New light on the use of Theobroma cacao by Late Classic Maya.

Cacao seeds, Theobroma cacao, provide the basis for a ceremonially important Mesoamerican food. Past efforts to identify cacao in ceramics focused on highly decorative vessel forms associated with elite ceremonial contexts, creating assumptions as to how cacao was distributed and who could access it. This study examines 54 archaeological ceramic sherds from El Pilar (Belize/Guatemala) of Late Classic (600 to 900 CE) residential and civic contexts representing a cross-section of ancient Maya inhabitants. Identification of cacao in ancient sherds has depended on the general presence of theobromine; we used the discrete presence of theophylline, a unique key biomarker for cacao in the region. Analysis was done by grinding off all outside surfaces to reduce contamination, pulverizing the inner clay matrix, extracting absorbed molecules, and concentrating the extractions. In order to obtain especially high selectivity and low limits of detection, our study utilized the technique of resonance-enhanced multiphoton ionization coupled with laser-desorption jet-cooling mass spectrometry. This technique isolates molecules in the cold gas phase where they can be selectively ionized through a resonant two-photon process. Of the sherds analyzed, 30 samples (56%) were found to contain significant amounts of theophylline and thus test positive for cacao. Importantly, cacao is present in all contexts, common to all Maya residents near and far from centers.

A combination of conserved and diverged responses underlies Theobroma cacao's defense response to Phytophthora palmivora.

BACKGROUND: Plants have complex and dynamic immune systems that have evolved to resist pathogens. Humans have worked to enhance these defenses in crops through breeding. However, many crops harbor only a fraction of the genetic diversity present in wild relatives. Increased utilization of diverse germplasm to search for desirable traits, such as disease resistance, is therefore a valuable step towards breeding crops that are adapted to both current and emerging threats. Here, we examine diversity of defense responses across four populations of the long-generation tree crop Theobroma cacao L., as well as four non-cacao Theobroma species, with the goal of identifying genetic elements essential for protection against the oomycete pathogen Phytophthora palmivora. RESULTS: We began by creating a new, highly contiguous genome assembly for the P. palmivora-resistant genotype SCA 6 (Additional file 1: Tables S1-S5), deposited in GenBank under accessions CP139290-CP139299. We then used this high-quality assembly to combine RNA and whole-genome sequencing data to discover several genes and pathways associated with resistance. Many of these are unique, i.e., differentially regulated in only one of the four populations (diverged 40 k-900 k generations). Among the pathways shared across all populations is phenylpropanoid biosynthesis, a metabolic pathway with well-documented roles in plant defense. One gene in this pathway, caffeoyl shikimate esterase (CSE), was upregulated across all four populations following pathogen treatment, indicating its broad importance for cacao's defense response. Further experimental evidence suggests this gene hydrolyzes caffeoyl shikimate to create caffeic acid, an antimicrobial compound and known inhibitor of Phytophthora spp. CONCLUSIONS: Our results indicate most expression variation associated with resistance is unique to populations. Moreover, our findings demonstrate the value of using a broad sample of evolutionarily diverged populations for revealing the genetic bases of cacao resistance to P. palmivora. This approach has promise for further revealing and harnessing valuable genetic resources in this and other long-generation plants.

The cacao gene atlas: a transcriptome developmental atlas reveals highly tissue-specific and dynamically-regulated gene networks in Theobroma cacao L.

BACKGROUND: Theobroma cacao, the cocoa tree, is a tropical crop grown for its highly valuable cocoa solids and fat which are the basis of a 200-billion-dollar annual chocolate industry. However, the long generation time and difficulties associated with breeding a tropical tree crop have limited the progress of breeders to develop high-yielding disease-resistant varieties. Development of marker-assisted breeding methods for cacao requires discovery of genomic regions and specific alleles of genes encoding important traits of interest. To accelerate gene discovery, we developed a gene atlas composed of a large dataset of replicated transcriptomes with the long-term goal of progressing breeding towards developing high-yielding elite varieties of cacao. RESULTS: We describe the creation of the Cacao Transcriptome Atlas, its global characterization and define sets of genes co-regulated in highly organ- and temporally-specific manners. RNAs were extracted and transcriptomes sequenced from 123 different tissues and stages of development representing major organs and developmental stages of the cacao lifecycle. In addition, several experimental treatments and time courses were performed to measure gene expression in tissues responding to biotic and abiotic stressors. Samples were collected in replicates (3-5) to enable statistical analysis of gene expression levels for a total of 390 transcriptomes. To promote wide use of these data, all raw sequencing data, expression read mapping matrices, scripts, and other information used to create the resource are freely available online. We verified our atlas by analyzing the expression of genes with known functions and expression patterns in Arabidopsis (ACT7, LEA19, AGL16, TIP13, LHY, MYB2) and found their expression profiles to be generally similar between both species. We also successfully identified tissue-specific genes at two thresholds in many tissue types represented and a set of genes highly conserved across all tissues. CONCLUSION: The Cacao Gene Atlas consists of a gene expression browser with graphical user interface and open access to raw sequencing data files as well as the unnormalized and CPM normalized read count data mapped to several cacao genomes. The gene atlas is a publicly available resource to allow rapid mining of cacao gene expression profiles. We hope this resource will be used to help accelerate the discovery of important genes for key cacao traits such as disease resistance and contribute to the breeding of elite varieties to help farmers increase yields.

Volatile Organic Compounds from Ceratocystis cacaofunesta, a Causal Agent of Ceratocystis Wilt of Cacao.

Fungi of the genus Ceratocystis are aggressive tree pathogens that cause serious diseases in several crops around the world. Ceratocystis wilt disease caused by C. cacaofunesta has been shown to be responsible for severe reductions in cacao production. In this study, headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS) was used in combination with chemometric analysis for monitoring volatile organic compounds (VOCs) released from C. cacaofunesta. Low-molecular-weight esters, alcohols, ketones, and sulphur compounds were identified in the liquid broth. Monitoring the volatile profile over five days of fungal growth revealed that the concentrations of alcohol and esters were inversely proportional. Acetate esters were responsible for the intense fruity aroma of the C. cacaofunesta culture produced within the first hours after fungal inoculation, which decreased over time, and are likely associated with the attraction of insect vectors to maintain the life cycle of the pathogen. PCA revealed that 3-methylbutyl acetate was the metabolite with the highest factor loading for the separation of the VOC samples after 4 h of fungal growth, whereas ethanol and 3-methylbutan-1-ol had the highest factor loadings after 96 and 120 h. 3-Methylbutan-1-ol is a phytotoxic compound that is likely associated with host cell death since C. cacaofunesta is a necrotrophic fungus. Fungal VOCs play important roles in natural habitats, regulating developmental processes and intra- and interkingdom interactions. This is the first report on the volatiles released by C. cacaofunesta.

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.

Genomic structural variants constrain and facilitate adaptation in natural populations of Theobroma cacao, the chocolate tree.

Genomic structural variants (SVs) can play important roles in adaptation and speciation. Yet the overall fitness effects of SVs are poorly understood, partly because accurate population-level identification of SVs requires multiple high-quality genome assemblies. Here, we use 31 chromosome-scale, haplotype-resolved genome assemblies of Theobroma cacao-an outcrossing, long-lived tree species that is the source of chocolate-to investigate the fitness consequences of SVs in natural populations. Among the 31 accessions, we find over 160,000 SVs, which together cover eight times more of the genome than single-nucleotide polymorphisms and short indels (125 versus 15 Mb). Our results indicate that a vast majority of these SVs are deleterious: they segregate at low frequencies and are depleted from functional regions of the genome. We show that SVs influence gene expression, which likely impairs gene function and contributes to the detrimental effects of SVs. We also provide empirical support for a theoretical prediction that SVs, particularly inversions, increase genetic load through the accumulation of deleterious nucleotide variants as a result of suppressed recombination. Despite the overall detrimental effects, we identify individual SVs bearing signatures of local adaptation, several of which are associated with genes differentially expressed between populations. Genes involved in pathogen resistance are strongly enriched among these candidates, highlighting the contribution of SVs to this important local adaptation trait. Beyond revealing empirical evidence for the evolutionary importance of SVs, these 31 de novo assemblies provide a valuable resource for genetic and breeding studies in Tcacao.

Moniliophthora perniciosa in Angola, Africa-the First Record in the Eastern Hemisphere.

Moniliophthora perniciosa causes a destructive disease known as witches' broom disease of cacao (WBDC). WBDC has been responsible for major reductions in production or even total abandonment of cacao plantations in most countries that it has invaded. To date, however, the disease is known only from the cacao-producing regions of South America and a few Central American and Caribbean countries. It is not known from the Eastern Hemisphere and remains a major threat should it invade West Africa or Southeast Asia, where the majority of the world's chocolate production now occurs. In 2019, a pink pigmented mushroom was found fruiting from unidentified twigs in the Serra Vamba of Angola. The specimen was identified as M. perniciosa based on morphological and molecular analyses. Although Angola is not a major cacao-producing country, the presence of the fungus in the Eastern Hemisphere could be of global concern and may indicate the need for quarantine in Angola and vigilance in neighboring countries.

Metabolome profiling of cacao (Theobroma cacao L.) callus under drought stress conditions induced by polyethylene glycol (PEG) as osmoticant.

INTRODUCTION: The cacao tree (Theobroma cacao), a perennial crop that serves as a source of cacao beans, can suffer from drastic climate changes such as irregular rainfall and shorter rainy seasons. The search for hybrids which are capable of producing specific metabolites favoring adaptation in new climatic conditions is a challenge in cacao farming. OBJECTIVES: We aimed to (1) analyze the metabolic changes in calli of three cacao genotypes during water deficit induced by incubation with polyethylene glycol and (2) assess their response to water deficit stress with regard to somatic embryo differentiation. METHODS: Metabolic profiling was carried out using 1H-NMR spectroscopy and multivariate data analysis was applied to crude extracts of calli grown in non-stress or water deficit stress conditions. RESULTS: Water deficit stress influences the capacity of calli to produce embryos. The SCA12 genotype exhibited the best conversion capacity under severe conditions and was considered as tolerant to drought, followed by the SCA6 genotype (mid-tolerant) and the MA12 genotype (sensitive). Fifty-four metabolites were identified in the three cacao genotypes and discriminant metabolites were identified. Metabolites involved in water stress tolerance such as fructose, trans-aconitic acid, leucine, and hydroxybenzene derivatives were observed in SCA12, the tolerant genotype. CONCLUSION: These results demonstrate the utility of 1H-NMR metabolomics as an essential tool for the analysis of the drought tolerance characteristics of T. cacao.

Assessing the cadmium content of cacao crops in Arauca, Colombia.

The district of Arauca is the second-largest producer of cacao in Colombia. However, despite its quality, it faces issues for export due to levels of cadmium (Cd) higher than the regulatory thresholds. A central question is how it may impact agricultural performance in the presence of Cd in cacao and chocolates. This study quantified Cd in cacao plantations from Arauca. Thus, 180 farms were assessed in the municipalities of Arauquita, Fortul, Saravena, and Tame. Five sample types (soil, irrigation channel sediment, soil litter, cacao seeds, and chocolates) were assessed for Cd. As a technological innovation, the new MXRF technology was used for Cd in chocolates. The sequence of Cd content was soil litter > chocolate > soils > cacao seeds > irrigation-channel sediment. A gradient north-south of Cd content in soil was observed, where highest content was found in farms near the Arauca River, and lower farther away. In irrigation channel sediment, Cd levels averaged 0.07 mg kg-1. The Cd content in cacao seeds was 0.78 mg kg-1 on average. Cd content in chocolates was above the threshold (1.10 mg kg-1 on average, including several cacao mass percentages). These artisanal chocolate bars produced by single farms were near the limit of Cd set by the European Union (up to 0.8 mg kg-1). Therefore, mixing beans from different farms could reduce their Cd content. The present study underscores the complexity of Cd distribution, emphasizing the importance of integrating soil, crop, and landscape features in managing and mitigating Cd levels in cacao.

Birds and bats enhance cacao yield despite suppressing arthropod mesopredation.

Bird- and bat-mediated biocontrol benefits the productivity of tropical commodity crops such as cacao, but the ecological interactions driving these ecosystem services remain poorly understood. Whereas birds and bats prey on herbivorous arthropods, they may also prey on arthropod mesopredators such as ants, with poorly understood consequences for pest biocontrol. We used a full-factorial experiment excluding birds, bats, and ants to assess their effects on (a) the abundance of multiple arthropod groups; (b) predation pressure on arthropods evaluated through artificial sentinel caterpillars; and (c) cacao yield over 1 year in shaded agroforestry systems of native cacao varieties in Peru. Birds and bats increased cacao yield by 118%, which translates in smallholder benefits of ca. US $959 ha-1 year-1 . Birds and bats decreased predation by ants and other arthropods, but contributed to the control of phytophagous taxa such as aphids and mealybugs. By contrast, ant presence increased the abundance of these sap-sucking insects, with negative impacts for cacao yield. Notably, high abundances of the dominant ant Nylanderia sp., known to attend sap-sucking insects, were associated with lower cacao yield along a distance gradient from the closest forest edge. According to these results, arthropod predation by birds and bats, rather than mesopredation by arthropods, was most responsible for increases in cacao yield. Moving forward, detailed research about their trophic interactions will be necessary to identify the cause of such benefits. Retaining and restoring the large benefits of birds and bats as well as minimizing disservices by other taxa in cacao agroforests can benefit from management schemes that prioritize preservation of shade trees and adjacent forests within agroforestry landscapes.

The immunostimulatory effects of Theobroma cacao L. pod husk extract via injection and dietary administrations on Macrobrachium rosenbergii and its resistance against Lactococcus garvieae.

The extract from Theobroma cacao L, pod husk served as the immunostimulant to enhance the immunity and resistance against Lactococcus garvieae of Macrobrachium rosenbergii. In this study, we employed the injection method and dietary administration method to determine the effect of cocoa pod husk (CPH) on M. rosenbergii. The non-specific immune parameters and disease resistance were evaluated after the prawn injected with 1 µg prawn-1 CPH extract (C1), 3 µg prawn-1 CPH extract (C3), and 5 µg prawn-1 CPH extract (C5) for 1, 3, and 7 days. The results showed a significant increase of total haemocyte count (THC), differential haemocyte count (DHC), phenoloxidase (PO) activity, respiratory bursts (RBs), and phagocytic activity and clearance efficiency to L. garvieae. The non-specific immune parameters, physiological parameters, and disease resistance and growth performance were evaluated after the prawn fed with 1 g kg-1 CPH extract diet (CD1), 3 g kg-1 CPH extract diet (CD3) and 5 g kg-1 CPH extract diet (CD5). The results showed a significant increase in all immune parameters and showed a significant decrease in physiological parameters. No significant difference was observed in growth performance of prawn fed with the CPH containing diet. Both injection and dietary method showed a significant increase in disease resistance against to L. garvieae. We therefore recommend that CPH extract can be used as a immunostimulant for prawn by dietary administration to regulate immune responses, and carbohydrate metabolism lead to enhance resistance against pathogen.

Effectiveness of various cacao pod husk extraction byproducts in promoting growth and immunocompetence in Litopenaeus vannamei.

Extracts from plant products can promote growth, can act as immunostimulants, and have antibacterial and antiparasitic properties. These extracts can be used as alternatives to the chemical treatments commonly used to prevent and control disease in aquatic species. Research on the subject has focused on identifying invasive plants or agricultural waste products that can be used as immunostimulants. The present study further identified an optimal means of extracting pectin from cacao pod husks to promote growth performance and immunocompetence in Litopenaeus vannamei that would both reduce production costs and enable waste recycling. The byproducts of pectin extraction from cacao pod husks, that is, dried cacao pod husk powder (DCP), steamed DCP (sDCP), hot water-treated cacao pod husk powder (HCP), hot water-treated cacao pod husk supernatant (HCS), and cacao pod husk pectin (CPH pectin), were used to create five experimental diets, which were administered to five groups. The control group was fed a basal diet. The growth and immunocompetence of the shrimp were determined after 30, 60, 90 and 120 days of feeding. To identify the most cost-effective means of obtaining dried cacao pod husks, this study firstly determined the costs and effectiveness of the sun-drying, dehumidification, and heated-wind drying techniques. According to the results of growth performance, the CPH pectin group had higher survival but lower weight gain than the DCP, sDCP, HCP, and HCS groups did. At 30, 60, and 90 days, the clearance efficiency of the experimental groups was higher than that of the control group. At 60 days, the experimental groups had significantly higher phagocytic activity than the control group did. However, at 30 and 90 days the HCP, HCS and CPH pectin group had higher phagocytic activity. The total hemocyte count, differential hemocyte count, phenoloxidase activity, and respiratory bursts of the CPH pectin group were higher at 30 days but the same as those of the control group at 60 and 90 days. After 120 days of feeding trial, the resistance of L. vannamei fed with diets containing byproducts of pectin extraction from cacao pod husks significantly enhanced compared to that in BD group when they were infected with Vibrio aliginolyticus for 168 h, and the related higher survival rate can be observed in HCP, HCS and CPH pectin groups. The study findings suggest that diet-administered HCP and HCS have long-term immunostimulant potential and that CPH pectin has potential in the early stages of feeding. In addition, when heated air drying was employed, a moisture level of below 10% was obtained within 12 h. The results of this study indicate that adding HCP obtained from heated air-dried cacao pod husks to the feed of L. vannamei is the most cost-effective and sustainable means of promoting long-term growth performance and immunocompetence in the species.

Viability of Moniliophthora roreri on Cocoa Beans Under Microfermentation and Long-Term Survival on Carrier Materials.

The viability of Moniliophthora roreri inoculum was evaluated during the microfermentation process of diseased and healthy pulp-seed masses and on a range of carrier materials: aluminum, cloth, glass, paper, plastic, raffia, and rubber tire. Fungal survival was assessed before the microfermentation (0 h) and every 24 to 96 h by the growth of colonies in potato-dextrose-agar (PDA) and sporulation in seed shells. Colonies of M. roreri and sporulation on seed shells were observed from seeds not submitted to microfermentation. No growth was recovered from diseased cocoa beans after 48 h under the microfermentation. The viability of M. roreri spores recovered from carrier materials was evaluated at 7, 15, 30, 45, and 100 days after inoculation (DAI) by collecting spores and plating them on Sabouraud dextrose yeast extract agar amended with chloramphenicol (50 mg l1). The viability was determined by counting germinated and ungerminated spores under a light microscope (40×) after incubating in a moist chamber at 26 ± 2°C for 72 h. Spores maintained long-term viability on all tested carrier materials toward the end of the experiment (overall 26%) with significant differences (<0.05) among them. Maximum spore viability occurred at 7 and 15 DAI, with cloth and plastic carrier materials considered at high risk of acting as vehicles for the fungal spread. Mathematical models of spore viability over time were fit to the data using the Bayesian information criterion. Findings confirmed the importance of the fermentation process to hamper M. roreri growth and the potential of carrier materials for fungal dispersal.

Cacao Swollen Shoot Viruses in Ghana.

Cacao swollen shoot virus causes cacao swollen shoot disease of Theobroma cacao (cacao) plants. At least six cacao-infecting Badnavirus species-Cacao swollen shoot Togo A virus, Cacao swollen shoot Togo B virus (previously known as Cacao swollen shoot virus), Cacao swollen shoot CE virus, Cacao swollen shoot Ghana M virus, Cacao swollen shoot Ghana N virus, and Cacao swollen shoot Ghana Q virus-are responsible for the swollen shoot disease of cacao in Ghana. Each of these species consists of a multiplicity of strains. The New Juaben strain, the most virulent cacao swollen shoot virus strain in Ghana, belongs to the Cacao swollen shoot Togo B virus species, and is a commonly used strain in laboratory transmission assays. Infection of cacao trees with multiple strains of the virus is common and new evidence suggests that these coinfections may have resulted in the emergence of recombinant strains of the virus. The impact of these emerging recombinant strains on disease severity is uncertain. This review focuses largely on the discovery of cacao swollen shoot virus in Ghana, diversity of the virus strains, molecular characterization, propagation of virus infection in cacao plants, emergence of recombinant virus strains, vector-mediated transmission of the virus, and the management of the cacao swollen shoot disease in Ghana. It also contains sections on the botany and origin of the cacao tree, its introduction to Ghana, the role of cacao swollen shoot disease in facilitating Ghana's independence from Britain, and a brief history of chocolate.

BASIDIN as a New Protein Effector of the Phytopathogen Causing Witche's Broom Disease in Cocoa.

The fungus Moniliophthora perniciosa secretes protein effectors that manipulate the physiology of the host plant, but few effectors of this fungus have had their functions confirmed. We performed functional characterization of a promising candidate effector of M. perniciosa. The inoculation of rBASIDIN at 4 µmol L-1 in the mesophyll of leaflets of Solanum lycopersicum caused symptoms of shriveling within 6 h without the presence of necrosis. However, when sprayed on the plant at a concentration of 11 µmol L-1, it caused wilting symptoms only 2 h after application, followed by necrosis and cell death at 48 h. rBASIDIN applied to Theobroma cacao leaves at the same concentration caused milder symptoms. rBASIDIN caused hydrogen peroxide production in leaf tissue, damaging the leaf membrane and negatively affecting the photosynthetic rate of Solanum lycopersicum plants. Phylogenetic analysis indicated that BASIDIN has orthologs in other phytopathogenic basidiomycetes. Analysis of the transcripts revealed that BASIDIN and its orthologs are expressed in different fungal species, suggesting that this protein is differentially regulated in these basidiomycetes. Therefore, the results of applying BASIDIN allow the inference that it is an effector of the fungus M. perniciosa, with a strong potential to interfere in the defense system of the host plant.

Roasting and Cacao Origin Affect the Formation of Volatile Organic Sulfur Compounds in 100% Chocolate.

Chocolate is a highly appreciated food that develops its characteristic flavors in large part during the roasting of cacao beans. Many functional classes have been noted for their importance to chocolate flavor, including volatile organic sulfur compounds (VSCs). Despite this, the effect of roasting on the concentration of VSCs has never been thoroughly assessed. Here, we studied the effects of roasting temperature, time, and cacao origin on the formation of VSCs. Twenty-seven 100% chocolate samples made from cacao from three different origins and roasted according to an I-optimal experimental design were analyzed by comprehensive gas chromatography with sulfur-selective detection (GCxGC-SCD). For two compounds, dimethyl disulfide and dimethyl trisulfide, the effects of roasting time, roasting temperature, and cacao origin were modelled using response surface methodology and semi-quantified relative concentration. Overall, roasting increased the number of sulfur-containing volatiles present in chocolate, with a total of 28 detected, far more than previously thought. Increased roasting time and especially roasting temperature were found to significantly increase the concentration of VSCs (p < 0.05), while cacao origin effects were only seen for dimethyl disulfide (p < 0.05). The identity of most VSCs remains tentative, and more research is needed to unravel the impact of these volatiles on flavor perception in chocolate.

Microwave-Assisted Extraction Optimization and Effect of Drying Temperature on Catechins, Procyanidins and Theobromine in Cocoa Beans.

Cocoa beans (Theobroma cacao L.) are an important source of polyphenols. Nevertheless, the content of these compounds is influenced by post-harvest processes. In this sense, the concentration of polyphenols can decrease by more than 50% during drying. In this study, the process of procyanidins extraction was optimized and the stability of catechins, procyanidins, and theobromine to different drying temperatures was evaluated. First, the effectiveness of methanol, ethanol, acetone, and water as extract solvents was determined. A Box-Behnken design and response surface methodology were used to optimize the Microwave-Assisted Extraction (MAE) process. The ratios of methanol-water, time, and temperature of extraction were selected as independent variables, whereas the concentration of procyanidins was used as a response variable. Concerning the drying, the samples were dried using five temperatures, and a sample freeze-dried was used as a control. The quantitative analyses were carried out by HPLC-DAD-ESI-IT-MS. The optimal MAE conditions were 67 °C, 56 min, and 73% methanol. Regarding the drying, the maximum contents of procyanidins were obtained at 40 °C. To our knowledge, this is the first time that the stability of dimers, trimers, and tetramers of procyanidins on drying temperature was evaluated. In conclusion, drying at 40 °C presented better results than the freeze-drying method.

Physiological and growth responses of cacao to glyphosate exposure.

Herbicide drift phytotoxicity is a problem in plantation crops due to application failures and unfavorable spray conditions. With the increased use of glyphosate in cacao plantations in Ghana, there are concerns about the effect on cacao growth and productivity from doses that potentially could be expected from drift. The aim of this study was to evaluate the physiological and growth response of young cacao plants exposed to glyphosate. Two field experiments were conducted in randomized blocks, with four replications. Glyphosate was applied at rates 0 to 720 g a.e. ha-1. Crop injury, shikimate accumulation, chlorophyll content, quantum efficiency of PSІІ (Fv/Fm), height, and stem diameter were evaluated. Increased glyphosate rates increased crop injury and shikimate accumulation and decreased chlorophyll content, quantum efficiency of PSІІ (Fv/Fm), and plant growth. Glyphosate rates 360 g a.e. ha-1 or higher resulted in >60% foliar injury and more than 10-fold increase in shikimate accumulation. Glyphosate reduced chlorophyll content to <10 and Fv/Fm to <0.35 at the highest rates. Glyphosate rates ≥180 g a.e. ha-1 reduced height and stem diameter of plants and caused reductions in stand count. Thus, cacao showed sensitivity to glyphosate, and severe injury impaired plant growth.

Infection by Moniliophthora perniciosa reprograms tomato Micro-Tom physiology, establishes a sink, and increases secondary cell wall synthesis.

Witches' broom disease of cacao is caused by the pathogenic fungus Moniliophthora perniciosa. By using tomato (Solanum lycopersicum) cultivar Micro-Tom (MT) as a model system, we investigated the physiological and metabolic consequences of M. perniciosa infection to determine whether symptoms result from sink establishment during infection. Infection of MT by M. perniciosa caused reductions in root biomass and fruit yield, a decrease in leaf gas exchange, and down-regulation of photosynthesis-related genes. The total leaf area and water potential decreased, while ABA levels, water conductance/conductivity, and ABA-related gene expression increased. Genes related to sugar metabolism and those involved in secondary cell wall deposition were up-regulated upon infection, and the concentrations of sugars, fumarate, and amino acids increased. 14C-glucose was mobilized towards infected MT stems, but not in inoculated stems of the MT line overexpressing CYTOKININ OXIDASE-2 (35S::AtCKX2), suggesting a role for cytokinin in establishing a sugar sink. The up-regulation of genes involved in cell wall deposition and phenylpropanoid metabolism in infected MT, but not in 35S::AtCKX2 plants, suggests establishment of a cytokinin-mediated sink that promotes tissue overgrowth with an increase in lignin. Possibly, M. perniciosa could benefit from the accumulation of secondary cell walls during its saprotrophic phase of infection.

Chemical and biological properties of cocoa beans affected by processing: a review.

Cocoa (Theobroma cacao L.) is widely cultivated in tropical countries. The cocoa beans are a popular ingredient of confectionery. Cocoa beans contain various chemicals that contribute to their bioactivity and nutritional properties. There has been increasing interest in developing cocoa beans for "healthy" food products. Cocoa beans have special combination of nutrients such as lipids, carbohydrates, proteins and other compounds of biological activities. The bioactive phytochemicals include methylxanthines, polyphenols, biogenic amines, melanoidins, isoprostanoids and oxalates. These phytochemicals of cocoa are related to various in vivo and in vitro biological activities such as antioxidation, anti-cancer, anti-microbial, anti-inflammation, anti-diabetes, cardiovascular protection, physical improvement, anti-photoaging, anti-depression and blood glucose regulation. The potential of bioactive compounds in cocoa remains to be maximized for food and nutritional applications. The current processing technology promotes the degradation of beneficial bioactive compounds, while maximizing the flavors and its precursors. It is not optimized for the utilization of cocoa beans for "healthy" product formulations. Modifications of the current processing line and non-conventional processing are needed to better preserve and utilize the beneficial bioactive compounds in cocoa beans.