[Correction to article: Determination of theobromin in cacao; theobromin, caffeine & mineral content of cacao].
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Cacao↗
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Cacao polyphenolic substances. 5. The structure of cacao 'leucocyanidin 1.
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Cacao↗
Selection of international molecular standards for DNA fingerprinting of Theobroma cacao.
A collaborative international program was initiated to identify and describe the genetic diversity of living germplasm collections of Theobroma cacao genotypes that are maintained in several international collections scattered throughout tropical cacao growing countries of the world. Simple sequence repeat (SSR) DNA analysis was identified as the most appropriate molecular tool for DNA fingerprinting these collections during an international forum representing academic, government and industry scientists in the cacao community. Twenty-five SSR primers, which had been previously described, were evaluated as potential candidates to define an efficient, standardized, molecular fingerprinting protocol for T. cacao accessions. These primers have been evaluated for reliability, widespread distribution across the cacao genome, number of alleles produced by the SSR primers in cacao and their ability to discriminate between cacao accessions. Approximately 690 cacao accessions were used to evaluate the utility of these SSR primers as international molecular standards, and a small number of test samples of T. cacao were sent to two other independent laboratories for verification. DNA fragments were selectively amplified by PCR, using the SSR primers labeled with fluorescent dyes, and separated by capillary electrophoresis. Based on this study, the 15 SSR primers that had the highest reproducibility and consistency within a common genotype, while allowing the differentiation of separate divergent genotypes, were selected as international molecular standards for DNA fingerprinting of T. cacao.
Alleles↗
Fungal and plant gene expression during the colonization of cacao seedlings by endophytic isolates of four Trichoderma species.
Endophytic isolates of Trichoderma species are being considered as biocontrol agents for diseases of Theobroma cacao (cacao). Gene expression was studied during the interaction between cacao seedlings and four endophytic Trichoderma isolates, T. ovalisporum-DIS 70a, T. hamatum-DIS 219b, T. harzianum-DIS 219f, and Trichoderma sp.-DIS 172ai. Isolates DIS 70a, DIS 219b, and DIS 219f were mycoparasitic on the pathogen Moniliophthora roreri, and DIS 172ai produced metabolites that inhibited growth of M. roreri in culture. ESTs (116) responsive to endophytic colonization of cacao were identified using differential display and their expression analyzed using macroarrays. Nineteen cacao ESTs and 17 Trichoderma ESTs were chosen for real-time quantitative PCR analysis. Seven cacao ESTs were induced during colonization by the Trichoderma isolates. These included putative genes for ornithine decarboxylase (P1), GST-like proteins (P4), zinc finger protein (P13), wound-induced protein (P26), EF-calcium-binding protein (P29), carbohydrate oxidase (P59), and an unknown protein (U4). Two plant ESTs, extensin-like protein (P12) and major intrinsic protein (P31), were repressed due to colonization. The plant gene expression profile was dependent on the Trichoderma isolate colonizing the cacao seedling. The fungal ESTs induced in colonized cacao seedlings also varied with the Trichoderma isolate used. The most highly induced fungal ESTs were putative glucosyl hydrolase family 2 (F3), glucosyl hydrolase family 7 (F7), serine protease (F11), and alcohol oxidase (F19). The pattern of altered gene expression suggests a complex system of genetic cross talk occurs between the cacao tree and Trichoderma isolates during the establishment of the endophytic association.
Cacao↗
Intersterility, morphology and taxonomy of Ceratocystis fimbriata on sweet potato, cacao and sycamore.
Ceratocystis fimbriata is a large, diverse complex of species that cause wilt-type diseases of many economically important plants. Previous studies have shown that isolates in three monophyletic lineages within the Latin American clade of C. fimbriata are host-specialized to cacao (Theobroma cacao), sweet potato (Ipomoea batatas) and sycamore (Platanus spp.), respectively. We paired testers of opposite mating type from isolates of these lineages to find intersterility groups. Two intersterility groups corresponded to the sweet potato and sycamore lineages, respectively. The cacao lineage contained two intersterility groups, corresponding to two genetic sublineages centered in western Ecuador and Brazil/Costa Rica/Colombia. Six isolates from cacao that were not members of the cacao lineage and were not pathogenic to cacao in an earlier study also were intersterile with members of the two cacao intersterility groups. Some pairings between testers from different lineages or sublineages yielded perithecia from which a few abnormal progeny could be recovered, typical of interspecific hybrids. These progeny showed abnormal segregation of the MAT-2 gene and mycelial morphology, showing that they were indeed the result of crosses. Isolates of the sweet potato, cacao, and sycamore lineages were indistinguishable morphologically except for the presence or absence of a doliform (barrel-shaped) conidial state and minor differences in size of perithecial bases and necks and ascospores. C. fimbriata originally was described from sweet potato. We describe the cacao pathogen as a new species, Ceratocystis cacaofunesta and we raise the sycamore pathogen from a form to species Ceratocystis platani.
Ascomycota↗
Isolation and purification of functional total RNA from different organs of cacao tree during its interaction with the pathogen Crinipellis perniciosa.
Witches' broom disease, caused by Crinipellis perniciosa, is one of the major fungal diseases causing severe losses to cacao tree (Theobroma cacao L.) plantations in South America. One of the challenges associated with the understanding of the cacao and Crinipellis interaction in genomic studies is the isolation of intact nucleic acids. In this report, we describe a new, successful, and reliable procedure for the isolation of RNA from tissues of cacao tree, both infected and uninfected by Crinipellis. This protocol overcomes the problems associated with the very high amount of polyphenols and polysaccharides present in cacao organs that are not easily removed by conventional extraction procedures. The protocol requires few reagents, uses ultracentrifugation and inexpensive consumables, and can be easily applied in any laboratory. This method produced high-quality RNA that was suitable for subsequent purposes, such as reverse transcription PCR and cDNA library construction. We also report the first evidence of RNA isolation from cacao organs infected by C. perniciosa such as meristems and fruits.
Basidiomycota↗
Evaluation of a cacao drink as a simple oral stimulus to assess gallbladder contraction.
UNLABELLED: Gallbladder contractility plays an important role in the pathogenesis of gallstones and in the course of cholelithiasis. Furthermore, a functioning gallbladder is an important condition for performing a successful disolution of gallstones by bile acids. Therefore, a reliable simple physiological test is desired to assess gallbladder contractility. In ten volunteers gallbladder contraction was stimulated by 50 g chocolate, 330 ml cacao drink or in comparison by intramuscular injection of 0.3 microgram/kg ceruletide. Gallbladder volume was measured sonographically and CCK in serum was determined by radioimmunoassay (RIA) after 0, 15, 30 and 45 min. Additionally gallbladder contraction was determined in 20 patients with symptomatic gallstones using cacao drink on ceruletide. In health volunteers remaining gallbladder volume after 30 min was 28% +/- 5% using ceruletide and 37% +/- 7% using cacao. Stimulation by chocolate resulted in a remaining volume of 59% +/- 12% after 45 min only. Simultaneously to gallbladder contraction an increase of CCK in serum was registered. 30 min after cacao CCK had increased from 0.9 to 3.3 pmol/l. Using chocolate an increase of CCK amounted to 2.1 pmol/l after 45 min only. In patients with gallstones the positive predictive value of the cacao test for a functioning gallbladder was 91% and the negative predictive value was 78% in comparison to the unphysiologic stimulation by ceruletide injection. CONCLUSION: Cacao test but not chocolate is suitable and reliable to assess gallbladder contraction in patients with symptomatic gallstones.
Adult↗
Molecular and biochemical characterisation of two aspartic proteinases TcAP1 and TcAP2 from Theobroma cacao seeds.
Aspartic proteinase (EC 3.4.23) activity plays a pivotal role in the degradation of Theobroma cacao L. seed proteins during the fermentation step of cacao bean processing. Therefore, this enzyme is believed to be critical for the formation of the peptide and amino acid cocoa flavor precursors that occurs during fermentation. Using cDNA cloning and northern blot analysis, we show here that there are at least two distinct aspartic proteinase genes ( TcAP1 and TcAP2) expressed during cacao seed development. Both genes are expressed early during seed development and their mRNA levels decrease towards the end of seed maturation. TcAP2 is expressed at a much higher level than TcAP1, although the expression of TcAP1 increases slightly during germination. The proteins encoded by TcAP1 and TcAP2 are relatively different from each other (73% identity). This, and the fact that the two corresponding genes have different expression patterns, suggests that the TcAP1 and TcAP2 proteins may have different functions in the maturing seeds and during germination. Because the TcAP2 gene is expressed at a much higher level during seed development than TcAP1, it is likely that the TcAP2 protein is primarily responsible for the majority of the industrially important protein hydrolysis that occurs during cacao bean fermentation. Finally, TcAP2 has been functionally expressed in the yeast Yarrowia lipolytica. The secreted recombinant protein is able to hydrolyse bovine haemoglobin at acidic pH and is sensitive to pepstatin A, confirming that TcAP2 encodes an aspartic proteinase, and strongly suggests that this gene encodes the well-characterized aspartic proteinase of mature cacao seeds.
Amino Acid Sequence↗
Analyses of polyphenols in cacao liquor, cocoa, and chocolate by normal-phase and reversed-phase HPLC.
The antioxidant polyphenols in cacao liquor, a major ingredient of chocolate and cocoa, have been characterized as flavan-3-ols and proanthocyanidin oligomers. In this study, various cacao products were analyzed by normal-phase HPLC, and the profiles and quantities of the polyphenols present, grouped by molecular size (monomers to approximately oligomers), were compared. Individual cacao polyphenols, flavan-3-ols (catechin and epicatechin), and dimeric (procyanidin B2), trimeric (procyanidin C1), and tetrameric (cinnamtannin A2) proanthocyanidins, and galactopyranosyl-ent-(-)-epicatechin (2alpha-->7, 4alpha-->8)-(-)-epicatechin (Gal-EC-EC), were analyzed by reversed-phase HPLC and/or HPLC/MS. The profile of monomers (catechins) and proanthocyanidin in dark chocolate was similar to that of cacao liquor, while the ratio of flavan-3-ols to the total amount of monomeric and oligomeric polyphenols in the case of pure cocoa powder was higher than that in the case of cacao liquor or chocolate.
Anthocyanins↗
A new cacao linkage map based on codominant markers: development and integration of 201 new microsatellite markers.
A linkage map of cacao based on codominant markers has been constructed by integrating 201 new simple sequence repeats (SSR) developed in this study with a number of isoenzymes, restriction fragment length polymorphisms (RFLP), microsatellite markers and resistance and defence gene analogs (Rgenes-RFLP) previously mapped in cacao. A genomic library enriched for (GA)(n) and (CA)(n) was constructed, and 201 new microsatellite loci were mapped on 135 individuals from the same mapping population used to establish the first reference maps. This progeny resulted from a cross between two heterozygous cacao clones: an Upper-Amazon Forastero (UPA 402) and a Trinitario (UF 676). The new map contains 465 markers (268 SSRs, 176 RFLPs, five isoenzymes and 16 Rgenes-RFLP) arranged in ten linkage groups corresponding to the haploid chromosome number of cacao. Its length is 782.8 cM, with an average interval distance between markers of 1.7 cM. The new microsatellite markers were distributed throughout all linkage groups of the map, but their distribution was not random. The length of the map established with only SSRs was 769.6 cM, representing 94.8% of the total map. The current level of genome coverage is approximately one microsatellite every 3 cM. This new reference map provides a set of useful markers that is transferable across different mapping populations and will allow the identification and comparison of the most important regions involved in the variation of the traits of interest and the development of marker-assisted selection strategies.
Cacao↗
Analysis of catechins in Theobroma cacao beans by cyclodextrin-modified micellar electrokinetic chromatography.
A micellar electrokinetic chromatography (MEKC) method was developed for the quantitation of polyphenols (+)-catechin and (-)-epicatechin (catechin monomers) and the methylxanthine theobromine in Theobroma cacao beans. Owing to the poor stability of catechin monomers in alkaline conditions, a 50 mM Britton-Robinson buffer at a pH 2.50 was preferred as the background electrolyte. Under these conditions, the addition of hydroxypropyl-beta-cyclodextrin (HP-beta-CD) at a concentration of 12 mM to the SDS micellar solution (90 mM), resulted in a cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC) endowed with two peculiar advantages compare to the conventional MEKC: (i) strong improvement of separation of the most important phytomarkers of T. cacao and (ii) enantioselectivity toward (+/-)-catechin. In particular, separation of methylxanthines (theobromine and caffeine), procyanidin dimers B1 and B2, and catechins (epicatechin and catechin) was obtained simultaneously to the enantioseparation of racemic catechin within 10min. The enantioselectivity of the method makes it suitable in evaluation of possible epimerisation at the C-2 position of epicatechin monomer potentially occurring during heat processing and storage of T. cacao beans. The extraction procedure of the phytomarkers from the beans was approached using ultrasonic bath under mild conditions optimized by a multivariate strategy. The method was validated for robustness, selectivity, sensitivity, linearity, range, accuracy and precision and it was applied to T. cacao beans from different countries; interestingly, the native enantiomer (+)-catechin was found in the beans whereas, for the first time we reported that in chocolate, predominantly (-)-catechin is present, probably yielded by epimerisation of (-)-epicatechin occurred during the manufacture of chocolate.
Cacao↗
Trichoderma theobromicola and T. paucisporum: two new species isolated from cacao in South America.
Trichoderma theobromicola and T. paucisporum spp. nov. are described. Trichoderma theobromicola was isolated as an endophyte from the trunk of a healthy cacao tree (Theobroma cacao, Malvaceae) in Amazonian Peru; it sporulates profusely on common mycological media. Trichoderma paucisporum is represented by two cultures that were obtained in Ecuador from cacao pods partially infected with frosty pod rot, Moniliophthora roreri; it sporulates sporadically and most cultures remain sterile on common media and autoclaved rice. It sporulates more reliably on synthetic low-nutrient agar (SNA) but produces few conidia. Trichoderma theobromicola was reintroduced into cacao seedlings through shoot inoculation and was recovered from stems but not from leaves, indicating that it is an endophytic species. Both produced a volatile/diffusable antibiotic that inhibited development of M. roreri in vitro and on-pod trials. Neither species demonstrated significant direct in vitro mycoparasitic activity against M. roreri.
Base Sequence↗
Emission tomography with a large-hole collimator (CACAO): a possible new way to improve radionuclide imaging.
This work aims to improve the quality of scintigraphy. It evaluates the use of a large-hole collimator, the Computer Aided Collimation Gamma Camera Project (CACAO), in SPECT. Acquisition data from the same object were simulated for CACAO and for a conventional collimator. Better signal-to-noise ratios were found for CACAO images, whatever the number of emitted photons. This work demonstrates that high-resolution images may be obtained with large-hole collimators. The combination of CACAO and pixilated detectors may further improve radionuclide imaging.
Algorithms↗
Abscisic Acid and the maturation of cacao embryos in vitro.
Abscisic acid (ABA) was tested for its ability to affect development of immature zygotic embryos of cacao (Theobroma cacao) in vitro, by adding exogenous ABA, fluridone, or mefluidide to cultured embryos. Endogenous ABA levels, measured by enzyme-linked immunosorbent assay, were increased by exogenous ABA or by culture on sucrose increasing to 21%, and were decreased by fluridone and, to a lesser extent, by mefluidide. The effects of these on maturation were measured as effects on anthocyanins, lipids, and fatty acid saturation, all of which increase with maturation of the cacao embryo. Maturation was stimulated by increasing sucrose and, to a lesser degree, the addition of ABA, but decreasing endogenous ABA by treating with fluridone significantly inhibited all maturation parameters. Although desiccation tolerance does not develop in cacao embryos, these results suggest that ABA and sucrose are both needed for the initiation of events associated with maturation in vitro.
Journal Article↗
Antimicrobial effects of ionizing radiation on artificially and naturally contaminated cacao beans.
With an initial microbial level of ca. 10 microorganisms per g of Ivory Coast cacao beans, 5 kGy of gamma radiation under an atmosphere of air reduced the microflora per g by 2.49 and 3.03 logs at temperatures of 35 and 50 degrees C, respectively. Bahia cacao beans were artificially contaminated with dried spores of Aspergillus flavus and Penicillium citrinum, giving initial fungal levels of 1.9 x 10 and 1.4 x 10 spores per g of whole Bahia cacao beans, respectively. The average D(10) values for A. flavus and P. citrinum spores on Bahia cacao beans were 0.66 and 0.88 kGy, respectively.
Journal Article↗
STATUS OF CACAO WITCHES' BROOM: biology, epidemiology, and management.
Origins of Theobroma cacao and Crinipellis perniciosa occurred in the Amazon Basin region of South America, and their interaction, the witches' broom disease, was first described in the late 1700s. The 100 years of scientific investigations of witches' broom of cacao that began in the 1890s developed the present state of knowledge of the biology and epidemiology of witches' broom that are discussed. Recommended management to reduce the deleterious effects of witches' broom on cacao production include the use of phytosanitation (removal of diseased plant parts), applications of chemical fungicides, and the use of host resistance. At present, there is a paucity of resistant planting materials, and efforts to evaluate germplasm for resistance to witches' broom are described. Research topics to augment present knowledge about witches' broom of cacao are presented with the hope that disease management can be improved.
Journal Article↗
The genome of the tropical tree Theobroma cacao L.
2C values for angiosperms vary over 2500-fold and a positive correlation exists between C-value and latitude in herbaceous plants. Woody plants differ from herbaceous plants in chromosome size and C-value. In addition, tropical hardwoods have smaller chromosomes than other tropical plants and do not share the correlation of minimum generation time with genome size seen in herbaceous plants. Theobroma cacao is a tropical hardwood cultivated for its beans, which are used to make chocolate and cocoa butter. Its cytology is typical of the pantropical and subtropical family Sterculiaceae. Its small chromosomes, single secondary constriction, and lack of C-banding suggest a small genome. The genome size of T. cacao, measured by reassociation kinetics, is 2.01 x 10(8), which is small compared to both temperate and tropical plants previously studied. We also provide data on the melting point, base composition, and relative extent of methylation (at sites most commonly methylated in higher plants), of T. cacao DNA.
Arabidopsis↗
Resistance gene homologues in Theobroma cacao as useful genetic markers.
Resistance gene homologue (RGH) sequences have been developed into useful genetic markers for marker-assisted selection (MAS) of disease resistant Theobroma cacao. A plasmid library of amplified fragments was created from seven different cultivars of cacao. Over 600 cloned recombinant amplicons were evaluated. From these, 74 unique RGHs were identified that could be placed into 11 categories based on sequence analysis. Primers specific to each category were designed. The primers specific for a single RGH category amplified fragments of equal length from the seven different cultivars used to create the library. However, these fragments exhibited single-strand conformational polymorphism (SSCP), which allowed us to map six of the RGH categories in an F(2) population of T. cacao. RGHs 1, 4 and 5 were in the same linkage group, with RGH 4 and 5 separated by less than 4 cM. As SSCP can be efficiently performed on our automated sequencer, we have developed a convenient and rapid high throughput assay for RGH alleles.
Amino Acid Sequence↗