Cacao Cultivars Research Articles

Ca-oxalate crystals are involved in cadmium storage in a high Cd accumulating cultivar of cacao

Cadmium (Cd) concentrations in cacao often exceed food limits. A better understanding of the molecular mechanisms of Cd accumulation in cacao trees is necessary to advance Cd mitigation strategies. This study explores Cd distribution and speciation (i.e., chemical form) within the vegetative organs of a high Cd accumulating cacao cultivar growing in soil with background Cd concentrations (0.28 mg Cd kg−1) by synchrotron radiation-based micro- and nano-X-ray fluorescence imaging and X-ray absorption spectroscopy on duplicate samples of roots, young branches, mature branches, and leaves. In both the roots and branches, the incorporation of Cd in Ca-oxalate crystals with binding to oxygen-ligands was identified as a major mechanism of Cd regulation. The Ca-oxalate crystals were more abundant in the branches than in the roots. We suggest that the incorporation of Cd into the Ca-oxalate crystals may limit the transfer of Cd to other organs like the leaves and beans. The roots also featured Cd-sulfur species in dilated cells of the wood, probably for the retention of Cd in the roots by strong binding with Cd to thiol-containing ligands. A Cd hotspot in large, secreting channels of the branches suggested that mucilage may play a role in the transport of Cd within cacao trees. The leaves had local Cd enrichments in the parenchymatous tissues and displayed a higher Cd concentration in the mesophyll compared to the epidermis. These insights in Cd distribution and speciation underscore that Theobroma cacao L. develops original Cd detoxification strategies, strongly different from non-tolerant species such as cereals.

Rootstock, scion, and microbiome contributions to cadmium mitigation in five Indonesian cocoa cultivars.

Reducing levels of heavy metals such as cadmium (Cd) in cacao beans has become an important priority for cacao production in Indonesia. Current mitigation strategies revolve around breeding and the use of soil ameliorants, and in the future, the soil microbiome may also have the potential to reduce Cd bioavailability and uptake by cacao trees. However, there remains a need for locally specific recommendations for low-Cd-accumulating cacao cultivars and knowledge of native beneficial bacteria and fungi. In a greenhouse study using field soil supplemented with Cd, five cacao clones (MCC02, M01, S1, S2, and ICCRI 9) were grafted in a fully factorial design to the same hybrid half sibling rootstocks, plant uptake was measured before and after the addition of cadmium nitrate, and rhizosphere microbial communities were characterized. Rootstock, scion, and graft combinations all significantly affected plant Cd levels, but the ranking of clones differed between low and high Cd soils. Twenty-six bacterial taxa and one fungal taxon were associated with Cd uptake. These results highlight the continued importance of breeding as a cadmium mitigation strategy and support the potential for the soil microbiome to contribute to reducing cadmium uptake in cacao.

Integrating new variables into a framework to support cacao denomination of origin: a case study in Southwest Colombia.

Cocoa quality plays a pivotal role in establishing denominations of origin, with genotypes, geography, climate and soil conditions being key variables. However, these factors have not been comprehensively explored in defining cacao denominations of origin. The present study addresses this gap by laying the foundation for cacao denomination of origin, focusing on the Buenaventura region on Colombia's Pacific coast. Our goal is to provide a holistic understanding of the elements underpinning cacao denomination of origin, emphasizing Buenaventura's unique cocoa quality and geographical significance. Through the Buenaventura case, we propose a robust framework applicable to other cacao-producing regions, elevating the recognition and value of cacao denomination of origin. Our framework encompasses geography, agronomy, genetics, microbial diversity, pests and diseases and cocoa quality. In a pioneering move, we propose a cacao denomination of origin in Colombia, specifically examining Bajo Calima, Sabaletas and Cisneros within Buenaventura region. Buenaventura stands out for its cocoa quality, characterized by fruity flavors attributed to the rich biodiversity of the lowland rainforest. Our analysis indicates specific geographical indicators for each of the study zones, with Buenaventura identified as a region with natural characteristics to produce fine flavour cocoa products. Each zone exhibited a high differentiation and diversity of cacao cultivars. Buenaventura has the potential to be designated as a future denomination of origin for cacao from the Pacific region of Colombia, characterized by its unique fruity-aroma chocolates. Our framework is adaptable to other cacao-producing regions, facilitating the establishment of denominations of origin within the cocoa industry and agriculture. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Phyllosphere microbial diversity and specific taxa mediate within-cultivar resistance to Phytophthora palmivora in cacao.

The oomycete pathogen Phytophthora palmivora, which causes black pod rot (BPR) on cacao (Theobroma cacao L.), is responsible for devastating yield losses worldwide. Genetic variation in resistance to Phytophthora spp. is well documented among cacao cultivars, but variation has also been observed in the incidence of BPR even among trees of the same cultivar. In light of evidence that the naturally occurring phyllosphere microbiome can influence foliar disease resistance in other host-pathogen systems, it was hypothesized that differences in the phyllosphere microbiome between two field accessions of the cultivar Gainesville II 164 could be responsible for their contrasting resistance to P. palmivora. Bacterial alpha diversity was higher but fungal alpha diversity was lower in the more resistant accession MITC-331, and the accessions harbored phyllosphere microbiomes with distinct community compositions. Six bacterial and 82 fungal amplicon sequence variants (ASVs) differed in relative abundance between MITC-333 and MITC-331, including bacterial putative biocontrol agents and a high proportion of fungal pathogens, and nine fungal ASVs were correlated with increased lesion development. The roles of contrasting light availability and host mineral nutrition, particularly potassium, are also discussed. Results of this preliminary study can be used to guide research into microbiome-informed integrated pest management strategies effective against Phytophthora spp. in cacao. IMPORTANCE Up to 40% of the world's cacao is lost each year to diseases, the most devastating of which is black pod rot, caused by Phytophthora palmivora. Though disease resistance is often attributed to cacao genotypes (i.e., disease-resistant rootstocks), this study highlights the role of the microbiome in contributing to differences in resistance even among accessions of the same cacao cultivar. Future studies of plant-pathogen interactions may need to account for variation in the host microbiome, and optimizing the cacao phyllosphere microbiome could be a promising new direction for P. palmivora resistance research.

From soil to cacao bean: Unravelling the pathways of cadmium translocation in a high Cd accumulating cultivar of Theobroma cacao L.

The research on strategies to reduce cadmium (Cd) accumulation in cacao beans is currently limited by a lack of understanding of the Cd transfer pathways within the cacao tree. Here, we elucidated the transfer of Cd from soil to the nib (seed) in a high Cd accumulating cacao cultivar. Here, we elucidated the transfer of Cd from soil to the nib (seed) in a high Cd accumulating cacao cultivar through Cd stable isotope fractionation, speciation (X-Ray Absorption Spectroscopy), and localization (Laser Ablation Inductively Coupled Plasma Mass Spectrometry). The plant Cd concentrations were 10-28 higher than the topsoil Cd concentrations and increased as placenta< nib< testa< pod husk< root< leaf< branch. The retention of Cd in the roots was low. Light Cd isotopes were retained in the roots whilst heavier Cd isotopes were transported to the shoots (Δ 114/110 Cd shoot-root = 0.27 ± 0.02 ‰ (weighted average ± standard deviation)). Leaf Cd isotopes were heavier than Cd in the branches (Δ 114/110 Cd IF3 leaves-branch = 0.18 ± 0.01 ‰), confirming typical trends observed in annual crops. Nibs and branches were statistically not distinguishable (Δ 114/110 Cd nib-branch = -0.08‰ ± 0.06 ‰), contrary to the leaves and nibs (Δ 114/110 Cd nib-IF3 leaves = -0.25‰ ± 0.05 ‰). These isotope fractionation patterns alluded to a more direct transfer from branches to nibs rather than from leaves to nibs. The largest fraction (57%) of total plant Cd was present in the branches where it was primarily bound to carboxyl-ligands (60-100%) and mainly localized in the phloem rays and phelloderm of the bark. Cadmium in the nibs was mainly bound to oxygen ligands (60-90%), with phytate as the most plausible ligand. The weight of evidence suggested that Cd was transferred like other nutrients from root to shoot and accumulated in the phloem rays and phelloderm of the branches to reduce the transfer to foliage. Finally, the data indicated that the main contribution of nib Cd was from the phloem tissues of the branch rather than from leaf remobilization. This study extended the limited knowledge on Cd accumulation in perennial, woody crops and revealed that the Cd pathways in cacao are markedly different than in annual crops.

Screening of cultivars for tissue culture response and establishment of genetic transformation in a high-yielding and disease-resistant cultivar of Theobroma cacao

A highly efficient transformation protocol is a prerequisite to developing genetically modified and genome-edited crops. A tissue culture system spanning culture initiation from floral material to conversion of embryos to plants has been tested and improved in Theobroma cacao. Nine cultivars were screened for their tissue culture response and susceptibility to Agrobacterium transfer-DNA delivery as measured through transient expression. These key factors were used to determine the genetic transformability of various cultivars. The high-yielding, disease-resistant cultivar INIAPG-038 was selected for stable transformation and the method was further optimized. Multiple transgenic events were produced using two vectors containing both yellow fluorescent protein and neomycin phosphotransferase II genes. A two-fold strategy to improve both T-DNA delivery and secondary somatic embryogenesis rates was conducted to improve overall transformation frequency. The use of Agrobacterium strain AGL1 and cotyledon tissue derived from secondary somatic embryos ranging in size between 4 to 10 mm resulted in the highest T-DNA delivery efficiency. Furthermore, the use of higher concentrations of basal salts and cupric sulfate in the medium increased the frequency of explants producing greater than ten embryos by five-fold and four-fold during secondary somatic embryogenesis, respectively. Consequently, an optimal combination of all these components resulted in a successful transformation of INIAPG-038 with 3.7% frequency at the T0 plant-level. Grafting transgenic scions with undeveloped roots to non-transgenic seedlings with healthy roots helped make plantlets survive and facilitated quick transplantation to the soil. The presented strategy can be applied to improve tissue culture response and transformation frequency in other Theobroma cacao cultivars.

Cadmium distribution in soils, soil litter and cacao beans: a case study from Colombia

Cadmium is a toxic non-essential metal for almost all living systems. It is one of the biggest challenges for farmlands and the food chain because of its toxic effect on human health. This research aims to determine the Cd content in soils, litter and cacao beans, following the Cd fluxes within each cacao system using the two-dimensional electrical resistivity tomography technique. The study was carried out in four farms located in the Magdalena basin in Antioquia, Colombia. The farms showed a heterogeneity in relation to cacao cultivars, altitude, topography and geology. The soil cation electrical capacity, pH and soil organic matter levels, as well as the Al, Al3+H+, Ca, K, Mg and P contents were measured at different depths. Moreover, the Cd content was correlated to the resistivity values of samples taken in situ using ERT. Soil Al3+H+ and the altitude of the farms fitted as the best predictors of the beans’ Cd content. Furthermore, the Cd content in soils from the assessed farms ranged between 1.22 and 2.03 mg kg−1. The Cd content in cocoa beans ranged from 0.07 to 1.44 mg kg−1, with a value of 0.40 mg kg−1 on average. The resistivity values obtained in field showed a high correlation with the soil Cd content determined (R2 = 0.82). The predictive tomography plots highlighted topsoil Cd dynamics between litter, amendments and fertilizers. Therefore, these results underlie the utility of the combined geophysical techniques and soil chemical properties including the analysis of fertilizer amendments to improve the understanding of Cd dynamic.

The evaluation of java fine flavor cocoa propagation through somatic embryogenesis technique for germplasm preservation

Somatic embryogenesis is one of the newest technology that applied for the mass production of cocoa. This research aims to evaluate the regeneration rate of somatic embryos through somatic embryogenesis propagation techniques on java fine flavor cocoa. Cultivars in this study are ICCRI 01, ICCRI 02, DR 1, DR 2, DRC 16, DR 38, PNT 16, and PNT 30. Observations include parameters to determine the percentage of primary callus and embryogenic callus formation and the number of somatic embryos produced. Based on data, the ability of callus to produce primary embryos is highly dependent on plant cultivars and explant sources. Five cultivars showed a higher regeneration rate using explants from the petal part, while the rest showed a higher regeneration rate using explants from the staminode section. Embryogenic callus from each cacao cultivar has the same basic structure: a nodular friable structure consisting of many embryonic cells. Some fine flavor cacao cultivars that were able to produce callus and primary somatic embryos could not produce secondary somatic embryos and plantlets. However, two cultivars, which had low potential in producing primary embryos, had the high ability to produce secondary somatic embryos and develop into plantlets.

Growth and nutritional responses of wild and domesticated cacao genotypes to soil Cd stress

Cadmium (Cd), a toxic non-essential metal, is easily accumulated in cacao tissues. This represents a risk for cacao exportation, and consequently it affects the economic well-being of the resource-poor-small-producers in Latin America. A greenhouse experiment was conducted with 53 wild and domesticated cacao genotypes to determine their response to Cd in terms of growth and Cd and essential nutrients accumulation. Cacao seedlings were grown for 6 months in an acidic soil with or without added Cd. The total concentration of macro (Ca, K, Mg, N and P) and micronutrients (B, Cu, Fe, Mn and Zn) as well as Cd were measured in shoots along with growth (biometric) parameters after harvest. The results revealed that even if there was a wide range of Cd concentrations among genotypes, there was a reduction in the concentration of essential nutrients in genotypes grown in Cd spiked soils, however these concentrations were not significantly different from the control. In the case of growth parameters, the effects of Cd were diverse across all genotypes some of them being more tolerant to Cd stress than others. Thus, different growth responses to Cd stress are related to a genotype effect. Based on their lower Cd concentration, a total of 11 cacao genotypes (AYP-22, PAS-105, UGU-126, ICT-1026, ICT-1087, ICT-1189, ICT-1292, PH-17, CCN-51, ICS-39 and TSH-565) are proposed here as low Cd-accumulating genotypes. Therefore, these genotypes are potentially useful as rootstock to reduce uptake and transport of Cd, especially in economically important cacao cultivars.

Cloning and in silico analysis revealed a genetic variation in osmotin-encoding genes in an Indonesian local cacao cultivar

Theobroma cacao L. is an important Indonesian estate crop, which suffers from biotic and abiotic stresses. TcOSM, which encodes osmotin as a response to pathogens and environmental stresses, is, therefore, a focus of interest in this research, aiming to characterize TcOSM in an Indonesian local cacao cultivar. Bioinformatics queries for putative TcOSM were performed against the reference genome of a Criollo-type cacao cultivar. Based on nucleotide sequence determination, our results revealed two genes, TcOSM1 and TcOSM2, which have the highest similarity (≥ 90\%) to the cacao reference genes. Heterozygosity was detected in the TcOSM1-encoding gene, which contained two overlapping peaks in Sanger-sequencing chromatograms. One of the alleles resulted from a single nucleotide change (G to A), leading to a same-sense mutation that did not substitute corresponding alanine residue. Homology modeling using Phyre2 and structural alignment (superimposition) was conducted to examine the influence of genetic variations in TcOSM sequences upon the global protein structures. The result showed no significant changes (RMSD ≤ 0.206 Å, TM-score &gt; 0.5) in tertiary protein structures. Altogether, this research succeeded in characterizing TcOSM while providing a fundamental study for future cacao biotechnology endeavors.

Shade tree species affect gas exchange and hydraulic conductivity of cacao cultivars in an agroforestry system.

Shade tolerance is a widespread strategy of rainforest understory plants. Many understory species have green young stems that may assimilate CO2 and contribute to whole-plant carbon balance. Cacao commonly grows in the shaded understory and recent emphasis has been placed on diversifying the types of trees used to shade cacao plants to achieve additional ecosystem services. We studied three agricultural cacao cultivars growing in the shade of four timber species (Cedrela odorata L., Cordia thaisiana Agostini, Swietenia macrophylla King and Tabebuia rosea (Bertol) A.D.C.) in an agroforestry system to (i) evaluate the timber species for their effect on the physiological performance of three cacao cultivars; (ii) assess the role of green stems on the carbon economy of cacao; and (iii) examine coordination between stem hydraulic conductivity and stem photosynthesis in cacao. Green young stem photosynthetic CO2 assimilation rate was positive and double leaf CO2 assimilation rate, indicating a positive contribution of green stems to the carbon economy of cacao; however, green stem area is smaller than leaf area and its relative contribution is low. Timber species showed a significant effect on leaf gas exchange traits and on stomatal conductance of cacao, and stem water-use efficiency varied among cultivars. There were no significant differences in leaf-specific hydraulic conductivity among cacao cultivars, but sapwood-specific hydraulic conductivity varied significantly among cultivars and there was an interactive effect of cacao cultivar × timber species. Hydraulic efficiency was coordinated with stem-stomatal conductance, but not with leaf-stomatal conductance or any measure of photosynthesis. We conclude that different shade regimes determined by timber species and the interaction with cacao cultivar had an important effect on most of the physiological traits and growth variables of three cacao cultivars growing in an agroforestry system. Results suggested that C. odorata is the best timber species to provide partial shade for cacao cultivars in the Barlovento region in Venezuela, regardless of cultivar origin.

Rapid And Non-Destructive Evaluation On Cacao Pigments, Flavonoids And Nitrogen Contents During Pod Development And Maturity Using A Fluorescence Sensor

The detection of pigments in cacao pods together with colourless flavonoids serves as a useful indicator for pod maturity using a fast and non-destructive multiparametric fluorescence sensor. In this study, the contents of anthocyanin, flavonol, chlorophyll and nitrogen balance were determined monthly (1-5 months) after flower fertilization as pod developed and matured using a fluorescence-based portable sensor on cacao pods from five different clones of DESA1, KKM22, KKM25, MCBC1 and PBC221. There were significant differences (P≤0.05) observed between the interaction of five different cacao clones and pod development periods in flavonol, chlorophyll and nitrogen balance contents. As pods developed, anthocyanin and flavonol accumulated while the content of chlorophyll decreased only when pod matured with nitrogen balance showed a decreasing trend in cacao pods. Among these clones, as expected, natural red appearance in cacao pods of DESA1 showed significantly highest index of anthocyanin (0.637), following by KKM22 (0.255). In addition, there was no significant difference observed in KKM25, MCBC1 and PBC221 for anthocyanin content. During pod development, MCBC1 showed the least content in flavonol (P≤0.05) and the chlorophyll contents in KKM22 and MCBC1 were lower compared to other clones. As a conclusion, non-destructive fluorescence-based indices can be used to measure the pigments and flavonoids in cacao which can provide valuable non-destructive indicators for cacao pod maturity across different cacao cultivars.

Genetic classification of Vietnamese cacao cultivars assessed by SNP and SSR markers

Vietnam has the potential to become a larger cocoa producer as the country experiences climatic conditions favorable to the growth of the crop. However, to enable a high cacao quality, genetic methods are required to identify and conserve promising cacao cultivars. Previous research as discussed by Everaert et al. (2017), using 14 microsatellite (SSR) markers, showed a genetic characterization of Vietnamese cacao, but additional research is required to further clarify the genetic background of the different Vietnamese cacao cultivars. Therefore, in this study, the Vietnamese dataset was enlarged, and a classification model was developed. Firstly, we compared the classification strength of the 14 previously applied SSR markers and 42 additional single nucleotide polymorphism (SNP) markers, either separately or combined, using 53 international reference cacao cultivars. Secondly, the most classifying marker set was used to examine the genetic associations of the Vietnamese cultivars to the reference cultivars, using Bayesian clustering and principal coordinate analysis (PCoA). The classification method based on the combined marker set performed best and could subdivide the Vietnamese cultivars in three clusters. The first cluster was strongly related to Nanay, the second to Criollo/Trinitario, and the third predominantly to Amelonado and Criollo/Trinitario. For all marker sets, an apparent high genetic diversity could be observed for the total Vietnam collection. The obtained knowledge provides a strong genetic basis for the Vietnamese cacao industry. In addition, the proposed marker set and the analysis workflow could be applied worldwide for future breeding programs and further production of origin chocolate.

Cadmium accumulation and allocation in different cacao cultivars

Cadmium (Cd) is a biologically non-essential heavy metal that can cause toxic effects in plants, animals and humans already at low concentrations compared to other metals. After Cd concentrations in cacao beans of various provenances, particularly from Latin America, were found to exceed the new regulations enforced by the European Union in 2019, there is an urgent need to find measures to lower Cd accumulation in cacao beans to acceptable values. In this research, the long-term cacao cultivar trial CEDEC-JAS in northern Honduras was used to investigate differences between 11 cultivars in Cd uptake and translocation. Sampling of various plant parts, including rootstocks, scions, leaves and beans, from three replicate trees per cultivar and the soil around each tree was conducted at this site. Results indicate that concentrations of available soil Cd were more closely correlated with Cd concentrations of the rootstocks (R2 = 0.56), scions (R2 = 0.59) and leaves (R2 = 0.46) than with bean Cd concentrations (R2 = 0.26). In addition, Cd concentrations of rootstocks, scions and leaves showed close relationships to available soil Cd concentrations, with no significant differences between the cultivars. In contrast, bean Cd concentrations showed only weak correlations to available soil Cd and Cd concentrations in the vegetative plant parts, but significant variation among cultivars. Three cultivars, which were analysed in more detail, showed significant differences in Cd concentrations of mature beans, but not of immature beans. These results suggest that cultivar-related differences in bean Cd concentrations primarily result from differences in Cd loading during bean maturation, possibly due to cultivar-specific differences in the xylem-to-phloem transfer of Cd. The results show that selection of cultivars with low Cd transfer from vegetative parts into the beans has high potential to keep Cd accumulation in cacao beans at levels that are safe for consumption.

Resistant and susceptible cacao genotypes exhibit defense gene polymorphism and unique early responses to Phytophthora megakarya inoculation

Key genes potentially involved in cacao disease resistance were identified by transcriptomic analysis of important cacao cultivars. Defense gene polymorphisms were identified which could contribute to pathogen recognition capacity. Cacao suffers significant annual losses to the water mold Phytophthora spp. (Oomycetes). In West Africa, P. megakarya poses a major threat to farmer livelihood and the stability of cocoa production. As part of a long-term goal to define key disease resistance genes in cacao, here we use a transcriptomic analysis of the disease-resistant cacao clone SCA6 and the susceptible clone NA32 to characterize basal differences in gene expression, early responses to infection, and polymorphisms in defense genes. Gene expression measurements by RNA-seq along a time course revealed the strongest transcriptomic response 24h after inoculation in the resistant genotype. We observed strong regulation of several pathogenesis-related genes, pattern recognition receptors, and resistance genes, which could be critical for the ability of SCA6 to combat infection. These classes of genes also showed differences in basal expression between the two genotypes prior to infection, suggesting that prophylactic expression of defense-associated genes could contribute to SCA6's broad-spectrum disease resistance. Finally, we analyzed polymorphism in a set of defense-associated receptors, identifying coding variants between SCA6 and NA32 which could contribute to unique capacities for pathogen recognition. This work is an important step toward characterizing genetic differences underlying a successful defense response in cacao.

Unraveling the genetic background of the Yangambi Research Center cacao germplasm collection, DR Congo

The Democratic Republic of the Congo (DR Congo or DRC) has some of the most fertile soils in Africa to cultivate cacao, an important cash crop and source of income for many smallholder farmers. Although cacao is currently produced there on small scale, DRC has the potential to grow as a cacao-producing country, thereby supplying the increasing cacao demand in the global market. Since the introduction of cacao varieties in the late nineteenth century, selection and breeding experiments have been carried out based on phenotype, without any knowledge on the genetic background of the cultivars. Therefore, this study analyzes 62 Congolese accessions of the Centre de Recherche de Yangambi (CRY) and 51 accessions of international collections representing 10 reference groups and Trinitario cultivars using 14 microsatellite markers. Descriptive statistics revealed a high gene diversity and polymorphic information content (PIC) for most of the markers, of which mTcCIR 12, 37, and 60 were the most discriminative. Both Bayesian clustering and principal coordinate analysis (PCoA) revealed high-admixed ancestry of the CRY cultivars. The collection was divided in two clusters, of which the first formed a hybrid population, linked to Amelonado, Trinitario, Maranόn, and Nanay, and the second was assigned to predominantly Amelonado, followed by Nanay, Contamana, and Nacional. This high admixture level resulted from numerous hybridization and recombination events that took place in the previous century. The obtained knowledge is essential for efficient conservation, utilization, and selection of high-quality cacao cultivars, which are agronomical favored, disease resistant, and of superior flavor quality.

Mode of Inheritance and Combining ability studies on Epicuticular wax Production in Resistance to Black pod disease in Cacao (Theobroma cacao L.)

Abstract Black pod caused by Phytophthora species is a devastating disease of cacao (Theobroma cacao L.) in production regions worldwide. Breeding for cacao genotypes resistant to black pod disease is crucial for sustainable cocoa production and profitability. Although breeding programmes in the past have made considerable efforts in improving resistance of cocoa genotypes with diverse genetic background, the disease continues to cause unacceptable yield losses in cacao production. To understand the mode of inheritance of epicuticular wax on the surface of cocoa pod known to play a major role in resistance to black pod disease, a 6 × 6 full diallel and a M x N (6 × 4) mating designs were used and data was analysed using Griffing’s method I model I approach and the North Carolina design –II approach, respectively. The results of the two mating designs were consistent and showed that epicuticular wax on leaf and pod surfaces of cacao were under the control of additive genetic effects. The non-additive effects were generally much smaller compared to the additive genetic effects. The significant association between GCA estimates of parents and their means indicates that parental values could be used as indicators of progeny performance. The best general combiners for epicuticular wax load on leaf and pod surfaces were Pa7/808, T60/887 and Pa 150. The larger additive components and heritability observed in this study, indicates that pedigree selection could be used to develop cacao cultivars with high amount of epicuticular wax on pod surface to enhance resistance to black pod disease.

Sensitivity of Yeast Mutants Deficient in Mitochondrial or Vacuolar ABC Transporters to Pathogenesis-Related Protein TcPR-10 of Theobroma cacao.

Pathogenesis-related proteins (PRs) are induced in plants after infection by pathogens and/or abiotic stress. Among these proteins, the family 10 (PR-10) influences the biosynthesis of secondary metabolites and shows antimicrobial ribonuclease activity. TcPR-10p (Pathogenesis-related Protein 10 of Theobroma cacao) was isolated from resistant and susceptible Moniliophthora perniciosa cacao cultivars. Cell survival with Saccharomyces cerevisiae mutant lines deficient in ATP-binding cassette (ABC) transporter proteins indicated the influence on resistance to TcPR-10p. Proteins of the ABC transport type are considered important in the process of resistance to antimicrobials and toxins. Thus, the objective of this work was to observe the sensitivity of ABC transporter yeast mutants in the presence of the TcPR-10p. Chronic exposure of S. cerevisiae mitochondrial (BYatm1Δ and BYmdl1Δ) and vacuole (BYnft1Δ, BYvmr1Δ, BYybt1Δ, BYycf1Δ and BYbpt1Δ) ABC transporter mutants to TcPR-10p (3 μg/mL, 0, 6, 12 and 24 h) was performed. Two TcPR-10p sensitive strains (BYmdl1Δ and BYnft1Δ) were submitted to a fluorescence test with the fluorogenic dihydroethidium (DHE), to visualize the presence of oxidative stress in the cells. Oxidative stress-increased sensitivity was confirmed by flow cytometry indicating induced cell death either via apoptosis or necrosis. This yeast data combined with previous data of literature (of M. perniciosa sensitivity to TcPR-10p) show that increased sensitivity to TcPR-10p in these mutants could be due to the TcPR10p-generated higher levels of intracellular reactive oxygen species (ROS), leading to increased cell death either via necrosis or apoptosis.

Entomofauna Associated with Agroforestry Systems of Timber Species and Cacao in the Southern Region of the Maracaibo Lake Basin (Mérida, Venezuela).

Agroforestry systems are environment-friendly production systems which help to preserve biodiversity while providing people with a way of earning a living. Cacao is a historically important crop in Venezuela that traditionally has been produced in agroforestry systems. However, few studies have evaluated how different trees used in those systems affect the dynamics and abundance of insects. The present study evaluated the entomofauna assemblages associated with different combinations of four timber-yielding trees and four Criollo cacao cultivars established in a lowland tropical ecosystem in Venezuela. A randomized block design with two replicates was used, each block having 16 plots which included all 16 possible combinations of four native timber trees (Cordia thaisiana, Cedrela odorata, Swietenia macrophylla, and Tabebuia rosea) and four Criollo cacao cultivars (Porcelana, Guasare, Lobatera and Criollo Merideño). Insects were collected with yellow pan traps and sorted to order. Coleoptera and parasitoid Hymenoptera were determined to the family level. In total, 49,538 individuals of seven orders were collected, with Hymenoptera, Diptera, and Hemiptera being the most abundant, although only Lepidoptera and Coleoptera abundances were significantly influenced by the timber tree species. Twenty-three families of parasitoid Hymenoptera and 26 of Coleoptera were found. Significant differences in insects’ assemblages were found both in parasitoid Hymenoptera and Coleoptera families associated to every shade tree, with the families Eulophidae and Lycidae being indicators for Cordia, and Chalcididae for Swietenia. The entomofauna relationship with the cacao cultivar was barely significant, although Scydmaenidae and Scarabaeidae were indicators for Lobatera and Merideño, respectively. No significant effects were found for interaction with cacao cultivars and native trees. We concluded that the particular insect assemblages found in Cedrela odorata and Cordia thaisiana, together with their high growing rates, make these two species an optimal choice for cacao agroforestry systems.

Protease prospecion and determination of its isoenzymes activity in cocoa cultivars (Theobroma cacao L.)

Our objective was to characterize the protease enzymatic activity and its isoenzymes on cacao cultivars PH 16 and TSH 1188, produced in southern Bahia, linking it to the conditions of the fermentation process. Proteases were extracted and semi-purified, their activities determined changing substrate, pH and temperature, and the values ​​compared with the parameters of fermentation (pH and temperature), and yet determined the kinetic parameters and the activity of its isoenzymes: aminopeptidase, carboxypeptidase and endoprotease. In the experimental conditions, differences in protease activities were shown as to different cocoa cultivars on various conditions. The albumin stands out as the preferred enzyme substrate, in a pH range between 3 and 6 and temperatures between 29 ° C and 50 ° C. As for the isoenzymes activity, an increased activity in these seeds and in the cultivar TSH 1188 was observed. When correlated with the fermentative parameters, the conditions for enzymatic activity are not the best determined, with emphasis on farming pH 16 presenting its fermentation conditions far from those found as optimal, especially in pH evaluation, since temperature varies very little between the two cultivars.