Cocoa Apoplastome Contains Defense Proteins Against Pathogens.

The apoplast performs important functions in the plant, such as defense against stress, and compounds present form the apoplastic washing fluid (AWF). The fungus Moniliophthora perniciosa, the causal agent of witches' broom disease (WBD) in Theobroma cacao, initially colonizes the apoplast in its biotrophic phase. In this period, the fungus can remain for approximately 60 days, until it changes to its second phase, causing tissue death and consequently large loss in the production of beans. To better understand the importance of the apoplast in the T. cacao-M. perniciosa interaction, we performed the first apoplastic proteomic mapping of two contrasting genotypes for WBD resistance (CCN51-resistant and Catongo-susceptible). Based on two-dimensional gel analysis, we identified 36 proteins in CCN-51 and 15 in Catongo. We highlight PR-proteins, such as peroxidases, ß-1,3-glucanases, and chitinases. A possible candidate for a resistance marker of the CCN-51 genotype, osmotin, was identified. The antioxidative metabolism of the superoxide dismutase (SOD) enzyme showed a significant increase (P < 0.05) in the AWF of the two genotypes under field conditions (FD). T. cacao AWF inhibited the germination of M. perniciosa basidiospores (>80%), in addition to causing morphological changes. Our results shed more light on the nature of the plant's defense performed by the apoplast in the T. cacao-M. perniciosa interaction in the initial (biotrophic) phase of fungal infection and therefore make it possible to expand WBD control strategies based on the identification of potential targets for resistance markers and advance scientific knowledge of the disease.

Chemical Characterization of Trichoderma spp. Extracts with Antifungal Activity against Cocoa Pathogens.

Ecuador is one of the major cocoa producers worldwide, but its productivity has lately been affected by diseases. Endophytic biocontrol agents have been used to minimize pathogenic effects; however, compounds produced by endophytes are minimally understood. This work presents the chemical characterization of the Trichoderma species extracts that proved inhibition against cocoa pathogens. Solid-liquid extraction was performed as a partitioning method using medium with the fungal mycelia of Trichoderma reesei (C2A), Trichoderma sp. (C3A), Trichoderma harzianum (C4A), and Trichoderma spirale (C10) in ethyl acetate individually. The extract of T. spirale (C10) exhibited the growth inhibition (32.97-47.02%) of Moniliophthora perniciosa at 10 µg/mL, while a slight stimulation of Moniliophthora roreri was shown by the extracts of T. reesei (C2A) and T. harzianum (C4A) at higher concentrations. The inhibitory activity could be related to alkaloids, lactones, quinones, flavonoids, triterpenes, and sterols, as indicated by chemical screening and antifungal compounds, such as widdrol, ß-caryophyllene, tyrosol, butyl isobutyrate, sorbic acid, palmitic acid, palmitelaidic acid, linoleic acid, and oleic acid, which were identified by gas chromatography-mass spectrometry (GC-MS). The results showed that the extracts, particularly T. spirale (C10), have the potential as biocontrol agents against witches' broom disease; however, further studies are needed to confirm their effectiveness.

ß-Glucosidase produced by Moniliophthora perniciosa: Characterization and application in the hydrolysis of sugarcane bagasse.

ß-Glucosidases (BGLs) belong to the group of enzymes of cellulases and act in the last stage of cellulose degradation, releasing glucose molecules, eliminating the inhibitory effect of cellobiose. This study focused on the production, characterization, and application of BGL from Moniliophthora perniciosa in the hydrolysis of pretreated sugarcane bagasse (3% NaOH + 6% Na2 SO3 ), with varying enzymatic loads and reaction times. The enzyme showed an optimum pH of 4.5 and 60°C. It was stable at all temperatures analyzed (50-90°C) and retained about 100% of its activity at 50°C after 60 min of incubation. Among the ions analyzed, BaCl2 increased BGL activity 9.04 ± 1.41 times. The maximum production of reducing sugars (89.15%) was achieved after 48 h with 10 mg of protein.

Population Structure of Moniliophthora perniciosa in the Main Cacao Producing Departments of Colombia.

The witches' broom (Moniliophthora perniciosa) is considered as one of the main threats for cacao production and, consequently, for chocolate production worldwide. In this work, the genetic diversity and population structure of M. perniciosa were analyzed for 59 isolates collected in five departments of Colombia and using 10 microsatellite markers. Analyses revealed 35 multilocus genotypes and clonal populations structure according to linkage disequilibrium analysis. One of the objectives of this study was to determine whether populations were differentiated by geographic origin or Theobroma cacao host genotype. Analysis of molecular variance, discriminant analysis of principal components, and Bruvo genetic distance suggested that the genetic structure was driven by geographic origin and not by T. cacao genotype. The results of this study were consistent with previous findings obtained in other cocoa-producing countries. Important insights were discussed regarding the dispersal patterns of the pathogen in Colombia and the genetic change of its populations because of different environmental conditions.

Antifungal Activity of Chemical Constituents from Piper pesaresanum C. DC. and Derivatives against Phytopathogen Fungi of Cocoa.

In this study, the antifungal potential of chemical constituents from Piper pesaresanum and some synthesized derivatives was determined against three phytopathogenic fungi associated with the cocoa crop. The methodology included the phytochemical study on the aerial part of P. pesaresanum, the synthesis of some derivatives and the evaluation of the antifungal activity against the fungi Moniliophthora roreri, Fusarium solani and Phytophthora sp. The chemical study allowed the isolation of three benzoic acid derivatives (1-3), one dihydrochalcone (4) and a mixture of sterols (5-7). Seven derivatives (8-14) were synthesized from the main constituents, of which compounds 9, 10, 12 and 14 are reported for the first time. Benzoic acid derivatives showed strong antifungal activity against M. roreri, of which 11 (3.0 ± 0.8 µM) was the most active compound with an IC50 lower compared with positive control Mancozeb® (4.9 ± 0.4 µM). Dihydrochalcones and acid derivatives were active against F. solani and Phytophthora sp., of which 3 (32.5 ± 3.3 µM) and 4 (26.7 ± 5.3 µM) were the most active compounds, respectively. The preliminary structure-activity relationship allowed us to establish that prenylated chains and the carboxyl group are important in the antifungal activity of benzoic acid derivatives. Likewise, a positive influence of the carbonyl group on the antifungal activity for dihydrochalcones was deduced.

Heterologous expression of an alternative oxidase from Moniliophthora perniciosa in Saccharomyces cerevisiae: Antioxidant function and in vivo platform for the study of new drugs against witches' broom disease.

The fungus Moniliophthora perniciosa is the causal agent of witches' broom disease (WBD), one of the most devastating diseases of cacao, the chocolate tree. Many strategies to control WBD have been tested so far, including the use of agrochemicals such as the strobilurins. Strobilurins are fungicides of the QoI family, and they are used in the control of a wide array of fungal diseases in many different crops, including cereals, field crops, fruits, tree nuts, and vegetables. These drugs act by specifically inhibiting fungal respiration at the Qo site of complex III, which is a component of the main mitochondrial respiratory chain. However, M. perniciosa is resistant to this family of chemicals. It has been postulated that this resistant phenotype is, at least in part, a result of the strong ability of this fungus to counteract the oxidative stress generated by the impairment of the main mitochondrial respiratory chain, through the activation of an alternative oxidase (Mp-AOX). To test this hypothesis, we expressed functional mitochondria-localized Mp-AOX in the model yeast Saccharomyces cerevisiae. We demonstrated that heterologous expression of Mp-AOX strongly inhibits hydrogen peroxide production by mitochondria. It also diminishes the total cell amount of oxidized glutathione (GSSG), resulting in a fifty-fold higher GSH/GSSG ratio in cells expressing Mp-AOX than in wild type cells. In addition, Mp-AOX activity decreases yeast growth rate and leads to low biomass production. Therefore, we propose the use of this heterologous expression system to direct the development of new inhibitors of fungal AOX by comparing the differences in optical density of Mp-AOX-expressing cells in the presence and absence of potential AOX inhibitors. Together, our results confirm the antioxidant role of Mp-AOX and provide an in vivo platform to be used in the screening of new fungicides based on Mp-AOX inhibition.

Major phytopathogens and strains from cocoa (Theobroma cacao L.) are differentiated by MALDI-MS lipid and/or peptide/protein profiles.

Phytopathogens are the main disease agents that promote attack of cocoa plantations in all tropical countries. The similarity of the symptoms caused by different phytopathogens makes the reliable identification of the diverse species a challenge. Correct identification is important in the monitoring and management of these pests. Here we show that matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) in combination with multivariate data analysis is able to rapidly and reliably differentiate cocoa phytopathogens, namely Moniliophthora perniciosa, Phytophthora palmivora, P. capsici, P. citrophthora, P. heveae, Ceratocystis cacaofunesta, C. paradoxa, and C. fimbriata. MALDI-MS reveals unique peptide/protein and lipid profiles which differentiate these phytopathogens at the level of genus, species, and single strain coming from different hosts or cocoa tissues collected in several plantations/places. This fast methodology based on molecular biomarkers is also shown to be sufficiently reproducible and selective and therefore seems to offer a suitable tool to guide the correct application of sanitary defense approaches for infected cocoa plantations. International trading of cocoa plants and products could also be efficiently monitored by MALDI-MS. It could, for instance, prevent the entry of new phytopathogens into a country, e.g., as in the case of Moniliophthora roreri fungus that is present in all cocoa plantations of countries bordering Brazil, but that has not yet attacked Brazilian plantations. Graphical Abstract Secure identification of phytopathogens attacking cocoa plantations has been demonstrated via typical chemical profiles provided by mass spectrometric screening.

Genomic analyses and expression evaluation of thaumatin-like gene family in the cacao fungal pathogen Moniliophthora perniciosa.

Thaumatin-like proteins (TLPs) are found in diverse eukaryotes. Plant TLPs, known as Pathogenicity Related Protein (PR-5), are considered fungal inhibitors. However, genes encoding TLPs are frequently found in fungal genomes. In this work, we have identified that Moniliophthora perniciosa, a basidiomycete pathogen that causes the Witches' Broom Disease (WBD) of cacao, presents thirteen putative TLPs from which four are expressed during WBD progression. One of them is similar to small TLPs, which are present in phytopathogenic basidiomycete, such as wheat stem rust fungus Puccinia graminis. Fungi genomes annotation and phylogenetic data revealed a larger number of TLPs in basidiomycetes when comparing with ascomycetes, suggesting that these proteins could be involved in specific traits of mushroom-forming species. Based on the present data, we discuss the contribution of TLPs in the combat against fungal competitors and hypothesize a role of these proteins in M. perniciosa pathogenicity.

Apoplastic and intracellular plant sugars regulate developmental transitions in witches' broom disease of cacao.

Witches' broom disease (WBD) of cacao differs from other typical hemibiotrophic plant diseases by its unusually long biotrophic phase. Plant carbon sources have been proposed to regulate WBD developmental transitions; however, nothing is known about their availability at the plant-fungus interface, the apoplastic fluid of cacao. Data are provided supporting a role for the dynamics of soluble carbon in the apoplastic fluid in prompting the end of the biotrophic phase of infection. Carbon depletion and the consequent fungal sensing of starvation were identified as key signalling factors at the apoplast. MpNEP2, a fungal effector of host necrosis, was found to be up-regulated in an autophagic-like response to carbon starvation in vitro. In addition, the in vivo artificial manipulation of carbon availability in the apoplastic fluid considerably modulated both its expression and plant necrosis rate. Strikingly, infected cacao tissues accumulated intracellular hexoses, and showed stunted photosynthesis and the up-regulation of senescence markers immediately prior to the transition to the necrotrophic phase. These opposite findings of carbon depletion and accumulation in different host cell compartments are discussed within the frame of WBD development. A model is suggested to explain phase transition as a synergic outcome of fungal-related factors released upon sensing of extracellular carbon starvation, and an early senescence of infected tissues probably triggered by intracellular sugar accumulation.

Apoplastomes of contrasting cacao genotypes to witches' broom disease reveals differential accumulation of PR proteins.

Witches' broom disease (WBD) affects cocoa trees (Theobroma cacao L.) and is caused by the fungus Moniliophthora perniciosa that grows in the apoplast in its biotrophic phase and later progresses into the tissues, causing serious losses in the production of cocoa beans. Therefore, the apoplast of T. cacao can provide important defense responses during the interaction with M. perniciosa. In this work, the protein profile of the apoplast of the T. cacao genotypes Catongo, susceptible to WBD, and CCN-51, resistant one, was evaluated. The leaves of T. cacao were collected from asymptomatic plants grown in a greenhouse (GH) and from green witches' brooms grown under field (FD) conditions for extraction of apoplastic washing fluid (AWF). AWF was used in proteomic and enzymatic analysis. A total of 14 proteins were identified in Catongo GH and six in Catongo FD, with two proteins being common, one up-accumulated, and one down-accumulated. In CCN-51, 19 proteins were identified in the GH condition and 13 in FD, with seven proteins being common, one up-accumulated, and six down-accumulated. Most proteins are related to defense and stress in both genotypes, with emphasis on pathogenesis-related proteins (PR): PR-2 (ß-1,3-glucanases), PR-3 and PR-4 (chitinases), PR-5 (thaumatine), PR-9 (peroxidases), and PR-14 (lipid transfer proteins). Furthermore, proteins from microorganisms were detected in the AWF. The enzymatic activities of PR-3 showed a significant increase (p < 0.05) in Catongo GH and PR-2 activity (p < 0.01) in CCN-51 FD. The protein profile of the T. cacao apoplastome offers insight into the defense dynamics that occur in the interaction with the fungus M. perniciosa and offers new insights in exploring future WBD control strategies.

Optimization of Paenibacillus sp. NMA1017 Application as a Biocontrol Agent for Phytophthora tropicalis and Moniliophthora roreri in Cacao-Growing Fields in Chiapas, Mexico.

In Mexico, cacao production is endangered by pathogenic fungi, such as Phytophthora spp. and Moniliophthora rorei, that cause black pod rot and moniliasis, respectively. In this study the biocontrol agent Paenibacillus sp. NMA1017 was tested in cacao fields against the previous diseases. The treatments applied were shade management, inoculation of the bacterial strain with or without an adherent, and use of chemical control. The statistical analysis showed that the incidence of black pod rot in tagged cacao trees diminished when the bacterium was applied (reduction of 44.24 to 19.11%). The same result was observed with moniliasis when the pods were tagged (reduction of 66.6 to 27%). The use of Paenibacillus sp. NMA1017 with an integrated management might be a solution to cacao diseases and to having a sustainable production of cacao in Mexico.

Genomic and Pathogenicity Mechanisms of the Main Theobroma cacao L. Eukaryotic Pathogens: A Systematic Review.

A set of diseases caused by fungi and oomycetes are responsible for large losses in annual world cocoa production. Managing the impact caused by these diseases is very complex because a common solution has yet to be found for different pathogens. In this context, the systematic knowledge of Theobroma cacao L. pathogens' molecular characteristics may help researchers understand the possibilities and limitations of cocoa disease management strategies. This work systematically organized and summarized the main findings of omics studies of T. cacao eukaryotic pathogens, focusing on the plant-pathogen interaction and production dynamics. Using the PRISMA protocol and a semiautomated process, we selected papers from the Scopus and Web of Science databases and collected data from the selected papers. From the initial 3169 studies, 149 were selected. The first author's affiliations were mostly from two countries, Brazil (55%) and the USA (22%). The most frequent genera were Moniliophthora (105 studies), Phytophthora (59 studies) and Ceratocystis (13 studies). The systematic review database includes papers reporting the whole-genome sequence from six cocoa pathogens and evidence of some necrosis-inducing-like proteins, which are common in T. cacao pathogen genomes. This review contributes to the knowledge about T. cacao diseases, providing an integrated discussion of T. cacao pathogens' molecular characteristics, common mechanisms of pathogenicity and how this knowledge is produced worldwide.

Expression of a Cutinase of Moniliophthora roreri with Polyester and PET-Plastic Residues Degradation Activity.

Cutinases are enzymes produced by phytopathogenic fungi like Moniliophthora roreri. The three genome-located cutinase genes of M. roreri were amplified from cDNA of fungi growing in different induction culture media for cutinase production. The mrcut1 gene was expressed in the presence of a cacao cuticle, while the mrcut2 and mrcut3 genes were expressed when an apple cuticle was used as the inducer. The sequences of all genes were obtained and analyzed by bioinformatics tools to determine the presence of signal peptides, introns, glycosylation, and regulatory sequences. Also, the theoretical molecular weight and pI were obtained and experimentally confirmed. Finally, cutinase 1 from M. roreri (MRCUT1) was selected for heterologous expression in Escherichia coli. Successful overexpression of MRCUT1 was observed with the highest enzyme activity of 34,036 U/mg under the assay conditions at 40°C and pH 8. Furthermore, the degradation of different synthetic polyesters was evaluated; after 21 days, 59% of polyethylene succinate (PES), 43% of polycaprolactone (PCL), and 31% of polyethylene terephthalate (PET) from plastic residues were degraded. IMPORTANCE Plastic pollution is exponentially increasing; even the G20 has recognized an urgent need to implement actions to reduce it. In recent years, searching for enzymes that can degrade plastics, especially those based on polyesters such as PET, has been increasing as they can be a green alternative to the actual plastic degradation process. A promising option in recent years refers to biological tools such as enzymes involved in stages of partial and even total degradation of some plastics. In this context, the MRCUT1 enzyme can degrade polyesters contained in plastic residues in a short time. Besides, there is limited knowledge about the biochemical properties of cutinases from M. roreri. Commonly, fungal enzymes are expressed as inclusion bodies in E. coli with reduced activity. Interestingly, the successful expression of one cutinase of M. roreri in E. coli with enhanced activity is described.

Functional and morphological analysis of isolates of phylloplane and rhizoplane endophytic bacteria interacting in different cocoa production systems in the Amazon.

Endophytic bacteria colonize different internal tissues of plants without damaging their cells. They can establish themselves in the same niche as other microorganisms and develop antagonistic activities against phytopathogens. There is little research on the functional and morphological characterization of these bacteria in production systems in the Amazon. Thus, the objective of this work was to functionally and morphologically characterize endophytic bacteria isolated from cocoa trees (Theobroma cacao L.) and evaluate their antagonistic potential against phytopathogens. A total of 197 endophytic bacteria isolates were obtained from leaves and roots of cocoa plants with different production systems and at different times of the year. The characterization of functional groups consisted of proteolytic, amylolytic and cellulolytic activity and ability to fix nitrogen and solubilize phosphate. Morphological diversity was evaluated mainly according to the following parameters: shape, color, size and elevation of the colony. Thirteen isolates of endophytic bacteria, selected by cluster analysis, were used to evaluate the antagonistic potential in paired trials against four species of phytopathogenic fungi. The largest amount of endophytic bacteria was isolated from the root (95.9%), in the dry season. The most expressive activities with regards to the enzyme index were amylolytic (71.9%), proteolytic (70.2%) and nitrogen fixing (38.6%), respectively. The similarity analysis formed two clusters with isolates CS R 2.4 and CS R 2.25 exhibiting 100% similarity. Five isolates displayed inhibitory activity against phytopathogenic fungi, most notably isolate TS R 2.19, which exhibited antagonistic activity against all fungi and mycelial growth inhibition rates between 25.7% and 50.7%. Understanding the interaction between endophytes in cocoa plants is important as a possible additional tool in biological control. Our studies are incipient and the first to be carried out in different cocoa production systems in the state of Pará, Brazil.

Moniliophthora perniciosa, the Causal Agent of Cacao Witches' Broom Disease Is Killed in vitro by Saccharomyces cerevisiae and Wickerhamomyces anomalus Yeasts.

Cacao plantations from South America have been afflicted with the severe fungal disease known as Witches' Broom Disease (WBD), caused by the basidiomycete Moniliophthora perniciosa. Yeasts are increasingly recognized as good fungal biocides, although their application is still mostly restricted to the postharvest control of plant and fruit decay. Their possible utilization in the field, in a preharvest phase, is nevertheless promising, particularly if the strains are locally adapted and evolved and if they belong to species considered safe for man and the environment. In this work, a group of yeast strains originating from sugarcane-based fermentative processes in Brazil, the cacao-producing country where the disease is most severe, were tested for their ability to antagonize M. perniciosa in vitro. Wickerhamomyces anomalus LBCM1105 and Saccharomyces cerevisiae strains LBCM1112 from spontaneous fermentations used to produce cachaça, and PE2 widely used in Brazil in the industrial production of bioethanol, efficiently antagonized six strains of M. perniciosa, originating from several South American countries. The two fastest growing fungal strains, both originating from Brazil, were further used to assess the mechanisms underlying the yeasts' antagonism. Yeasts were able to inhibit fungal growth and kill the fungus at three different temperatures, under starvation, at different culture stages, or using an inoculum from old yeast cultures. Moreover, SEM analysis revealed that W. anomalus and S. cerevisiae PE2 cluster and adhere to the hyphae, push their surface, and fuse to them, ultimately draining the cells. This behavior concurs with that classified as necrotrophic parasitism/mycoparasitism. In particular, W. anomalus within the adhered clusters appear to be ligated to each other through roundish groups of fimbriae-like structures filled with bundles of microtubule-sized formations, which appear to close after cells detach, leaving a scar. SEM also revealed the formation of tube-like structures apparently connecting yeast to hypha. This evidence suggests W. anomalus cells form a network of yeast cells connecting with each other and with hyphae, supporting a possible cooperative collective killing and feeding strategy. The present results provide an initial step toward the formulation of a new eco-friendly and effective alternative for controlling cacao WBD using live yeast biocides.

The glutathione peroxidase family of Theobroma cacao: Involvement in the oxidative stress during witches' broom disease.

The glutathione peroxidases (GPXs) are enzymes which are part of the cell antioxidant system inhibiting the ROS-induced damages of membranes and proteins. In cacao (Theobroma cacao L.) genome, five GPX genes were identified. Cysteine insertion codons (UGU) were found in TcPHGPX, TcGPX2, TcGPX4, TcGPX6 and tryptophan insertion codon (UGG) in TcGPX8. Multiple alignments revealed conserved domains between TcGPXs and other plants and human GPXs. Homology modeling was performed using the Populus trichocarpa GPX5 structure as template, and the molecular modeling showed that TcGPXs have affinity with selenometionine in their active site. In silico analysis of the TcGPXs promoter region revealed the presence of conserved cis-elements related to biotic stresses and hormone responsiveness. The expression analysis of TcGPXs in cacao plantlet meristems infected by M. perniciosa showed that TcGPXs are most expressed in susceptible variety than in resistant one, mainly in disease stages in which oxidative stress and programmed cell death occurred. This data, associated with phylogenetic and location analysis suggested that TcGPXs may play a role in protecting cells from oxidative stress as a try of disease progression reduction. To our knowledge, this is the first study of the overall GPX family from T. cacao.

Dimethyl Labeling-Based Quantitative Proteomics of Recalcitrant Cocoa Pod Tissue.

Dimethyl labeling is a type of stable-isotope labeling suitable for creating isotopic variants of peptides and thus be utilized for quantitative proteomics experiments. Labeling is achieved through a reductive amination/alkylation reaction using the low-cost reagents formaldehyde and cyanoborohydride, resulting in dimethylation of free amine groups of Lys and N-termini. Availability of isotopomeric forms of these reagents allows for the generation of up to six different isotopic variants. Here we describe the application of dimethylation to create two isotopic variants, light and heavy, differing in 4 Da, to label the total tryptic digest peptides of cocoa pod extracted from healthy pods from cultivars susceptible and resistant to the fungal disease called "frosty pod" caused by Moniliophthora roreri.

The glyceraldehyde-3-phosphate dehydrogenase gene of Moniliophthoraperniciosa, the causal agent of witches' broom disease of Theobroma cacao.

This report describes the cloning, sequence and expression analysis of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene of Moniliophthora perniciosa, the most important pathogen of cocoa in Brazil. Southern blot analysis revealed the presence of a single copy of the GAPDH gene in the M. perniciosa genome (MpGAPDH). The complete MpGAPDH coding sequence contained 1,461 bp with eight introns that were conserved in the GAPDH genes of other basidiomycete species. The cis-elements in the promoter region of the MpGAPDH gene were similar to those of other basidiomycetes. Likewise, the MpGAPDH gene encoded a putative 339 amino acid protein that shared significant sequence similarity with other GAPDH proteins in fungi, plants, and metazoans. Phylogenetic analyses clustered the MPGAPDH protein with other homobasidiomycete fungi of the family Tricholomataceae. Expression analysis of the MpGAPDH gene by real-time PCR showed that this gene was more expressed (~1.3X) in the saprotrophic stage of this hemibiotrophic plant pathogen than in the biotrophic stage when grown in cacao extracts.

Killer yeasts inhibit the growth of the phytopathogen Moniliophthora perniciosa, the causal agent of Witches' Broom disease.

Fruit and soil yeasts isolated from the Amazon, Atlantic Rainforests and an organic farm were screened for killer activity against yeasts. Killer yeasts were then tested against the phytopathogen Moniliophthora perniciosa (syn. Crinipellis perniciosa) and a Dipodascus capitatus strain and a Candida sp strain inhibited its growth.

The selenium-binding protein of Theobroma cacao: A thermostable protein involved in the witches' broom disease resistance.

The selenium-binding proteins are known to be inducers of apoptosis in human and animals, and have been studied as target for the treatment of various types of cancer. In plants, SBP expression has been related to abiotic and biotic stress resistance. The SBP from Theobroma cacao (TcSBP) was first identified from a cocoa-Moniliophthora perniciosa cDNA library. The present study provides details on the TcSBP gene and protein structure. Multiple alignments revealed conserved domains between SBP from plants, human and archea. Homology modeling and molecular docking were performed and showed that the TcSBP has affinity to selenite in the active CSSC site. This result was confirmed by circular dichroism of the recombinant TcSBP, which also presented thermostable behavior. RT-qPCR analysis showed that TcSBP was differentially expressed in resistant vs susceptible cacao varieties inoculated by M. perniciosa and its expression was probably due to hormone induction via cis-regulating elements present in its promotor. The presence of the CSSC domain suggested that TcSBP acted by altering oxidation/reduction of proteins during H2O2 production and programmed cell death in the final stages of the witches' broom disease. To our knowledge, this is the first in silico and in vitro analysis of the SBP from cacao.