasmiaceae. Mycologia 2005, 97, 1012–1022.
  38. Münch, S.; Lingner, U.; Floss, D.S.; Ludwig, N.; Sauer, N.; Deising, H.B. The Hemibiotrophic Lifestyle of Colletotrichum Species. J. Plant Physiol. 2008, 165, 41–51.
  39. Perfect, S.E.; Green, J.R. Infection Structures of Biotrophic and Hemibiotrophic Fungal Plant Pathogens. Mol. Plant Pathol. 2001, 2, 101–108.
  40. Sena, K.; Alemanno, L.; Gramacho, K.P. The Infection Process of Moniliophthora perniciosa in Cacao. Plant Pathol. 2014, 63, 1272–1281.
  41. de Oliveira, B.V.; Teixeira, G.S.; Reis, O.; Barau, J.G.; Teixeira, P.J.P.L.; do Rio, M.C.S.; Domingues, R.R.; Meinhardt, L.W.; Paes Leme, A.F.; Rincones, J.; et al. A Potential Role for an Extracellular Methanol Oxidase Secreted by Moniliophthora perniciosa in Witches’ Broom Disease in Cacao. Fungal Genet. Biol. 2012, 49, 922–932.
  42. Wei, Y.; Shen, W.; Dauk, M.; Wang, F.; Selvaraj, G.; Zou, J. Targeted Gene Disruption of Glycerol-3-Phosphate Dehydrogenase in Colletotrichum gloeosporioides Reveals Evidence That Glycerol Is a Significant Transferred Nutrient from Host Plant to Fungal Pathogen. J. Biol. Chem. 2004, 279, 429–435.
  43. Rio, M.C.S.D.; de Oliveira, B.V.; de Tomazella, D.P.T.; da Silva, J.A.F.; Pereira, G.A.G. Production of Calcium Oxalate Crystals by the Basidiomycete Moniliophthora perniciosa, the Causal Agent of Witches’ Broom Disease of Cacao. Curr. Microbiol. 2008, 56, 363–370.
  44. Dias, C.V.; Mendes, J.S.; dos Santos, A.C.; Pirovani, C.P.; da Silva Gesteira, A.; Micheli, F.; Gramacho, K.P.; Hammerstone, J.; Mazzafera, P.; de Mattos Cascardo, J.C. Hydrogen Peroxide Formation in Cacao Tissues Infected by the Hemibiotrophic Fungus Moniliophthora perniciosa. Plant Physiol. Biochem. 2011, 49, 917–922.
  45. Purdy, L.; Schmidt, R. STATUS OF CACAO WITCHES’ BROOM: Biology, Epidemiology, and Management. Annu. Rev. Phytopathol. 1996, 34, 573–594.
  46. Meinhardt, L.W.; Rincones, J.; Bailey, B.A.; Aime, M.C.; Griffith, G.W.; Zhang, D.; Pereira, G.A.G. Moniliophthora perniciosa, the Causal Agent of Witches’ Broom Disease of Cacao: What’s New from This Old Foe? Mol. Plant Pathol. 2008, 9, 577–588.
  47. Teixeira, P.J.P.L.; Thomazella, D.P.T.; Vidal, R.O.; do Prado, P.F.V.; Reis, O.; Baroni, R.M.; Franco, S.F.; Mieczkowski, P.; Pereira, G.A.G.; Mondego, J.M.C. The Fungal Pathogen Moniliophthora perniciosa Has Genes Similar to Plant PR-1 That Are Highly Expressed during Its Interaction with Cacao. PLoS ONE 2012, 7, e45929.
  48. Ferraz dos Santos, L.; Moreira Fregapani, R.; Falcão, L.L.; Togawa, R.C.; Costa, M.M.d.C.; Lopes, U.V.; Peres Gramacho, K.; Alves, R.M.; Micheli, F.; Marcellino, L.H. First Microsatellite Markers Developed from Cupuassu ESTs: Application in Diversity Analysis and Cross-Species Transferability to Cacao. PLoS ONE 2016, 11, e0151074.
  49. Brown, J.S.; Schnell, R.J.; Motamayor, J.C.; Lopes, U.; Kuhn, D.N.; Borrone, J.W. Resistance Gene Mapping for Witches’ Broom Disease in Theobroma cacao L. in an F2 Population Using SSR Markers and Candidate Genes. J. Am. Soc. Hortic. Sci. 2005, 130, 366–373.
  50. Faleiro, F.G.; Queiroz, V.T.; Lopes, U.V.; Guimarães, C.T.; Pires, J.L.; Yamada, M.M.; Araújo, I.S.; Pereira, M.G.; Schnell, R.; Filho, G.A.d.S.; et al. Mapping QTLs for Witches’ Broom (Crinipellis perniciosa) Resistance in Cacao (Theobroma cacao L.). Euphytica 2006, 149, 227–235.
  51. Santos, R.M.F.; Lopes, U.V.; Bahia, R.d.C.; Machado, R.C.R.; Ahnert, D.; Corrêa, R.X. Marcadores Microssatélites Relacionados Com a Resistência à Vassoura-de-Bruxa Do Cacaueiro. Pesqui. Agropecuária Bras. 2007, 42, 1137–1142.
  52. Lima, L.S.; Gramacho, K.P.; Gesteira, A.S.; Lopes, U.V.; Gaiotto, F.A.; Zaidan, H.A.; Pires, J.L.; Cascardo, J.C.M.; Micheli, F. Characterization of Microsatellites from Cacao–Moniliophthora perniciosa Interaction Expressed Sequence Tags. Mol. Breed. 2008, 22, 315–318.
  53. Lima, L.S.; Gramacho, K.P.; Pires, J.L.; Clement, D.; Lopes, U.V.; Carels, N.; da Silva Gesteira, A.; Gaiotto, F.A.; de Mattos Cascardo, J.C.; Micheli, F. Development, Characterization, Validation, and Mapping of SSRs Derived from Theobroma cacao L.–Moniliophthora perniciosa Interaction ESTs. Tree Genet. Genomes 2010, 6, 663–676.
  54. Lima, E.M.; Pereira, N.E.; Pires, J.L.; Barbosa, A.M.M.; Corrêa, R.X. Genetic Molecular Diversity, Production and Resistance to Witches’ Broom in Cacao Clones. Crop Breed. Appl. Biotechnol. 2013, 13, 127–135.
  55. Fouet, O.; Allegre, M.; Argout, X.; Jeanneau, M.; Lemainque, A.; Pavek, S.; Boland, A.; Risterucci, A.M.; Loor, G.; Tahi, M.; et al. Structural Characterization and Mapping of Functional EST-SSR Markers in Theobroma cacao. Tree Genet. Genomes 2011, 7, 799–817.
  56. Motilal, L.A.; Zhang, D.; Mischke, S.; Meinhardt, L.W.; Boccara, M.; Fouet, O.; Lanaud, C.; Umaharan, P. Association Mapping of Seed and Disease Resistance Traits in Theobroma cacao L. Planta 2016, 244, 1265–1276.
  57. Collard, B.C..; Mackill, D.J. Marker-Assisted Selection: An Approach for Precision Plant Breeding in the Twenty-First Century. Philos. Trans. R. Soc. B Biol. Sci. 2008, 363, 557–572.
  58. Elroy, M.S.; Navarro, A.J.R.; Mustiga, G.; Stack, C.; Gezan, S.; Peña, G.; Sarabia, W.; Saquicela, D.; Sotomayor, I.; Douglas, G.M.; et al. Prediction of Cacao (Theobroma cacao) Resistance to Moniliophthora spp. Diseases via Genome-Wide Association Analysis and Genomic Selection. Front. Plant Sci. 2018, 9, 343.
  59. Osorio-Guarín, J.A.; Berdugo-Cely, J.A.; Coronado-Silva, R.A.; Baez, E.; Jaimes, Y.; Yockteng, R. Genome-Wide Association Study Reveals Novel Candidate Genes Associated with Productivity and Disease Resistance to Moniliophthora Spp. in Cacao ( Theobroma cacao L.). G3 Genes|Genomes|Genetics 2020, 10, 1713–1725.
  60. Gupta, P.K.; Rustgi, S.; Mir, R.R. Array-Based High-Throughput DNA Markers for Crop Improvement. Heredity 2008, 101, 5–18.
  61. Davey, J.W.; Hohenlohe, P.A.; Etter, P.D.; Boone, J.Q.; Catchen, J.M.; Blaxter, M.L. Genome-Wide Genetic Marker Discovery and Genotyping Using next-Generation Sequencing. Nat. Rev. Genet. 2011, 12, 499–510.
  62. Gramacho, K.P.; Risterucci, A.M.; Lanaud, C.; Lima, L.S.; Lopes, U.V. Characterization of Microsatellites in the Fungal Plant Pathogen Crinipellis perniciosa. Mol. Ecol. Notes 2006, 7, 153–155.
  63. Silva, J.R.Q.; Figueira, A.; Pereira, G.A.G.; Albuquerque, P. Development of Novel Microsatellites from Moniliophthora perniciosa, Causal Agent of the Witches’ Broom Disease of Theobroma cacao. Mol. Ecol. Resour. 2008, 8, 783–785.
  64. Andebrhan, T.; Furtek, D.B. Random Amplified Polymorphic DNA (RAPD) Analysis of Crinipellis perniciosa Isolates from Different Hosts. Plant Pathol. 1994, 43, 1020–1027.
  65. Argout, X.; Martin, G.; Droc, G.; Fouet, O.; Labadie, K.; Rivals, E.; Aury, J.M.; Lanaud, C. The Cacao Criollo Genome v2.0: An Improved Version of the Genome for Genetic and Functional Genomic Studies. BMC Genom. 2017, 18, 730.
  66. de Wit, P.J.G.M.; van der Burgt, A.; Ökmen, B.; Stergiopoulos, I.; Abd-Elsalam, K.A.; Aerts, A.L.; Bahkali, A.H.; Beenen, H.G.; Chettri, P.; Cox, M.P.; et al. The Genomes of the Fungal Plant Pathogens Cladosporium Fulvum and Dothistroma Septosporum Reveal Adaptation to Different Hosts and Lifestyles But Also Signatures of Common Ancestry. PLoS Genet. 2012, 8, e1003088.
  67. Guimarães, R.L.; Stotz, H.U. Oxalate Production by Sclerotinia sclerotiorum Deregulates Guard Cells during Infection. Plant Physiol. 2004, 136, 3703–3711.
  68. Apel, K.; Hirt, H. Reactive Oxygen Species: Metabolism, Oxidative Stress, and Signal Transduction. Annu. Rev. Plant Biol. 2004, 55, 373–399.
  69. Meraz-Pérez, I.M.; Carvalho, M.R.; Sena, K.F.; Soares, Y.J.B.; Estrela Junior, A.S.; Lopes, U.V.; dos Santos Filho, L.P.; Araújo, S.A.; Soares, V.L.F.; Pirovani, C.P.; et al. The Moniliophthora perniciosa—Cacao Pod Pathosystem: Structural and Activated Defense Strategies against Disease Establishment. Physiol. Mol. Plant Pathol. 2021, 115, 101656.
  70. Darwiche, R.; El Atab, O.; Baroni, R.M.; Teixeira, P.J.P.L.; Mondego, J.M.C.; Pereira, G.A.G.; Schneiter, R. Plant Pathogenesis–Related Proteins of the Cacao Fungal Pathogen Moniliophthora perniciosa Differ in Their Lipid-Binding Specificities. J. Biol. Chem. 2017, 292, 20558–20569.
  71. Lanver, D.; Müller, A.N.; Happel, P.; Schweizer, G.; Haas, F.B.; Franitza, M.; Pellegrin, C.; Reissmann, S.; Altmüller, J.; Rensing, S.A.; et al. The Biotrophic Development of Ustilago maydis Studied by RNA-Seq Analysis. Plant Cell 2018, 30, 300–323.
  72. Basse, C.W.; Stumpferl, S.; Kahmann, R. Characterization of a Ustilago maydis Gene Specifically Induced during the Biotrophic Phase: Evidence for Negative as Well as Positive Regulation. Mol. Cell. Biol. 2000, 20, 329–339.
  73. Bailey, B.A. Purification of a Protein from Culture Filtrates of Fusarium Oxysporum That Induces Ethylene and Necrosis in Leaves of Erythroxylum Coca. Phytopathology 1995, 85, 1250.
  74. Verica, J.A.; Maximova, S.N.; Strem, M.D.; Carlson, J.E.; Bailey, B.A.; Guiltinan, M.J. Isolation of ESTs from Cacao (Theobroma cacao L.) Leaves Treated with Inducers of the Defense Response. Plant Cell Rep. 2004, 23, 404–413.
  75. Bailey, B.A.; Bae, H.; Strem, M.D.; Antúnez de Mayolo, G.; Guiltinan, M.J.; Verica, J.A.; Maximova, S.N.; Bowers, J.H. Developmental Expression of Stress Response Genes in Theobroma cacao Leaves and Their Response to Nep1 Treatment and a Compatible Infection by Phytophthora Megakarya. Plant Physiol. Biochem. 2005, 43, 611–622.
  76. Arazoe, T.; Miyoshi, K.; Yamato, T.; Ogawa, T.; Ohsato, S.; Arie, T.; Kuwata, S. Tailor-Made CRISPR/Cas System for Highly Efficient Targeted Gene Replacement in the Rice Blast Fungus. Biotechnol. Bioeng. 2015, 112, 2543–2549.
  77. Matsu-ura, T.; Baek, M.; Kwon, J.; Hong, C. Efficient Gene Editing in Neurospora crassa with CRISPR Technology. Fungal Biol. Biotechnol. 2015, 2, 4.
  78. Liu, R.; Chen, L.; Jiang, Y.; Zhou, Z.; Zou, G. Efficient Genome Editing in Filamentous Fungus Trichoderma Reesei Using the CRISPR/Cas9 System. Cell Discov. 2015, 1, 15007.
  79. Schuster, M.; Schweizer, G.; Reissmann, S.; Kahmann, R. Genome Editing in Ustilago maydis Using the CRISPR–Cas System. Fungal Genet. Biol. 2016, 89, 3–9.
  80. Liu, M.; Kang, H.; Xu, Y.; Peng, Y.; Wang, D.; Gao, L.; Wang, X.; Ning, Y.; Wu, J.; Liu, W.; et al. Genome-wide Association Study Identifies an NLR Gene That Confers Partial Resistance to Magnaporthe oryzae in Rice. Plant Biotechnol. J. 2020, 18, 1376–1383.
  81. dos Santos, L.B.P.R.; Oliveira-Santos, N.; Fernandes, J.V.; Jaimes-Martinez, J.C.; De Souza, J.T.; Cruz-Magalhães, V.; Loguercio, L.L. Tolerance to and Alleviation of Abiotic Stresses in Plants Mediated by Trichoderma spp. In Advances in Trichoderma Biology for Agricultural Applications; Springer: Cham, Switzerland, 2022; pp. 321–359.
  82. Neves, D.M.; Almeida, L.A.d.H.; Santana-Vieira, D.D.S.; Freschi, L.; Ferreira, C.F.; Soares Filho, W.d.S.; Costa, M.G.C.; Micheli, F.; Coelho Filho, M.A.; Gesteira, A.d.S. Recurrent Water Deficit Causes Epigenetic and Hormonal Changes in Citrus Plants. Sci. Rep. 2017, 7, 13684.
  83. Santos, A.S.; Neves, D.M.; Santana-Vieira, D.D.S.; Almeida, L.A.H.; Costa, M.G.C.; Soares Filho, W.S.; Pirovani, C.P.; Coelho Filho, M.A.; Ferreira, C.F.; Gesteira, A.S. Citrus Scion and Rootstock Combinations Show Changes in DNA Methylation Profiles and ABA Insensitivity under Recurrent Drought Conditions. Sci. Hortic. 2020, 267, 109313.
  84. Rodrigues da Silva, A.; da Costa Silva, D.; dos Santos Pinto, K.N.; Santos Filho, H.P.; Coelho Filho, M.A.; dos Santos Soares Filho, W.; Ferreira, C.F.; da Silva Gesteira, A. Epigenetic Responses to Phytophthora citrophthora Gummosis in Citrus. Plant Sci. 2021, 313, 111082.
  85. Martínez-Soto, D.; González-Prieto, J.M.; Ruiz-Herrera, J. Transcriptomic Analysis of the GCN5 Gene Reveals Mechanisms of the Epigenetic Regulation of Virulence and Morphogenesis in Ustilago maydis. FEMS Yeast Res. 2015, 15, fov055.
  86. Camillo, L.R.; Filadelfo, C.R.; Monzani, P.S.; Corrêa, R.X.; Gramacho, K.P.; Micheli, F.; Pirovani, C.P. Tc-CAPX, a Cytosolic Ascorbate Peroxidase of Theobroma cacao L. Engaged in the Interaction with Moniliophthora perniciosa, the Causing Agent of Witches’ Broom Disease. Plant Physiol. Biochem. 2013, 73, 254–265.
  87. Mehta, A.; Brasileiro, A.C.M.; Souza, D.S.L.; Romano, E.; Campos, M.A.; Grossi-De-Sá, M.F.; Silva, M.S.; Franco, O.L.; Fragoso, R.R.; Bevitori, R.; et al. Plant-Pathogen Interactions: What Is Proteomics Telling Us? FEBS J. 2008, 275, 3731–3746.
  88. Yang, Z.; Li, M.; Sun, Q. RHON1 Co-Transcriptionally Resolves R-Loops for Arabidopsis Chloroplast Genome Maintenance. Cell Rep. 2020, 30, 243–256.e5.
  89. de O. Barsottini, M.R.; de Oliveira, J.F.; Adamoski, D.; Teixeira, P.J.P.L.; do Prado, P.F.V.; Tiezzi, H.O.; Sforça, M.L.; Cassago, A.; Portugal, R.V.; de Oliveira, P.S.L.; et al. Functional Diversification of Cerato-Platanins in Moniliophthora perniciosa as Seen by Differential Expression and Protein Function Specialization. Mol. Plant-Microbe Interact. 2013, 26, 1281–1293.
  90. Zaparoli, G.; Cabrera, O.G.; Medrano, F.J.; Tiburcio, R.; Lacerda, G.; Pereira, G.G. Identification of a Second Family of Genes in Moniliophthora Perniciosa, the Causal Agent of Witches’ Broom Disease in Cacao, Encoding Necrosis-Inducing Proteins Similar to Cerato-Platanins. Mycol. Res. 2009, 113, 61–72.
  91. Monzani, P.S.; Pereira, H.M.; Melo, F.A.; Meirelles, F.V.; Oliva, G.; Cascardo, J.C.M. A New Topology of ACBP from Moniliophthora perniciosa. Biochim. Biophys. Acta-Proteins Proteom. 2010, 1804, 115–123.
  92. Fiorin, G.L.; Sanchéz-Vallet, A.; Thomazella, D.P.d.T.; do Prado, P.F.V.; do Nascimento, L.C.; Figueira, A.V.d.O.; Thomma, B.P.H.J.; Pereira, G.A.G.; Teixeira, P.J.P.L. Suppression of Plant Immunity by Fungal Chitinase-like Effectors. Curr. Biol. 2018, 28, 3023–3030.e5.
  93. Villela-Dias, C.; Camillo, L.R.; de Oliveira, G.A.P.; Sena, J.A.L.; Santiago, A.S.; de Sousa, S.T.P.; Mendes, J.S.; Pirovani, C.P.; Alvim, F.C.; Costa, M.G.C. Nep1-like Protein from Moniliophthora perniciosa Induces a Rapid Proteome and Metabolome Reprogramming in Cells of Nicotiana benthamiana. Physiol. Plant. 2014, 150, 1–17.
  94. Mares, J.H.; Gramacho, K.P.; Dos Santos, E.C.; Santiago, A.D.S.; Silva, E.M.D.A.; Alvim, F.C.; Pirovani, C.P. Protein Profile and Protein Interaction Network of Moniliophthora Perniciosa Basidiospores. BMC Microbiol. 2016, 16, 120.
  95. Mares, J.H.; Gramacho, K.P.; Santana, J.O.; Oliveira de Souza, A.; Alvim, F.C.; Pirovani, C.P. Hydrosoluble Phylloplane Components of Theobroma cacao Modulate the Metabolism of Moniliophthora perniciosa Spores during Germination. Fungal Biol. 2020, 124, 73–81.
  96. Birner, R.; Bürgermeister, M.; Schneiter, R.; Daum, G. Roles of Phosphatidylethanolamine and of Its Several Biosynthetic Pathways in Saccharomyces cerevisiae. Mol. Biol. Cell 2001, 12, 997–1007.
  97. Gsell, M.; Mascher, G.; Schuiki, I.; Ploier, B.; Hrastnik, C.; Daum, G. Transcriptional Response to Deletion of the Phosphatidylserine Decarboxylase Psd1p in the Yeast Saccharomyces cerevisiae. PLoS ONE 2013, 8, e77380.
  98. Pirovani, C.P.; da Silva Santiago, A.; dos Santos, L.S.; Micheli, F.; Margis, R.; da Silva Gesteira, A.; Alvim, F.C.; Pereira, G.A.G.; de Mattos Cascardo, J.C. Theobroma cacao Cystatins Impair Moniliophthora perniciosa Mycelial Growth and Are Involved in Postponing Cell Death Symptoms. Planta 2010, 232, 1485–1497.
  99. Menezes, S.P.; Santos, J.L.; Cardoso, T.H.S.; Pirovani, C.P.; Micheli, F.; Noronha, F.S.M.; Alves, A.C.; Faria, A.M.C.; da Silva Gesteira, A. Evaluation of the Allergenicity Potential of TcPR-10 Protein from Theobroma cacao. PLoS ONE 2012, 7, e37969.
  100. Britto, D.S.; Pirovani, C.P.; Andrade, B.S.; dos Santos, T.P.; Pungartnik, C.; Cascardo, J.C.M.; Micheli, F.; Gesteira, A.S. Recombinant β-1,3-1,4-Glucanase from Theobroma cacao Impairs Moniliophthora perniciosa Mycelial Growth. Mol. Biol. Rep. 2013, 40, 5417–5427.
  101. Pereira Menezes, S.; de Andrade Silva, E.M.; Matos Lima, E.; Oliveira de Sousa, A.; Silva Andrade, B.; Santos Lima Lemos, L.; Peres Gramacho, K.; da Silva Gesteira, A.; Pirovani, C.P.; Micheli, F. The Pathogenesis-Related Protein PR-4b from Theobroma cacao Presents RNase Activity, Ca2+ and Mg2+ Dependent-DNase Activity and Antifungal Action on Moniliophthora perniciosa. BMC Plant Biol. 2014, 14, 161.
  102. Falcao, L.L.; Silva-Werneck, J.O.; Ramos, A.d.R.; Martins, N.F.; Bresso, E.; Rodrigues, M.A.; Bemquerer, M.P.; Marcellino, L.H. Antimicrobial Properties of Two Novel Peptides Derived from Theobroma cacao Osmotin. Peptides 2016, 79, 75–82.
  103. Cardoso, T.H.S.; Freitas, A.C.O.; Andrade, B.S.; de Sousa, A.O.; Santiago, A.d.S.; Koop, D.M.; Gramacho, K.P.; Alvim, F.C.; Micheli, F.; Pirovani, C.P. TcCYPR04, a Cacao Papain-Like Cysteine-Protease Detected in Senescent and Necrotic Tissues Interacts with a Cystatin TcCYS4. PLoS ONE 2015, 10, e0144440.
  104. do Amaral, M.; Freitas, A.C.O.; Santos, A.S.; dos Santos, E.C.; Ferreira, M.M.; da Silva Gesteira, A.; Gramacho, K.P.; Marinho-Prado, J.S.; Pirovani, C.P. TcTI, a Kunitz-Type Trypsin Inhibitor from Cocoa Associated with Defense against Pathogens. Sci. Rep. 2022, 12, 698.
  105. Almeida, D.S.M.; Gramacho, K.P.; Cardoso, T.H.S.; Micheli, F.; Alvim, F.C.; Pirovani, C.P. Cacao Phylloplane: The First Battlefield against Moniliophthora perniciosa, Which Causes Witches’ Broom Disease. Phytopathology 2017, 107, 864–871.
  106. Freire, L.; Santana, J.O.; Oliveira de Sousa, A.; Bispo dos Santos, J.; Barbosa de Oliveira, I.; Alvim, F.C.; Gramacho, K.P.; Costa, M.G.C.; Pirovani, C.P. Tc PHYLL, a Cacao Phylloplanin Expressed in Young Tissues and Glandular Trichomes. Physiol. Mol. Plant Pathol. 2017, 100, 126–135.
  107. dos Santos, E.C.; Pirovani, C.P.; Correa, S.C.; Micheli, F.; Gramacho, K.P. The Pathogen Moniliophthora perniciosa Promotes Differential Proteomic Modulation of Cacao Genotypes with Contrasting Resistance to Witches´ Broom Disease. BMC Plant Biol. 2020, 20, 1.
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