Effect of soil characteristics on cadmium absorption and plant growth of Theobroma cacao L. seedlings.

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 (r2  = -0.56) and real density (r2  = 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 (r2  = 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.

Effects of Maturity at Harvest and Fermentation Conditions on Bioactive Compounds of Cocoa Beans.

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.

Assessment of cocoa (Theobroma cacao L.) butter content and composition throughout fermentations.

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.

Governance Options to Enhance Ecosystem Services in Cocoa, Soy, Tropical Timber and Palm Oil Value Chains.

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.

Efecto de inoculantes bacterianos edáficos mixtos en el desarrollo temprano de cultivares mejorados de cacao (Theobroma cacao L.) en un sistema agroforestal tradicional del norte de Oaxaca, México / Effect of mixed edaphic bacterial inoculants in the early development of improved cocoa cultivars (Theobroma cacao L.) in a traditional agroforestry system of Oaxaca, Mexico

El cacao (Theobroma cacao L.) es nativo de América del Sur y representa uno de los recursos «bioculturales¼ más significativos de Mesoamérica, ya que es una región donde se domesticó y tuvo relevancia como bebida ritual y como moneda en muchas culturas prehispánicas hasta la llegada de los españoles, quienes difundieron su uso en el mundo y lo convirtieron en una de las mercancías «commodity¼ más consumidas. Mediante este trabajo se propone una alternativa para atender la problemática de las plantaciones a través de la introducción de una diversidad amplia de cultivares de cacao en sistemas agroforestales tradicionales, en sinergia con la inoculación de bacterias edáficas fijadoras de nitrógeno y solubilizadoras de fósforo insoluble. En una parcela agroforestal tradicional se introdujeron plantas de cacao injertadas de cuatro cultivares y se dispusieron 3 tratamientos: aplicación de biofertilizante, aplicación de fertilizante químico y testigo. Se registraron la altura, el diámetro basal, el número de hojas y el número de ramas a los 2 y 12 meses, y se caracterizó la población de microorganismos asociada alrededor del tallo bajo la copa de las plantas. Los resultados de crecimiento muestran un buen potencial para los 4 cultivares estudiados y se observó que la biofertilización generó efectos significativos en algunos de los indicadores de crecimiento de las plantas de cacao. Así, las asociaciones vegetales en un sistema agroforestal podrían ser favorables para potenciar el desarrollo de frutos y resistencia a plagas y enfermedades.

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.

Streptomyces cameroonensis sp. nov., a Geldanamycin Producer That Promotes Theobroma cacao Growth.

The taxonomy of an actinobacterial strain, designated JJY4T, was established using a polyphasic approach. JJY4T was isolated from the rhizosphere of Chromolaena odorata in Yaoundé (Cameroon) during a project for the selection of biological control agents. Strain JJY4T exhibited antimicrobial activities against bacteria, fungi, and oomycetes. Strain JJY4T 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 JJY4T 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 JJY4T is a typical feature of members found in the Streptomyces violaceusniger clade and, similar to some members of the clade, strain JJY4T produces geldanamycin. A phylogenetic analysis based on 16S rRNA gene sequences confirmed this classification and suggests that strain JJY4T be added to the subclade constituted of the type strains Streptomyces malaysiensis DSM 41697T and Streptomyces samsunensis DSM 42010T. However, DNA-DNA relatedness and physiological characteristics allowed for the differentiation of strain JJY4T from its closest phylogenetic relatives. Based on these results, strain JJY4T (=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.

Nutritional composition, bioactive compounds and volatile profile of cocoa beans from different regions of Cameroon.

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.

Chemical speciation of cadmium: An approach to evaluate plant-available cadmium in Ecuadorian soils under cacao production.

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.

Characterization of the legumains encoded by the genome of Theobroma cacao L.

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.

TcCYPR04, a Cacao Papain-Like Cysteine-Protease Detected in Senescent and Necrotic Tissues Interacts with a Cystatin TcCYS4.

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.

Análise comparativa de métodos de determinação de área foliar em genótipos de cacau / Comparative analysis of methods of determining leaf area in cocoa genotypes

O conhecimento da área foliar é uma ferramenta importante na análise do crescimento e desenvolvimento vegetal. Desse modo, objetivou-se comparar os métodos, integrador de área foliar, software DDA, software ImageJ e de medição das dimensões foliares, visando definir o fator de correção mais adequado para estimar a área foliar de genótipos cacau. O experimento foi desenvolvido na Estação Experimental Filogônio Peixoto no delineamento de blocos casualizados com quatro repetições em arranjo fatorial 4 x 8, sendo 4 métodos de avaliação de área foliar e oito genótipos de cacau (Variedade Comum; TSH 1188; Catongo; CCN 51; ICS 1; ESFIP 02; SJ 02 e PH 16). Os resultados foram submetidos a análises de variância e correlação e as médias comparadas pelo teste Tukey 5%. Não houve diferença entre os métodos de determinação de área foliar, sendo constatada correlação positiva entre os métodos DDA, ImageJ e Dimensões foliares com o método do integrador de área foliar (padrão), com valores dos coeficientes de correlação (r) superiores a 0,98. Os genótipos TSH 1188, Catongo e CCN 51 apresentaram maior área foliar média. Os métodos testados mostraram-se viáveis para estimar área foliar do cacaueiro com precisão, contudo, foram encontrados três grupos de fatores de correção, sendo K = 0,682 para os genótipos SJ 02 e Variedade Comum, K = 0,670 para os genótipos CCN 51, Catongo, TSH 1188 e ICS 01 e K = 0, 655 para os genótipos ESFIP 02 e PH 16.

The knowledge of the leaf area is an important tool in the analysis of plant growth and development. Thus, the objective was to compare the methods, integrating leaf area, software DDA, software ImageJ and measurement of leaf dimensions, aiming to define the most appropriate correction factor to estimate the leaf area of cacao genotypes. The experiment was conducted at the Experimental Station Filogônio Peixoto in a randomized block design with four replications in a factorial arrangement of 4 x 8, being 4 assessment methods of leaf area and eight cacao genotypes (Common Variety; TSH 1188; Catongo; CCN 51; ICS 1; ESFIP 02; SJ 02 e PH 16). The results were submitted to analysis of variance and correlation and means were compared by Tukey test 5%. There was no difference between the methods of determining leaf area, being found positive correlation between the methods DDA, ImageJ and leaf dimensions with the integrative method of leaf area (default), with values of correlation coefficients (r) greater than 0,98. The genotypes TSH 1188, Catongo and CCN 51 have higher average leaf area. The methods tested were shown to be viable to estimate leaf area of cacao accurately, however, we found three groups of correction factors, being K = 0,682 for genotypes SJ 02 and Common Variety, K = 0,670 for genotypes CCN 51, Catongo, TSH 1188 and ICS 01 and K = 0, 655 for genotypes ESFIP 02 and PH 16.

Population densities and genetic diversity of actinomycetes associated to the rhizosphere of Theobroma cacao / Densidades populacionais e diversidade genética de actinomicetos associados a rizosfera de Theobroma cacao

In spite of the acknowledged importance of growth-promoting bacteria, only a reduced number of studies were conducted with these microorganisms on Theobroma cacao. The objectives of this work were to study the population densities and genetic diversity of actinomycetes associated with the rhizosphere of cacao as a first step in their application in plant growth promotion and biological control. The populations densities of actinomycetes in soil and cacao roots were similar, with mean values of 1,0 x 10(6) CFU/g and 9,6 x 10(5) CFU/g, respectively. All isolates selected and used in this study were identified through sequencing analyses of a fragment of the rpoB gene that encodes the [beta]-subunit of the RNA polymerase as species of the genus Streptomyces. In vitro cellulolytic, xilanolytic and chitinolytic activity, indolacetic acid production and phosphate solubilization activities were observed in most of the isolates tested. The data obtained in this study demonstrate that actinomycetes account for a higher percentage of the total population of culturable bacteria in soil than on cacao roots. Additionally, actinomycetes from the cacao rhizosphere are genetically diverse and have potential applications as agents of growth promotion.

Apesar da reconhecida importância das bactérias promotoras de crescimento, apenas um reduzido número de estudos foi conduzido com este grupo de microrganismos na cultura do cacaueiro. Os objetivos deste trabalho foram o estudo da densidade populacional e da diversidade genética de actinomicetos associados à rizosfera do cacaueiro como o primeiro passo para sua utilização na promoção de crescimento de mudas desta cultura e no controle biológico de doenças. As densidades populacionais de actinomicetos em amostras de solo e de raízes de cacaueiro foram semelhantes, com valores médios de 1,0 x 10(6) UFC/g e de 9,6 x 10(5) UFC/g, respectivamente. Todos os isolados selecionados para este estudo foram identificados através de análises de seqüências de um fragmento do gene rpoB, que codifica a beta-subunidade da RNA polimerase, como pertencentes ao gênero Streptomyces. Dentre os isolados testados, constatou-se in vitro, a produção de celulase, xilanase, quitinase, ácido indolacético e a capacidade de solubilização de fosfato. Os dados obtidos demonstram que os actinomicetos representam uma maior proporção da população total de bactérias cultiváveis em solo do que em raízes. Adicionalmente, os actinomicetos da rizosfera do cacaueiro são geneticamente diversos e apresentam potencial para atuarem como agentes de promoção de crescimento.

Localization and identification of phenolic compounds in Theobroma cacao L. somatic embryogenesis.

Cocoa breeders and growers continue to face the problem of high heterogeneity between individuals derived from one progeny. Vegetative propagation by somatic embryogenesis could be a way to increase genetic gains in the field. Somatic embryogenesis in cocoa is difficult and this species is considered as recalcitrant. This study was conducted to investigate the phenolic composition of cocoa flowers (the explants used to achieve somatic embryogenesis) and how it changes during the process, by means of histochemistry and conventional chemical techniques. In flowers, all parts contained polyphenolics but their locations were specific to the organ considered. After placing floral explants in vitro, the polyphenolic content was qualitatively modified and maintained in the calli throughout the culture process. Among the new polyphenolics, the three most abundant were isolated and characterized by 1H- and 13C-NMR. They were hydroxycinnamic acid amides: N-trans-caffeoyl-l-DOPA or clovamide, N-trans-p-coumaroyl-l-tyrosine or deoxiclovamide, and N-trans-caffeoyl-l-tyrosine. The same compounds were found also in fresh, unfermented cocoa beans. The synthesis kinetics for these compounds in calli, under different somatic embryogenesis conditions, revealed a higher concentration under non-embryogenic conditions. Given the antioxidant nature of these compounds, they could reflect the stress status of the tissues.

Stable transformation of Theobroma cacao L. and influence of matrix attachment regions on GFP expression.

We describe a protocol for Agrobacterium-mediated genetic transformation of Theobroma cacao L. using cotyledonary explants from primary somatic embryos (SEs) and A. tumefaciens strain AGL1. Transgenic plants carrying the visible marker, gene green fluorescent protein ( EGFP), the selectable marker gene neomycin phosphotransferase II ( NPTII), the class I chitinase gene from cacao ( Chi), and tobacco nuclear matrix attachment regions (MARs) in different combinations were successfully produced via regeneration of secondary SEs. The presence of the Chi gene or MARs did not influence the number of transgenic plants produced compared to the marker genes alone. However, the inclusion of MARs contributed to increased mean GFP expression in the population of transgenics. Additionally, the presence of MARs reduced the occurrence of gene silencing and stabilized high levels of GFP expression in lines of transgenic plants multiplied via reiterative somatic embryogenesis. Ninety-four transgenic plants were acclimated in a greenhouse and grown to maturity. Detailed growth analysis indicated that there were no differences in various growth parameters between transgenic and non-transgenic SE-derived plants. Seeds produced from two genetic crosses with one of the transgenic lines were analyzed for EGFP expression-a near-perfect 1:1 segregation was observed, indicating that this line resulted from the insertion of a single locus of T-DNA.