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Cacao and chocolate production is a global industry worth around $133 billion. Full sun cultivation is a modern approach aimed at increasing yields. We evaluated six cacao clones (PS 1319, CCN 10, CCN 51, PH 16, SJ 02, and CP 49) grown under full sun conditions to assess their leaf physiology, leaf structure, yield, and yield components. Leaf physiology was measured through seven gas exchange parameters, while leaf structure was analyzed using eight measurements. For fruit and seed, we evaluated seven yield components. The clones showed differences in gas exchange. Clones PH 16 and PS 1319 had higher net photosynthetic rates per unit of leaf area (A), transpiration rates, and lower leaf internal CO 2 concentrations. These A high values suggest the clones are well-acclimatized to full sun cultivation. Water availability, nutrient supply, and appropriate plant architecture also contributed to this acclimatization. Under high light intensity, the potential quantum yield of photosystem II indicated no photoinhibition, and adaptations in the photosynthetic apparatus were observed, such as lower pigment concentration in clone PH 16. Clones differed in specific leaf area (SLA) and stomatal density (SD). CCN 51 had a higher SLA, while SJ 02 had a higher SD. A significant negative correlation (-0.89) was found between dry bean yield and leaf-to-air water vapor pressure deficit (VpdL), suggesting that VpdL is a crucial parameter for selecting high-performance clones for fertigated full sun cultivation. Yields ranged from 1,220 kg/ha (CCN 10) to 2,900 kg/ha (CCN 51). Full sun cacao farms have high yield potential due to a combination of cloning, management practices, and adequate water and nutrient availability., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Some germination is known to occur during the process of fermentation in cocoa beans. The impact of this biological process on the course of cocoa fermentation is not known and was thus investigated. In order to determine the impact of germination at the molecular level as well as on flavor, an untargeted metabolomics approach using Ultra Performance Liquid Chromatography-Electrospray Ionization-Time of Flight-Mass Spectrometry (UPLC-ESI-ToF-MS) with simultaneous acquisition of low- and high-collision energy mass spectra (MS e ) was performed. Extracts of raw and germinated cocoa beans of the same origin were measured and compared for characteristic differences by unsupervised principal component analysis. OPLS-DA revealed 12-hydroxyjasmonic acid (HOJA) sulfate, (+)-catechin and (-)-epicatechin as most down-regulated compounds as well as two hydroxymethylglutaryl (HMG) glucosides A and B among others as decisive up-regulated compounds in the germinated material. Additionally, further HMG glucosides and 12-hydroxyjasmonic acid could be identified in cocoa for the first time by coelution with isolated and synthesized reference compounds. HOJA sulfate, which has been postulated in cocoa, and HOJA were revealed to impart bitter and astringent taste qualities.

A recently published untargeted metabolomics approach toward marker compounds of cocoa germination revealed and identified 12-hydroxyjasmonic acid sulfate, (+)-catechin, and (-)-epicatechin as the most downregulated compounds and two hydroxymethylglutaryl glucosides (HMG gluc) A and B, among others, as the decisive upregulated compounds in the germinated material. These findings were quantitatively evaluated using ultrahigh-performance liquid chromatography-tandem mass spectrometry not only in previously examined sample material but also in a vastly expanded array of cocoa samples of different provenience and process and in cocoa products such as cocoa liquor and chocolate. Hereby, yields of newly identified HMG gluc derivatives could be determined in raw, fermented, germinated, and alternatively processed cocoa, and isomers of HMG gluc A and B could be established as key process indicators. Based on unsupervised clustering and supervised classification, models could identify germinated samples in testing sets consisting of raw, fermented, and germinated samples.

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., (© 2024. The Author(s).)

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.

Cocoa is a plant with origins in northwestern South America with high relevance in the global economy. Evidence indicates that cocoa is sensitive to a dry climate, under which crop production is reduced. Projections for future climate change scenarios suggest a warmer and drier climate in the Amazon basin. In this paper, we quantify the potential effects in cocoa production due to its edaphoclimatic suitability changes to the Brazilian Amazon biome and account for regional differences in planning occupation territories. We modeled the suitability of cocoa's geographical distribution using an ensemble of 10 correlative models that were run in the "biomod2" library and projected to two future climate scenarios (RCPs 4.5 and 8.5) by 2050. Combining information on climate and soil suitability and installed infrastructure in the macro-regions of the Brazilian Amazon. We defined a zoning system to indicate how cocoa production may respond to climate change according to the current and future suitability model. Our results suggest that a reduction in precipitation and an increase in temperature may promote a reduction in the suitability of cocoa production in the Brazilian Amazon biome. In addition of the areas suitable for cocoa plantation, we found a 37.05% and 73.15% decrease in the areas suitable for intensification and expansion zones under RCP 4.5 and 8.5, respectively, compared with the current scenario. We conclude that there may be a need to expand land to cocoa production in the future, or else it will be necessary to plant a cocoa variety resistant to new climatic conditions. Besides, we recommend procedures to combat illegal deforestation to prevent the most critical climate change scenarios from occurring., Competing Interests: The authors have declared that no competing interests exist.

Cocoa pulp occurs as a by-product of cocoa bean production and can be repurposed to different food applications, such as jams, fruit preparations and beverages, improving the sustainability of cocoa production, as well as the livelihoods of cocoa farmers. In this work, aroma-active compounds of fresh cocoa fruit pulps from different origins were investigated by applying aroma extract dilution analyses in combination with gas chromatography-mass spectrometry/olfactometry for identification. In total, 65 aroma-active compounds were determined in four different pulps originating from Indonesia, Vietnam, Cameroon, and Nicaragua. Vietnamese pulp showed the highest number of aroma-active regions, while Cameroonian pulp accounted for the lowest. Moreover, Cameroonian cocoa pulp showed the lowest FD factors. Overall, the odorants with the highest FD factors were trans -4,5-epoxy-( E )-decenal, 2- and 3-methylbutanoic acid, 3-(methylthio)propanal, 2-isobutyl-3-methoxypyrazine, ( E , E )-2,4-nonadienal, ( E , E )-2,4-decadienal, 4-vinyl-2-methoxyphenol, δ -decalactone, 3-hydroxy-4,5-dimethylfuran-2(5 H )-one, dodecanoic acid, and linalool. This study provides insights into the aroma composition of fresh cocoa pulp from different origins for future food applications.

Background: Cadmium uptake by cacao plants can affect plant growth, consumer health and commercialisation. To develop mitigation strategies, it is essential to identify the soil characteristics that could influence this absorption. To determine the relationships between cadmium absorption and the soil characteristics of cacao areas, the responses at concentrations of 0, 2, 5, 10 and 20 μg g -1 of cadmium in three soils of these areas and an andisol were evaluated, using 120-day-old seedlings of four cultivars of Theobroma cacao L., Results: In the present study, several relationships were found between chemical and physical soil characteristics and available cadmium, such as real and bulk densities, as well as contents of iron, sand, magnesium, potassium, sodium and copper. Additionally, moderate to strong correlations between potassium (r 2  = -0.56) and real density (r 2  = 0.42), with foliar cadmium, were found. Moreover, a differential deleterious effect on cacao growth in variables such as biomass was corroborated in cadmium concentrations from 5 μg g -1 in soils. There were no statistical differences between cultivars with respect to cadmium uptake or plant growth. Finally, a multiple linear regression model is proposed to estimate the foliar cadmium content (r 2  = 0.878)., Conclusion: Some soil characteristics such as density, as well as sand, clay, aluminium, potassium and iron contents, should be considered before establishing cacao crops to avoid cadmium accumulation. The correlation between potassium with foliar cadmium indicated that potassium could be significant in cadmium uptake mitigation strategies. The high correlation between available cadmium and foliar cadmium indicates that the quantification methodology developed using ethylenediaminetetraacetic acid extractant may be a useful diagnostic tool. © 2021 Society of Chemical Industry., (© 2021 Society of Chemical Industry.)

Background: Cacao is important for the economy of many countries in the humid tropics. Its quality is affected when fruits are not collected at the optimal harvest point. The aim of this study was to obtain maturity indices for producers to facilitate the timely harvest and improve the development of fermentation and the sensorial quality of cacao. The growth and respiration processes, and the physicochemical changes during the maturation of three cacao genotypes were determined. Physiological follow-ups measuring fruit length and diameter were performed from 30 days after anthesis to fruit deterioration in the tree., Results: Growth equations were obtained, establishing four maturity stages based on days after anthesis, and fruit length and diameter. Nineteen descriptors were used for the characterization, and through Pearson's correlation and principal component analysis (PCA), five descriptors were identified as representative of the maturity stages of the cacao fruit. PCA results and respiration measurements established that stage 3 presented the highest substrate availability for obtaining good fermentation and quality cacao. This stage showed values between 124 and 197 days after anthesis with lengths between 167.7 and 249.73 mm, and diameters between 64.4 and 95.8 mm, according to the locality., Conclusion: Three growth phases of the cacao fruits were established considering days after anthesis. Both the edaphoclimatic conditions and the cacao genotype characteristics influenced this determination. Stage 3 of fruit maturation shows the best physicochemical conditions for good fermentation. © 2021 Society of Chemical Industry., (© 2021 Society of Chemical Industry.)

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 T cacao ., Competing Interests: The authors declare no competing interest.

Background: In angiosperms the transition to flowering is controlled by a complex set of interacting networks integrating a range of developmental, physiological, and environmental factors optimizing transition time for maximal reproductive efficiency. The molecular mechanisms comprising these networks have been partially characterized and include both transcriptional and post-transcriptional regulatory pathways. Florigen, encoded by FLOWERING LOCUS T (FT) orthologs, is a conserved central integrator of several flowering time regulatory pathways. To characterize the molecular mechanisms involved in controlling cacao flowering time, we have characterized a cacao candidate florigen gene, TcFLOWERING LOCUS T (TcFT). Understanding how this conserved flowering time regulator affects cacao plant's transition to flowering could lead to strategies to accelerate cacao breeding., Results: BLAST searches of cacao genome reference assemblies identified seven candidate members of the CENTRORADIALIS/TERMINAL FLOWER1/SELF PRUNING gene family including a single florigen candidate. cDNA encoding the predicted cacao florigen was cloned and functionally tested by transgenic genetic complementation in the Arabidopsis ft-10 mutant. Transgenic expression of the candidate TcFT cDNA in late flowering Arabidopsis ft-10 partially rescues the mutant to wild-type flowering time. Gene expression studies reveal that TcFT is spatially and temporally expressed in a manner similar to that found in Arabidopsis, specifically, TcFT mRNA is shown to be both developmentally and diurnally regulated in leaves and is most abundant in floral tissues. Finally, to test interspecies compatibility of florigens, we transformed cacao tissues with AtFT resulting in the remarkable formation of flowers in tissue culture. The morphology of these in vitro flowers is normal, and they produce pollen that germinates in vitro with high rates., Conclusion: We have identified the cacao CETS gene family, central to developmental regulation in angiosperms. The role of the cacao's single FT-like gene (TcFT) as a general regulator of determinate growth in cacao was demonstrated by functional complementation of Arabidopsis ft-10 late-flowering mutant and through gene expression analysis. In addition, overexpression of AtFT in cacao resulted in precocious flowering in cacao tissue culture demonstrating the highly conserved function of FT and the mechanisms controlling flowering in cacao.

Beans of cacao (Theobroma cacaoL.) are used to produce a variety of chocolate products. Bioaccumulation of metals at toxic levels through the consumption of contaminated products has been identified as a health concern in humans. Both metal diversity and concentration as well as their interactions in the soil influence essential and non-essential metal uptake in plants; but the effects of these on bioaccumulation of metals in cacao is not understood across diverse soil types. In this study eight metals (Cd, Cr, Cu, Fe, Mn, Ni, Pb, and Zn) were investigated in 12 soil subgroups belonging to four soil orders across 15 locations in Trinidad, with the aim to investigate the effect of soil metal diversity and concentration on metal bioaccumulation in cacao. Soil metals were extracted using five methods (aqua regia, DTPA, Mehlich 3, nitric acid, and water). Cacao leaf metal concentrations were determined using the USEPA 3052 method. Metal extraction efficiency ranged between methods with aqua regia ≥ nitric acid > Mehlich 3 ≥ DTPA ≥ water across all metals. The soil extraction method that best predicted cacao leaf metal concentrations varied with the metal - Mehlich 3 or DTPA for Cd, Ni, Zn; aqua regia, Mehlich 3, or nitric acid for Pb, and water for Mn. A stepwise regression analysis showed that plant metal concentration can be predicted using soil physicochemical characteristics as well as the concentration of metals in the soil. The importance of soil type on cacao leaf metal bioaccumulation is discussed., Competing Interests: Declaration of Competing Interest The authors declare that they have no know competing financial interest or personal relationships that could have appeared to influence the work reported in this Paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The average weight of cocoa beans is not generally taken into account during breeding processes, although it is a trait of interest. Several studies indicate that the weight of the beans has a high heritability in Theobroma cacao. However, the values obtained from different countries for the same clone often vary. In this study, we analyzed the effect of different factors on the weight of the beans. Apart from the clone effect, three main factors had an impact: i) the number of beans per pod: a good filling of the pod with beans tended to limit the weight of the beans, ii) the position of the beans in the pod: beans in the apical part of the pod were significantly lighter than the others and iii) the longer the duration of the fructification cycle the heavier the beans were (positive genetics correlation). These results lead us to propose protocols aimed at normalizing the phenotypic values of the genetic material. To obtain a reliable estimate of the bean weight, the following is proposed: either to use beans obtained from manual pollination to saturate the pods with beans, or to systematically use the number beans in the pods as a covariable.

Food poisoning and environmental pollution are products of excessive chemical usage in Agriculture. In Nigeria, cocoa farmers apply fungicides frequently to control black pod disease (BPD), this practice is life threatening and lethal to the environment. The development of a warning system to detect BPD outbreak can help minimize excessive usage of fungicide by farmers. 8 models (MRM1-MRM8) were developed and 5 (MRM1-MRM5) selected for optimization and performance check. MRM5 (ETAPOD) performed better than the other forecast models. ETAPOD had 100% performance rating for BPD prediction in Ekiti (2009, 2010, 2011 and 2015) with model efficiency of 95-100%. The performance of the model was rated 80% in 2010 and 2015 (Ondo) with model efficiency of 85-90%, 70% in 2011 (Osun) with model efficiency of 81-84%, 60% in 2010 (Ondo and Osun) and 2015 (Osun) with model efficiency of 75-80%, 40% in 2009 (Osun) with model efficiency of 65-69% and 0% 1n 2011 (Ondo) with model efficiency between 0 and 49%. ETAPOD is a simplified BPD detection device for the past, present and future., Competing Interests: The authors have declared that no competing interests exist.

Background: Colombia is known for its production of fine and aromatic cocoa; however, the lack of homogeneity in the ripeness stage of cocoa fruit affects the final quality of cocoa beans. Therefore, the aim of this work was to identify parameters that can be use as indicators of ripeness in cocoa fruit in order to homogenize the characteristics of raw cocoa used in the production of cocoa-products industry. The parameters evaluated were fruit, seed and pod weight, firmness, color, polar and equatorial diameters of the fruit, seed moisture content, total titratable acidity, pH, and total soluble solids of pulp., Results: Factors such as seed weight, firmness, diameters, total soluble solids, pH, and acidity were affected by the clone factor, whereas seed weight, pH, and total titratable acidity were affected by ripeness stage., Conclusion: Identification of indicators of ripeness for cocoa fruit is a complex task due to the influence of the clone on the evolution of the physicochemical characteristics of cocoa fruit during its maturation process. Thus, indicators must be developed for each clone, and at least two parameters among color, pH, and total titratable acidity should be used to determine the ripeness stage of cocoa fruit. © 2019 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., (© 2019 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)

Farmers expect yield reduction of cash crops like cocoa when growing in agroforestry systems compared to monocultures, due to competition for resources, e.g. nutrients and water. However, complementarities between species in the use of resources may improve resource use efficiency and result in higher system performance. Cocoa trees have a shallow rooting system while the rooting characteristics of the associated trees are mainly unknown. This work investigates fine root distribution and production in five cocoa production systems: two monocultures and two agroforestry systems under conventional and organic farming, and a successional agroforestry system. In the organic systems a perennial leguminous cover crop was planted and compost was added, while herbicides and chemical fertilizers were applied in the conventional ones. We measured cocoa fine root parameters in the top 10cm of soil and annual total fine root production at 0-25 and 25-50cm depth. We related the root data with both the aboveground performance (tree and herbaceous biomass), and the cocoa and system yields. Cocoa fine roots were homogenously distributed over the plot area. Around 80% of the total fine roots were located in the upper 25cm of soil. The total fine root production was 4-times higher in the agroforestry systems and the organic monoculture than in the conventional monoculture. The roots of the associated tree species were located in the same soil space as the cocoa roots and, in principle, competed for the same soil resources. The cocoa yield was lower in the agroforestry systems, but the additional crops generated a higher system yield and aboveground biomass than the conventional cocoa monocultures, implying effective resource exploitation. The leguminous cover crop in the organic monoculture competed with the cocoa trees for nutrients, which may explain the lower cocoa yield in this system in contrast with the conventional monoculture., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: A series of fluorescence indices (anthocyanin, flavonol, chlorophyll and nitrogen balance) were deployed to detect the pigments and colourless flavonoids in cacao pods of three commercial cacao (Theobroma cacao L.) genotypes (QH1003, KKM22 and MCBC1) using a fast and non-destructive multiparametric fluorescence sensor. The aim was to determine optimum harvest periods (either 4 or 5 months after pod emergence) of commercial cacao based on fluorescence indices of cacao development and bean quality., Results: As pod developed, cacao exhibited a rise with the peak of flavonol occurring at months 4 and 5 after pod maturity was initiated while nitrogen balance showed a decreasing trend during maturity. Cacao pods contained high chlorophyll as they developed but chlorophyll content declined significantly on pods that ripened at month 5., Conclusion: Cacao pods harvested at months 4 and 5 can be considered as commercially-ready as the beans have developed good quality and comply with the Malaysian standard on cacao bean specification. Thus, cacao pods can be harvested earlier when they reach maturity at month 4 after pod emergence to avoid germinated beans and over fermentation in ripe pods harvested at month 5. © 2018 Society of Chemical Industry., (© 2018 Society of Chemical Industry.)

Cocoa beans and cocoa products contain considerable amounts of bioactive compounds. Harvesting cocoa fruit too early or too late may have effects on the phenolic and alkaloid concentrations of the cocoa powder. Fermentation, a primary processing used to transform cocoa beans to cocoa powder, may also influence the contents of bioactive compounds. In this study, proanthocyanidins, the major compounds in cocoa polyphenols, caffeine and theobromine of cocoa beans, were evaluated at different maturities at harvest, and with different fermentation durations, with and without the addition of a commercial enzyme, Pectinex® Ultra SP-L. The amounts of proanthocyanidins, caffeine and theobromine, and the antioxidant capacities of the unfermented cocoa beans increased as the fruits matured. The values ranged from 16.12-27.28 g catechin equivalents (CE)/100 g dry weight (DW); 99.66-173.61 mg/100 g DW; 556.39-948.84 mg/100 g DW; 23.23-26.32 mol Trolox equivalents (TE)/100 g DW, respectively. Prolonged fermentation with or without the addition of pectinase, from three to seven days, significantly reduced the amounts of these compounds present. Fermentation using the enzyme significantly reduced the proanthocyanidin content and antioxidant capacity of the cocoa powder, with the overall means decreasing from 8.93-4.93 g CE/100 g DW and from 15.81-12.95 g mol TE/100 g DW, respectively. Two-way ANOVA analyses showed that the proanthocyanidins, caffeine, theobromine contents and the antioxidant capacity of cocoa beans were strongly dependet to their stages of maturity, fermentation methods and fermentation duration.

Cocoa agroforestry systems have the potential to conserve biodiversity and provide environmental or ecological benefits at various nested scales ranging from the plot to ecoregion. While integrating organic practices into cocoa agroforestry may further enhance these potentials, empirical and robust data to support this claim is lacking, and mechanisms for biodiversity conservation and the provision of environmental and ecological benefits are poorly understood. A field study was conducted in the Eastern Region of Ghana to evaluate the potential of organic cocoa agroforests to conserve native floristic diversity in comparison with conventional cocoa agroforests. Shade tree species richness, Shannon, Simpson's reciprocal and Margalef diversity indices were estimated from 84 organic and conventional cocoa agroforestry plots. Species importance value index, a measure of how dominant a species is in a given ecosystem, and conservation status were used to evaluate the conservation potential of shade trees on studied cocoa farms. Organic farms recorded higher mean shade tree species richness (5.10 ± 0.38) compared to conventional farms (3.48 ± 0.39). Similarly, mean Shannon diversity index, Simpson's reciprocal diversity index and Margalef diversity index were significantly higher on organic farms compared to conventional farms. According to the importance value index, fruit and native shade tree species were the most important on both organic and conventional farms for all the cocoa age groups but more so on organic farms. Organic farms maintained 14 native tree species facing a conservation issue compared to 10 on conventional cocoa farms. The results suggest that diversified organic cocoa farms can serve as reservoirs of native tree species, including those currently facing conservation concerns thereby providing support and contributing to the conservation of tree species in the landscape., Competing Interests: The authors have declared that no competing interests exist.

Theobroma cacao, the source of cocoa, is a crop of particular importance in many developing countries. Availability of elite planting material is a limiting factor for increasing productivity of Theobroma cacao; therefore, the development of new strategies for clonal propagation is essential to improve farmers' incomes and to meet increasing global demand for cocoa. To develop a more efficient embryogenesis system for cacao, tissue was transformed with a transgene encoding a fusion of Leafy Cotyledon 2 (TcLEC2) to a glucocorticoid receptor domain (GR) to control nuclear localization of the protein. Upon application of the glucocorticoid dexamethasone (dex), downstream targets of LEC2 involved in seed-development were up-regulated and somatic embryos (SEs) were successfully regenerated from TcLEC2-GR transgenic flower and leaf tissue in large numbers. Immature SEs regenerated from TcLEC2-GR leaves were smaller in size than immature SEs from floral tissue, suggesting a different ontogenetic origin. Additionally, exposure of TcLEC2-GR floral explants to dex increased the number of SEs compared to floral explants from control, non-transgenic trees or from TcLEC2-GR floral explants not treated with dex. Testing different durations of exposure to dex indicated that a three-day treatment produced optimal embryo regeneration. Leaf derived SEs were successfully grown to maturity, converted into plants, and established in the greenhouse, demonstrating that these embryos are fully developmentally competent. In summary, we demonstrate that regulating TcLEC2 activity offers a powerful new strategy for optimizing somatic embryogenesis pipelines for cacao., Competing Interests: The authors have the following interests: Andrew S. Fister is employed by Pairwise Plants and Yufan Zhang by Essenlix Corporation. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Cacao (Theobroma cacao L.) has traditionally been considered a crop that requires shade, and consequently it is implemented under agroforestry systems (AFs) in order to regulate the level of incident solar radiation. However, optimal shade levels for this tree crop may vary depending on the climate conditions of where it is grown. Here we analyzed the physiological performance of cacao under three different AFs in the Colombian Amazon that differed in solar radiation patterns: high (HPAR), medium (MPAR), or low (LPAR) mean daily incident radiation. The physiological performance was characterized using photosynthetic variables in leaves such as light- and CO2-response curves, chlorophyll a fluorescence parameters, and total chlorophyll and carotenoid contents, in conjunction with other leaf functional traits. Cacao trees exposed to HPAR showed an improved physiological performance as compared to those from the other two AFs. Compared to MPAR and LPAR, cacao trees in HPAR doubled the rate of net carbon assimilation and reached higher maximum rates of RuBisCO carboxylation and RuBP regeneration. Moreover, cacao trees in HPAR presented photoprotection mechanisms that avoided photoinhibition, which was accompanied by a greater non-photochemical quenching coefficient and biochemical and morphological adjustments (low chlorophyll but higher carotenoid contents and low specific leaf area) compared to cacao trees from the other AFs. Overall, our results show that, due to the high cloud cover in the Colombian Amazon, cacao plantations under conditions of sparse shade maximized their carbon use, showing an improved physiological performance as a result of higher photosynthetic rates and energy dissipation mechanisms. If the crop were managed with sparse shade, the paradigm that favors the cultivation of cacao under shade would be called into question in the Colombian Amazon and other regions with similar climatic conditions., Competing Interests: The authors have declared that no competing interests exist.

Cocoa black pod disease caused by Phytophthora megakarya and reduced soil fertility are major constraints to cocoa production resulting in high yield losses. In the absence of effective control measures and constraints related to the use of chemical fungicides and fertilizers, there is a need to develop additional and sustainable disease and fertilization management strategies. With the lack of studies related to the use of compost in cocoa cultivation, the present study aims to evaluate the potential of cocoa pod husk (CPH)-based compost as a soil amendment to reduce the severity of cocoa black pod disease and enhance plant growth. In vitro antagonism test showed that compost water extracts (CWE) reduced mycelial growth with inhibition rate reaching 100% associated with microorganisms. Disease score of cocoa plantlets grown on compost-amended soils significantly reduced compared to plantlets grown on non-amended soil (control). All compost rates tested significantly increased populations of actinomycetes and fungi and biological activity in the soil. Compost application increased soil pH and majority of the essential elements but decreased Al content, which is toxic to cocoa growth in acidic soils. Soil application of compost at the dose of 20% (v/v) significantly increased stem length and number of leaves compared to the control. This study shows that CPH-based compost can not only improve soil fertility and cocoa growth but also reduce cocoa black pod disease severity by direct effects on inoculums level in the soil and by inducing resistance in the plant.

Annual estimates of CO2 and dissolved carbon concentrations in the soil profile provide valuable insight into the dynamics of organic matter in soil and the effect of changes to vegetation cover. The aim of this study was to observe the spatial influence of litter decomposition in the first few centimeters of the soil for CO2 fluxes and to describe the processing of soil organic matter throughout the soil profile by comparing three small tropical watersheds. Data were collected biweekly for six months, from December 2015 to May 2016. CO2 was measured using an infrared gas analyzer in fixed chambers and the dissolved carbon of soil solution was analyzed in a TOC analyzer. No differences were found in the total soil CO2 fluxes (control flux treatments) between the three study areas. In both cacao agroforestry systems (managed and unmanaged), total CO2 fluxes were influenced by the decomposition of litter. CO2 emissions in the soil profile of the cacao agroforestry systems were highly variable, compared to the preserved forest, and highly dependent on the soil characteristics attributed to the type of vegetation cover. Although a definite pattern between the temperature and soil moisture was not identified, these parameters showed a strong relationship in controlling the release of CO2 between treatments. The organic and inorganic dissolved carbon patterns in the soil solution of the three areas revealed different responses of soil organic matter processing related to soil characteristics and vegetation. The results confirm the hypothesis that the top of soils (total CO2 fluxes) of both cacao agroforestry systems (managed and unmanaged) emits fluxes of CO2, which do not differ statistically from the preserved forest. However, depending on the soil characteristics, the cacao agroforestry system can result in an accumulation of CO2 and dissolved inorganic carbon in the soil profile that is prone to being transported by hydrological routes to groundwater and stream water., Competing Interests: The authors have declared that no competing interests exist.

Value chain analyses have focused mainly on collaboration between chain actors, often neglecting collaboration "beyond the chain" with non-chain actors to tackle food security, poverty and sustainability issues in the landscapes in which these value chains are embedded. Comparing conventional and advanced value chain collaborations involving small-scale cocoa farmers in Ghana, this paper analyzes the merits of a more integrated approach toward value chain collaboration. It particularly asks whether advanced value chain collaboration targeting cocoa-producing areas potentially offers an entry point for implementing a landscape approach. The findings detail current chain actors and institutions and show how advanced value chain collaboration has a greater positive impact than conventional value chain collaboration on farmers' social, human and natural capital. The paper concludes that the integrated approach, focus on learning, and stable relationships with small-scale farmers inherent in advanced value chain collaboration makes it both more sustainable and effective at the local level than conventional approaches. However, its scope and the actors' jurisdictional powers and self-organization are too limited to be the sole tool in negotiating land use and trade-offs at the landscape level. To evolve as such would require certification beyond the farm level, partnering with other landscape stakeholders, and brokering by bridging organizations.

Cocoa beans from different geographical and genetic origins show distinct fermentation dynamics which result in different chocolate qualities. In order to understand the effects of genetic improvement of cocoa plants, in this work volatile compounds and proteins profiles of beginning and end of the fermentation from different cocoa hybrids (CEPEC2004, PH15, PS1319, SJ02) were searched. Moreover, sensorial characterization of the produced chocolate from these hybrids was performed. According to the results obtained, different volatile compounds were identified in fermented beans and in the chocolate produced. Chocolate from CEPEC2004 was the most accepted by judges and correlated with sweet and bitter taste which can be explained by the presence of desired flavor compounds, such as 2,3-butanediol and 2-methyl-1-butanol. A higher presence of acids (undesirable compounds) was observed in chocolates samples from PS1319 hybrid, that have resulted in the low acceptance by judges. In addition, MALDI-TOF MS analysis showed that during fermentation the protein profile was different among the hybrids, which indicates this kind of compounds also contributes to the cocoa-specific flavor., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Dutch policies have advocated sustainable commodity value chains, which have implications for the landscapes from which these commodities originate. This study examines governance and policy options for sustainability in terms of how ecosystem services are addressed in cocoa, soy, tropical timber and palm oil value chains with Dutch links. A range of policies addressing ecosystem services were identified, from market governance (certification, payments for ecosystem services) to multi-actor platforms (roundtables) and public governance (policies and regulations). An analysis of policy narratives and interviews identified if and how ecosystem services are addressed within value chains and policies; how the concept has been incorporated into value chain governance; and which governance options are available. The Dutch government was found to take a steering but indirect role in all the cases, primarily through supporting, financing, facilitating and partnering policies. Interventions mainly from end-of-chain stakeholders located in processing and consumption countries resulted in new market governance, notably voluntary sustainability standards. These have been successful in creating awareness of some ecosystem services and bringing stakeholders together. However, they have not fully addressed all ecosystem services or stakeholders, thus failing to increase the sustainability of value chains or of the landscapes of origin. We argue that chains sourced in tropical landscapes may be governed more effectively for sustainability if voluntary, market policy tools and governance arrangements have more integrated goals that take account of sourcing landscapes and impacts along the entire value chain. Given the international nature of these commodities. These findings have significance for debates on public-private approaches to value chain and landscape governance.

Theobroma cacao is cultivated in the shade, in a so-called 'Cabruca' system, in intercropped with Erithryna or other tree species of economic value, and in full sun as a monoculture in irrigated or chemically-irrigated systems. Since it is a species quite intolerant to wind, it is practically impossible to implant cacao crops under full exposure to the sun, or in areas of frequent winds, without the protection of windbreaks, using arboreal species around the area of culture in the form of box. Wind can cause mechanical stimuli in plants, affecting their growth and development. The objective of this work was to evaluate the photosynthetic changes in mature leaves and the molecular, biochemical and ultrastructural changes in young and mature leaves of the CCN 51 cloned genotype of T. cacao subjected to intermittent (IW) and constant (CW) wind, with velocities of 2.5, 3.5 and 4.5 m s-1, during 3, 6 and 12 h of exposure. It was verified that CW and IW, considering different exposure times, interfered directly in stomatal conductance (gs), transpiration (E) and water use efficiency (WUE), causing a reduction of the photosynthetic rate (A) in mature leaves. In addition, the pulvinus and blade of young and mature leaves, exposed to IW and CW with different exposure times (3 and 12 h), showed marked macroscopic and microscopic mechanical injuries resulting from the constant leaf movement. At both speeds, there was rupture of the cell nuclear membrane in pulvinus and the mesophyll tissues, mainly in the young leaves. On the other hand, in young and mature leaves exposed to CW and IW at different speeds and exposure times, there was lipid peroxidation, increased activity of guaiacol (GPX) and ascorbate (APX) peroxidases in most treatments; and altered expression of transcripts of psba and psbo genes related to the phothosynthetic apparatus and Cu-Zn-sod and per genes related to antioxidative enzymes at the rate of 4.5 m s-1. Younger leaves were more intolerant to mechanical stress caused by the wind, since presented greater macro and microscopic damages and, consequently, greater molecular, biochemical and ultrastructural changes. High wind speeds can seriously compromise the development of young leaves of T. cacao plants and affect their productivity., Competing Interests: The authors have declared that no competing interests exist.

The use of ants for biological control of insect pests was the first reported case of conservation biological control. Direct and indirect community interactions between ants and pests lead to differential spatial pattern. We investigated spatial interactions between mirids, the major cocoa pest in West Africa and numerically dominant ant species, using bivariate point pattern analysis to identify potential biological control agents. We assume that potential biological control agents should display negative spatial interactions with mirids considering their niche overlap. The mirid/ant data were collected in complex cacao-based agroforestry systems sampled in three agroecological areas over a forest-savannah gradient in Cameroon. Three species, Crematogaster striatula Emery (Hymenoptera: Formicidae), Crematogaster clariventris Mayr (Hymenoptera: Formicidae), and Oecophylla longinoda Latreille (Hymenoptera: Formicidae) with high predator and aggressive behaviors were identified as dominant and showed negative spatial relationships with mirids. The weaver ant, O. longinoda was identified as the only potential biological control agent, considering its ubiquity in the plots, the similarity in niche requirements, and the spatial segregation with mirids resulting probably from exclusion mechanisms. Combining bivariate point pattern analysis to good knowledge of insect ecology was an effective method to identify a potentially good biological control agent.

Cocoa fermentation is a crucial step for the development of cocoa aroma and precursors of high quality cocoa and by-products. This bioprocess has been studied for years to understand cocoa chemical changes but some matters concerning changes in fat content remain that are investigated in this work. Changes in the quantity (free and total fat), extractability and composition of cocoa butter were assessed in samples from Madagascar, the Dominican Republic and Ecuador. Increases in free fat content were highlighted in samples from each origin thanks to the use of the 'soxtec' solvent method, which preserves the integrity of the butter. A 4.71% increase in free fat was measured in the Ecuadorian samples fermented for 144 h. Conversely, total fat content remained stable throughout fermentation. Protein and polyphenol contents decreases were linked to fat content augmentation by a strong negative interaction. Triglyceride and total and linked fatty acid kinetics (0 to 6 days) of the butter remained statistically stable during fermentation, as did unsaponifiable matter. The origin of fermentation had a predominant and significant impact on composition, revealed by PCA. This work underlines and explains the importance of fermentation process in improving yield of fat that can be extracted while preserving the composition of this cocoa butter. This study highlights an interaction in cocoa unfermented or partially fermented beans. This phenomenon causes butter content retention but is slowly broken after 72 h fermentation. Therefore, fermentation appears to be also necessary to enhance the cocoa butter content extracted from the nibs., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Drought is worldwide considered one of the most limiting factors of Theobroma cacao production, which can be intensified by global climate changes. In this study, we aimed to investigate the phenotypic correlation among morphological characteristics of cacao progenies submitted to irrigation and drought conditions and their partitions into direct and indirect effects. Path analysis with phenotypic plasticity index was used as criteria for estimation of basic and explanatory variables. The experiment was conducted in a greenhouse at the Cacao Research Center (CEPEC), Ilhéus, Bahia, Brazil, in a randomized block 21 x 2 factorial arrangement [21 cacao progenies obtained from complete diallel crosses and two water regimes (control and drought)] and six replications. In general, drought conditions influenced biomass production in most progenies, causing significant reductions in total leaf area, leaf number, leaf biomass, fine-roots length (diameter <1 mm), root volume and root area for considered drought intolerant. All progenies showed alterations in growth due to drought. Phenotypic plasticity was most strongly pronounced in root volume. Stem and root diameters, as well as stem dry biomass were the growth variables with the greatest direct effects on root volume under drought conditions, these characters being indicated in screening of cacao progenies drought tolerant.

Aim and Background: We present a typology of cacao agroforest systems in Colombian Amazonia. These systems had yet to be described in the literature, especially their potential in terms of biodiversity conservation. The systems studied are located in a post-conflict area, and a deforestation front in Colombian Amazonia. Cacao cropping systems are of key importance in Colombia: cacao plays a prime role in post conflict resolution, as cacao is a legal crop to replace illegal crops; cacao agroforests are expected to be a sustainable practice, promoting forest-friendly land use., Material and Methods: We worked in 50 x 2000 m2 agroforest plots, in Colombian Amazonia. A cluster analysis was used to build a typology based on 28 variables characterised in each plot, and related to diversity, composition, spatial structure and light availability for the cacao trees. We included variables related to light availability to evaluate the amount of transmitted radiation to the cacao trees in each type, and its suitability for cacao ecophysiological development., Main Results: We identified 4 types of cacao agroforests based on differences concerning tree species diversity and the impact of canopy spatial structure on light availability for the cacao trees in the understorey. We found 127 tree species in the dataset, with some exclusive species in each type. We also found that 3 out of the 4 types identified displayed an erosion of tree species diversity. This reduction in shade tree species may have been linked to the desire to reduce shade, but we also found that all the types described were compatible with good ecophysiological development of the cacao trees., Main Conclusions and Prospects: Cacao agroforest systems may actually be achieving biodiversity conservation goals in Colombian Amazonia. One challenging prospect will be to monitor and encourage the conservation of tree species diversity in cacao agroforest systems during the development of these cropping systems, as a form of forest-friendly management enhancing sustainable peace building in Colombia.

Cocoa seed storage proteins play an important role in flavour development as aroma precursors are formed from their degradation during fermentation. Major proteins in the beans of Theobroma cacao are the storage proteins belonging to the vicilin and albumin classes. Although both these classes of proteins have been extensively characterized, there is still limited information on the expression and abundance of other proteins present in cocoa beans. This work is the first attempt to characterize the whole cocoa bean proteome by nano-UHPLC-ESI MS/MS analysis using tryptic digests of cocoa bean protein extracts. The results of this analysis show that >1000 proteins could be identified using a species-specific Theobroma cacao database. The majority of the identified proteins were involved with metabolism and energy. Additionally, a significant number of the identified proteins were linked to protein synthesis and processing. Several proteins were also involved with plant response to stress conditions and defence. Albumin and vicilin storage proteins showed the highest intensity values among all detected proteins, although only seven entries were identified as storage proteins. A comparison of MS/MS data searches carried out against larger non-specific databases confirmed that using a species-specific database can increase the number of identified proteins, and at the same time reduce the number of false positives. The results of this work will be useful in developing tools that can allow the comparison of the proteomic profile of cocoa beans from different genotypes and geographic origins. Data are available via ProteomeXchange with identifier PXD005586., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cocoa beans are produced all across the humid tropics under different environmental conditions provided by the region but also by the season and the type of production system. Agroforestry systems compared to monocultures buffer climate extremes and therefore provide a less stressful environment for the understory cocoa, especially under seasonally varying conditions. We measured the element concentration as well as abiotic stress indicators (polyamines and total phenolic content) in beans derived from five different production systems comparing monocultures and agroforestry systems and from two harvesting seasons. Concentrations of N, Mg, S, Fe, Mn, Na, and Zn were higher in beans produced in agroforestry systems with high stem density and leaf area index. In the dry season, the N, Fe, and Cu concentration of the beans increased. The total phenolic content increased with proceeding of the dry season while other abiotic stress indicators like spermine decreased, implying an effect of the water availability on the chemical composition of the beans. Agroforestry systems did not buffer the variability of stress indicators over the seasons compared to monocultures. The effect of environmental growing conditions on bean chemical composition was not strong but can contribute to variations in cocoa bean quality.

Background: Using conventional pesticides in crop protection has raised serious environmental concerns and there is therefore a need for integrated pest management (IPM) methods. In this paper, we found that the spacing of trees can impact disease, which could result in a reduction in pesticide applications and may act as a potential IPM method. We studied Frosty Pod Rot (FPR) in 20 cacao agroforests in Costa Rica (Upala region)., Results: Using a generalized linear mixed model, we analyzed the impact of the neighborhood composition and distance from a studied cacao individual on its individual FPR incidence. We found that the number of cacao tree neighbors in a radius of 3.7 m and the number of fruit trees in a radius of 4.3 m had a significant negative influence on the incidence of FPR on individual cacao trees. Moreover, cacao tree neighbors had the most significant local influence compared to the neighborhood of other taller categories such as fruit or forest trees., Conclusion: The mechanisms involved are related to the barrier effect, due to the effectiveness of the cacao tree's architecture as an efficient barrier against FPR spore dispersal. This paper provides new insights into optimization of the spatial environment around each host as an original IPM method. © 2017 Society of Chemical Industry., (© 2017 Society of Chemical Industry.)

Cocoa plant (Theobroma cacao L.) is native from South America and it represents one of the most significant "bio-cultural" resources of Mesoamerica, since it is a region where it was domesticated and had a relevance as ritual drink and as currency in many pre-hispanic cultures until the arrival of the Spaniards who spread its use worldwide, and became it one of the most consumed commodity goods. Through this research, an alternative is proposed to address the problem of cultivars through the introduction of a wide variety of cocoa plants in traditional agroforestry systems, in synergy with the inoculation of nitrogen-fixing and insoluble phosphor solubilizing edaphic bacterial consortia. Four cultivars of improved grafted cocoa plants were introduced in a traditional agroforestry plot and three fertilization treatments were applied: application of biofertilizer, application of chemical fertilizer and control. Measurements of height, stem diameter, number of leaves and branches were recorded at 2 and 12 months after planting and rhizosphere microbial populations were characterized. Growth results showed good potential for all studied cultivars and it was observed that biofertilization foresees significant effects in some of the growth indicators of cocoa plant. Thereby, plant associations in an agroforestry system could be favorable to promote fruit development and resistance to pests and diseases., (Copyright © 2017 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.)

Pterospermum kingtungense C.Y.Wu ex Hsue is a typical tree species living in the relatively adverse limestone habitat. Due to its excellent wood quality and big size, it is an important timber resource which caused its endangered. We firstly provide the data resources by reporting an annotated transcriptome assembly. 203 million unique Illumina RNA-seq reads were produced with totally 50,333 transcripts, among which 48,778 transcripts were annotated. By a global comparison of homology between P. kingtungense and cacao, we identified 9,507 single copy orthologues and 990 P. kingtungense specific genes. GO enrichment analyses indicate that P. kingtungense specific genes are enriched in defense response, implying potential adaptation to limestone environment. As to cell compartment, the genes are enriched in thylakoid component. Consistently, KEGG enrichment indicates that genes are enriched in photosynthesis. In addition, we identified two genes under positive selection in P. kingtungense species. These results suggest that P. kingtungense have strong photosynthetic capacity, which related to vegetation growth. Our work provides the genomic resources of a limestone specific tree with economic importance to local society and suggests possible mechanism on its characteristics on the limestone adaption and excellent wood properties, which will be important for its conservation and sustainable utilization.

The unique benefits of wild pollinators to the productivity of agricultural crops have become increasingly recognized in recent decades. However, declines in populations of wild pollinator species, largely driven by the conversion of natural habitat to agricultural land and broad-spectrum pesticide use often lead reductions in the provision of pollination services and crop production. With growing evidence that targeted pollinator conservation improves crop yield and/or quality, particularly for pollination specialist crops, efforts are increasing to substitute agriculturally intensive practices with those that alleviate some of the negative impacts of agriculture on pollinators and the pollination services they provide, in part through the provision of suitable pollinator habitat. Further, similarities between the responses of some pollinators and predators to habitat management suggest that efforts to conserve pollinators may also encourage predator densities. We evaluated the effects of one habitat management practice, the addition of cacao fruit husks to a monoculture cacao farm, on the provision of pollination services and the densities of two groups of entomophagous predators. We also evaluated the impacts of cacao fruit husk addition on pollen limitation, by crossing this habitat manipulation with pollen supplementation treatments. The addition of cacao fruit husks increased the number of fruits per tree and along with hand pollination treatments, increased final yields indicating a promotion of the pollination ecosystem service provided by the specialist pollinators, midges. We also found that cacao fruit husk addition increased the densities of two predator groups, spiders and skinks. Further, the conservation of these predators did not inhibit pollination through pollinator capture or deterrence. The findings show that, with moderate habitat management, both pollinator and predator conservation can be compatible goals within a highly specialized plant-pollinator system. The effectiveness of this habitat manipulation may be attributable to the increased availability of alternative habitat and food resources for both pollinators and predators. The results exemplify a win-win relationship between agricultural production and biological conservation, whereby agricultural practices to support vital pollinators and pollination services can increase production as well as support species conservation., (© 2016 by the Ecological Society of America.)

The taxonomy of an actinobacterial strain, designated JJY4 T , was established using a polyphasic approach. JJY4 T was isolated from the rhizosphere of Chromolaena odorata in Yaoundé (Cameroon) during a project for the selection of biological control agents. Strain JJY4 T exhibited antimicrobial activities against bacteria, fungi, and oomycetes. Strain JJY4 T also exhibited the traits of plant growth-promoting rhizobacteria such as the solubilization of inorganic phosphate, production of siderophores and indole-3-acetic acid, and 1-aminocyclopropane-1-carboxylate deaminase activity. In planta assays performed on cocoa plantlets confirmed that strain JJY4 T exhibited strong abilities to promote plant growth and protect against Phytophthora megakarya, the main causal agent of cocoa pod rot. The formation of rugose-ornamented spores in spiral spore chains by strain JJY4 T is a typical feature of members found in the Streptomyces violaceusniger clade and, similar to some members of the clade, strain JJY4 T produces geldanamycin. A phylogenetic analysis based on 16S rRNA gene sequences confirmed this classification and suggests that strain JJY4 T be added to the subclade constituted of the type strains Streptomyces malaysiensis DSM 41697 T and Streptomyces samsunensis DSM 42010 T . However, DNA-DNA relatedness and physiological characteristics allowed for the differentiation of strain JJY4 T from its closest phylogenetic relatives. Based on these results, strain JJY4 T (=NRRL B-65369, =NBRC 112705) appears to represent a novel species in the S. violaceusniger clade for which the proposed name is Streptomyces cameroonensis sp. nov.

Cocoa agroforests like the cabrucas of Brazil's Atlantic forest are among the agro-ecosystems with greatest potential for biodiversity conservation. Despite a global trend for their intensification, cocoa agroforests are also being abandoned for socioeconomic reasons especially on marginal sites, because they are incorporated in public or private protected areas, or are part of mandatory set-asides under Brazilian environmental legislation. However, little is known about phylogenetic structure, the processes of forest regeneration after abandonment and the conservation value of former cabruca sites. Here we compare the vegetation structure and composition of a former cabruca 30-40 years after abandonment with a managed cabruca and mature forest in the Atlantic forest region of Espirito Santo, Brazil. The forest in the abandoned cabruca had recovered a substantial part of its original structure. Abandoned cabruca have a higher density (mean ± CI95 %: 525.0 ± 40.3 stems per ha), basal area (34.0 ± 6.5 m 2 per ha) and species richness (148 ± 11.5 species) than managed cabruca (96.0 ± 17.7; 24.15 ± 3.9 and 114.5 ± 16.0, respectively) but no significant differences to mature forest in density (581.0 ± 42.2), basal area (29.9.0 ± 3.3) and species richness (162.6 ± 15.5 species). Thinning (understory removal) changes phylogenetic structure from evenness in mature forest to clustering in managed cabruca, but after 30-40 years abandoned cabruca had a random phylogenetic structure, probably due to a balance between biotic and abiotic filters at this age. We conclude that abandoned cocoa agroforests present highly favorable conditions for the regeneration of Atlantic forest and could contribute to the formation of an interconnected network of forest habitat in this biodiversity hotspot.

This study aimed to estimate the combining ability, of T. cacao genotypes preselected for drought tolerance through diallel crosses. The experiment was conducted under greenhouse conditions at the Cacao Research Center (CEPEC), Ilhéus, Bahia, Brazil, in a completely randomized block design, in an experimental arrangement 21 x 2 [21 complete diallel crosses and two water regimes (control and stressed)]. In the control, soil moisture was kept close to field capacity, with predawn leaf water potential (ΨWL) ranging from -0.1 to -0.5 MPa. In the drought regime, the soil moisture was reduced gradually by decreasing the amount of water application until ΨWL reached -2.0 to -2.5 MPa. Significant differences (p < 0.05) were observed for most morphological attributes analyzed regarding progenies, water regime and their interactions. The results of the joint diallel analysis revealed significant effects between general combining ability (GCA) x water regimes and between specific combining ability (SCA) x water regimes. The SCA 6 genetic material showed high general combining ability for growth variables regardless of the water regime. In general, the water deficit influenced the production of biomass in most of the evaluated T. cacao crosses, except for SCA-6 x IMC-67, Catongo x SCA, MOC-01 x Catongo, Catongo x IMC-67 and RB-40 x Catongo. Multivariate analysis showed that stem diameter (CD), total leaf area (TLA), leaf dry biomass (LDB), stem dry biomass (SDB), root dry biomass (RDB), total dry biomass (TDB), root length (RL), root volume (RV), root diameter (RD) <1 mm and 1 <(RD) <2 mm were the most important growth parameters in the separation of T. cacao genotypes in to tolerant and intolerant to soil water deficit.

Analysis of the complex composition of cocoa beans provides fundamental information for evaluating the quality and nutritional aspects of cocoa-based food products, nutraceuticals and supplements. Cameroon, the world's fourth largest producer of cocoa, has been defined as "Africa in miniature" because of the variety it habitats. In order to evaluate the nutritional characteristics of cocoa beans from five different regions of Cameroon, we studied their polyphenolic content, volatile compounds and fatty acids composition. The High Performance Thin Layer Chromatography (HPTLC) analysis showed that the Mbalmayo sample had the highest content of theobromine (11.6 mg/g) and caffeic acid (2.1 mg/g), while the Sanchou sample had the highest level of (-)-epicatechin (142.9 mg/g). Concerning fatty acids, the lowest level of stearic acid was found in the Mbalmayo sample while the Bertoua sample showed the highest content of oleic acid. Thus, we confirmed that geographical origin influences the quality and nutritional characteristics of cocoa from these regions of Cameroon.

Elevated concentration of cadmium (Cd) in cacao beans has raised serious concerns about the chocolate consumption on human health. Accumulation of Cd in cacao bean in southern Ecuador has been related to soil contamination. In this study, soil fractionation approach was used to identify available Cd pools in the soils and to correlate these Cd pools with bean Cd concentration and soil test indexes. The distribution of soil Cd fractions decreased in the order: oxidizable > acid-soluble > residual > reducible >> water-soluble (+exchangeable). Oxidizable and acid-soluble fractions accounted for 59 and 68% of the total recoverable Cd for the 0-5 and 5-15 cm soil depth, respectively. Acid-soluble fraction was closely related to bean-Cd, with correlation coefficients (r) of 0.70 and 0.81 (P < 0.01) for the 0-5 and 5-15 cm soil depth, respectively. Acid-soluble Cd was significantly correlated with 0.01 M HCl- (r = 0.99, P < 0.01) or Mehlich 3- extractable Cd (r = 0.97, P < 0.01). These results indicate that acid-soluble Cd fraction is an important part of available Cd pool. Since approximately 60% of Cd in the cacao-growing soils is related to the acid-soluble fraction and bound to organic matter, remediation of the contaminated soils should consider to the dynamics of soil pH and organic matter content., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

One of the main drivers of tropical forest loss is their conversion to oil palm, soy or cacao plantations with low biodiversity and greatly reduced carbon storage. Southeast Asian cacao plantations are often established under shade tree cover, but are later converted to non-shaded monocultures to avoid resource competition. We compared three co-occurring cacao cultivation systems (3 replicate stands each) with different shade intensity (non-shaded monoculture, cacao with the legume Gliricidia sepium shade trees, and cacao with several shade tree species) in Sulawesi (Indonesia) with respect to above- and belowground biomass and productivity, and cacao bean yield. Total biomass C stocks (above- and belowground) increased fivefold from the monoculture to the multi-shade tree system (from 11 to 57 Mg ha-1), total net primary production rose twofold (from 9 to 18 Mg C ha-1 yr-1). This increase was associated with a 6fold increase in aboveground biomass, but only a 3.5fold increase in root biomass, indicating a clear shift in C allocation to aboveground tree organs with increasing shade for both cacao and shade trees. Despite a canopy cover increase from 50 to 93%, cacao bean yield remained invariant across the systems (variation: 1.1-1.2 Mg C ha-1 yr-1). The monocultures had a twice as rapid leaf turnover suggesting that shading reduces the exposure of cacao to atmospheric drought, probably resulting in greater leaf longevity. Thus, contrary to general belief, cacao bean yield does not necessarily decrease under shading which seems to reduce physical stress. If planned properly, cacao plantations under a shade tree cover allow combining high yield with benefits for carbon sequestration and storage, production system stability under stress, and higher levels of animal and plant diversity.

Legumains are cysteine proteases related to plant development, protein degradation, programmed cell death, and defense against pathogens. In this study, we have identified and characterized three legumains encoded by Theobroma cacao genome through in silico analyses, three-dimensional modeling, genetic expression pattern in different tissues and as a response to the inoculation of Moniliophthora perniciosa fungus. The three proteins were named TcLEG3, TcLEG6, and TcLEG9. Histidine and cysteine residue which are part of the catalytic site were conserved among the proteins, and they remained parallel in the loop region in the 3D modeling. Three-dimensional modeling showed that the propeptide, which is located in the terminal C region of legumains blocks the catalytic cleft. Comparing dendrogram data with the relative expression analysis, indicated that TcLEG3 is related to the seed legumain group, TcLEG6 is related with the group of embryogenesis activities, and protein TcLEG9, with processes regarding the vegetative group. Furthermore, the expression analyses proposes a significant role for the three legumains during the development of Theobroma cacao and in its interaction with M. perniciosa., (Copyright © 2015 Universidade Estadual de Santa Cruz, CNPJ: 40738999/0001-95. Published by Elsevier Masson SAS.. All rights reserved.)

The interaction amongst papain-like cysteine-proteases (PLCP) and their substrates and inhibitors, such as cystatins, can be perceived as part of the molecular battlefield in plant-pathogen interaction. In cacao, four cystatins were identified and characterized by our group. We identified 448 proteases in cacao genome, whereof 134 were cysteine-proteases. We expressed in Escherichia coli a PLCP from cacao, named TcCYSPR04. Immunoblottings with anti-TcCYSPR04 exhibited protein increases during leaf development. Additional isoforms of TcCYSPR04 appeared in senescent leaves and cacao tissues infected by Moniliophthora perniciosa during the transition from the biotrophic to the saprophytic phase. TcCYSPR04 was induced in the apoplastic fluid of Catongo and TSH1188 cacao genotypes, susceptible and resistant to M. perniciosa, respectively, but greater intensity and additional isoforms were observed in TSH1188. The fungal protein MpNEP induced PLCP isoform expression in tobacco leaves, according to the cross reaction with anti-TcCYSPR04. Several protein isoforms were detected at 72 hours after treatment with MpNEP. We captured an active PLCP from cacao tissues, using a recombinant cacao cystatin immobilized in CNBr-Sepharose. Mass spectrometry showed that this protein corresponds to TcCYSPR04. A homology modeling was obtained for both proteins. In order to become active, TcCYSPR04 needs to lose its inhibitory domain. Molecular docking showed the physical-chemical complementarities of the interaction between the cacao enzyme and its inhibitor. We propose that TcCYSPR04 and its interactions with cacao cystatins are involved in the senescence and necrosis events related to witches' broom symptoms. This molecular interaction may be the target for future interventions to control witches' broom disease.

Aim: To isolate and characterize rhizobacteria from Theobroma cacao with antagonistic activity against Phytophthora palmivora, the causal agent of the black pod rot, which is one of the most important diseases of T. cacao., Methods and Results: Among 127 rhizobacteria isolated from cacao rhizosphere, three isolates (CP07, CP24 and CP30) identified as Pseudomonas chlororaphis, showed in vitro antagonistic activity against P. palmivora. Direct antagonism tested in cacao detached leaves revealed that the isolated rhizobacteria were able to reduce symptom severity upon infection with P. palmivora Mab1, with Ps. chlororaphis CP07 standing out as a potential biocontrol agent. Besides, reduced symptom severity on leaves was also observed in planta where cacao root system was pretreated with the isolated rhizobacteria followed by leaf infection with P. palmivora Mab1. The production of lytic enzymes, siderophores, biosurfactants and HCN, as well as the detection of genes encoding antibiotics, the formation of biofilm, and bacterial motility were also assessed for all three rhizobacterial strains. By using a mutant impaired in viscosin production, derived from CP07, it was found that this particular biosurfactant turned out to be crucial for both motility and biofilm formation, but not for the in vitro antagonism against Phytophthora, although it may contribute to the bioprotection of T. cacao., Conclusions: In the rhizosphere of T. cacao, there are rhizobacteria, such as Ps. chlororaphis, able to protect plants against P. palmivora., Significance and Impact of the Study: This study provides a theoretical basis for the potential use of Ps. chlororaphis CP07 as a biocontrol agent for the protection of cacao plants from P. palmivora infection., (© 2015 The Society for Applied Microbiology.)

Criollo-type cacao trees are an important pool of genes with potential to be used in cacao breeding and selection programs. For that reason, we assessed the diversity and population structure of Criollo-type trees (108 cultivars with Criollo phenotypic characteristics and 10 Criollo references) using 12 simple sequence repeat (SSR) markers. Cultivars were selected from 7 demes in the Soconusco region of southern Mexico. SSRs amplified 74 alleles with an average of 3.6 alleles per population. The overall populations showed an average observed heterozygosity of 0.28, indicating heterozygote deficiency (average fixation index F = 0.50). However, moderate allelic diversity was found within populations (Shannon index for all populations I = 0.97). Bayesian method analysis determined 2 genetic clusters (K = 2) within individuals. In concordance, an assignment test grouped 37 multilocus genotypes (including 10 references) into a first cluster (Criollo), 54 into a second (presumably Amelonado), and 27 admixed individuals unassigned at the 90% threshold likely corresponding to the Trinitario genotype. This classification was supported by the principal coordinate analysis and analysis of molecular variance, which showed 12% of variation among populations (FST = 0.123, P < 0.0001). Sampled demes sites (1- 7) in the Soconusco region did not show any evidence of clustering by geographic location, and this was supported by the Mantel test (Rxy = 0.54, P = 0.120). Individuals with high Criollo lineage planted in Soconusco farms could be an important reservoir of genes for future breeding programs searching for fine, taste, flavor, and aroma cocoa.

Combining crop plants with other plant species in agro-ecosystems is one way to enhance ecological pest and disease regulation mechanisms. Resource availability and microclimatic variation mechanisms affect processes related to pest and pathogen life cycles. These mechanisms are supported both by empirical research and by epidemiological models, yet their relative importance in a real complex agro-ecosystem is still not known. Our aim was thus to assess the independent effects and the relative importance of different variables related to resource availability and microclimatic variation that explain pest and disease occurrence at the plot scale in real complex agro-ecosystems. The study was conducted in cacao (Theobroma cacao) agroforests in Cameroon, where cocoa production is mainly impacted by the mirid bug, Sahlbergella singularis, and black pod disease, caused by Phytophthora megakarya. Vegetation composition and spatial structure, resource availability and pest and disease occurrence were characterized in 20 real agroforest plots. Hierarchical partitioning was used to identify the causal variables that explain mirid density and black pod prevalence. The results of this study show that cacao agroforests can be differentiated on the basis of vegetation composition and spatial structure. This original approach revealed that mirid density decreased when a minimum number of randomly distributed forest trees were present compared with the aggregated distribution of forest trees, or when forest tree density was low. Moreover, a decrease in mirid density was also related to decreased availability of sensitive tissue, independently of the effect of forest tree structure. Contrary to expectations, black pod prevalence decreased with increasing cacao tree abundance. By revealing the effects of vegetation composition and spatial structure on mirids and black pod, this study opens new perspectives for the joint agro-ecological management of cacao pests and diseases at the plot scale, through the optimization of the spatial structure and composition of the vegetation.

Coconut, cocoa and arecanut are commercial plantation crops that play a vital role in the Indian economy while sustaining the livelihood of more than 10 million Indians. According to 2012 Food and Agricultural organization's report, India is the third largest producer of coconut and it dominates the production of arecanut worldwide. In this study, three Plant Growth Promoting Rhizobacteria (PGPR) from coconut (CPCRI-1), cocoa (CPCRI-2) and arecanut (CPCRI-3) characterized for the PGP activities have been sequenced. The draft genome sizes were 4.7 Mb (56% GC), 5.9 Mb (63.6% GC) and 5.1 Mb (54.8% GB) for CPCRI-1, CPCRI-2, CPCRI-3, respectively. These genomes encoded 4056 (CPCRI-1), 4637 (CPCRI-2) and 4286 (CPCRI-3) protein-coding genes. Phylogenetic analysis revealed that both CPCRI-1 and CPCRI-3 belonged to Enterobacteriaceae family, while, CPCRI-2 was a Pseudomonadaceae family member. Functional annotation of the genes predicted that all three bacteria encoded genes needed for mineral phosphate solubilization, siderophores, acetoin, butanediol, 1-aminocyclopropane-1-carboxylate (ACC) deaminase, chitinase, phenazine, 4-hydroxybenzoate, trehalose and quorum sensing molecules supportive of the plant growth promoting traits observed in the course of their isolation and characterization. Additionally, in all the three CPCRI PGPRs, we identified genes involved in synthesis of hydrogen sulfide (H2S), which recently has been proposed to aid plant growth. The PGPRs also carried genes for central carbohydrate metabolism indicating that the bacteria can efficiently utilize the root exudates and other organic materials as energy source. Genes for production of peroxidases, catalases and superoxide dismutases that confer resistance to oxidative stresses in plants were identified. Besides these, genes for heat shock tolerance, cold shock tolerance and glycine-betaine production that enable bacteria to survive abiotic stress were also identified.

Aims: To investigate the effects of the endophyte Bacillus subtilisALB629 on the growth of cacao seedlings at early developmental stage and to evaluate its antimicrobial properties., Methods and Results: Germinating cacao seeds were inoculated with ALB629, and seedlings growth was evaluated 30 days later. Significant increase (P < 0·05) was observed in the root system (up to 30%), leaf area (14%) and stem height (7·6%). ALB629 colonized the entire plant, prevailing over indigenous micro-organisms. In addition, it was tested in vitro, by pairing assays, and showed antagonistic effect against the phytopathogenic fungi Moniliophthora perniciosa, Colletotrichum sp. and C. gossypii. When tested in cacao-grafting procedure in the field, ALB629 increased the grafting success rate (24%), indicating its protective effect. In addition, this Bacillus secretes an antagonist compound, as shown by the antifungal activity of the cell-free culture., Conclusions: Bacillus subtilisALB629 promotes cacao root growth, besides promoting growth of the aerial part of cacao seedlings. It has antimicrobial properties and produces an antifungal compound., Significance and Impact of the Study: ALB629 presented beneficial characteristics for cacao cultivation, being a good biological control agent candidate. Furthermore, it is a potential source of antifungal compound with potential for commercial exploitation., (© 2014 The Society for Applied Microbiology.)