Studies of Fruit Development of Cacao (Theobroma cacao) in Relation to Cherelle Wilt: II. Auxins and Development of the Seeds

The auxins of cacao seeds were examined in relation to seeddevelopment by the wheat straight growth and the first internodeassays after separation by filter paper chromatography. Therewere four consistent growth-active zones on the chromatograms:two acid growth promoters, one acid inhibitor, and a neutralgrowth promoter. An acid promoter (cacao auxin I) was shownby two-way chromatography and its activity in the oat curvaturetest to be similar to indole-3-acetic acid. Cacao auxin 1 occurredin roughly equal amounts in seeds of different ages throughoutthe wilting phase. The other growth substances detected on thechromatograms were unlikely to be associated with wilting phenomena. Three stages can be recognized in the growth of the perispermand endosperm. The only measurable differences between wiltingand healthy seeds was in the perisperm, which was smaller inthe wilting seeds during the second stage, 50 to 80 days fromfertilization. No differences could be found in the dimensionsof the wilting and healthy seeds. The observations on seed developmentare discussed in relation to growth of the fruit and cacao auxin1. A summary of the major steps in the development of the healthyand wilting seeded fruit and the hypothetical pathway by whichseedless and partially seeded fruits arise is presented.     

Xylem Occlusions in the Fruit of Cacao (Theobroma cacao) and their Relation to Cherelle Wilt

The anatomy and distribution of the xylem of the fruit of cao(Theobroma cacao) has been investigated by injection and stainingtechniques. The physical symptoms of cherelle wilt (pod wilt), a physiologicalfruitthinning mechanism, have been shown to be caused by occlusionsin the xylem vessels of the fruit-stalk. The initiation of theseocclusions in thought to be associated with the oxidative activityof a cambial or meristermatic band of tissue, which forms newvascular tissue to the pericarp.     

Studies of Fruit Development of Cacao (Theobroma cacao) in Relation to Cherelle wilt: III. Effects of fruit-thinning

Cacao trees were manually thinned to 1, 2, or 10 pods per treein order to observe the effect of competition onwilting behaviour.The number of pods wilting and harvested were recorded weeklyfor I6 months. The percentage of pods that wilted on the thinnedtrees was less than the controls. However, yields were reducedon the thinned trees more than expected owing to the continuedwilting of those pods left on the trees. The results supportthe hypothesis that cherelle with is a fruit thinning mechanism,but that biotic factors can cause the same syndrome.     

Cherelle Wilt of Cacao: I. POD DEVELOPMENT AND ITS RELATION TO WILT

The growth and development of cacao pods of three differentselections was studied in relation to pod loss through cherellewilt. Pods were observed to be specially liable to wilt at twostages in their development. These periods were called ‘firstwilt’ and ‘second wilt‘ respectively. First wilt reaches its peak at 7 weeks after pollination andis shown to cease as cell walls are laid down in the endosperm.Second wilt reaches its peak at 10 weeks from pollination anddeclines in response to greatly increased pod metabolism. Podswhich wilt during second wilt have larger embryos and smallerpod stalks than comparable healthy pods. It is postulated thatboth types of wilt arise as a result of a lack of hormones producedby the endosperm, causing a decrease in the uptake of waterand food materials and thereby bringing on wilt. This theory is discussed with reference to previous work andto the possible use of growth substances as a control measure.     

Cherelle Wilt of Cacao: II. WILT IN RELATION TO YIELD

Experiments were carried out to study whether cherelle wiltis responsible for loss of yield in cacao. It was found thatincreasing or decreasing flower setting had no effect on yieldas the crop was unrelated to the number of cherelles formed.Preventing setting till after the April ‘flush’had no effect on yield. Variations in wilt between and withinselections were studied, and it was concluded that yield isdependent on the nutrient status of the tree and independentof fluctuations in cherelle wilt.     

Translocation of phosphorus-32 into wilting and healthy fruits of cacao (Theobroma Cacao)

Summary The uptake of phosphorus into young cacao fruits (cherelles) was studied by applying 1.0 mC of P-32 to tins of soil in which young cacao trees were growing. The radiation from the fruits was measured daily with a shielded end-window G-M tube until the fruit wilted. While the fruit remained healthy accumulation of P-32 was linear and uptake continued for about three days after the fruit stopped growing (the first sign of wilting); uptake of P-32 then ceased. An increase in the count rate as the fruit became flaccid was attributed to reduced self-absorption due to loss of water.The distribution of the radio-active phosphorus in the seeds and pericarp was investigated and comparative analyses of phosphate in wilting and healthy fruits are presented.The results provide additional evidence for the hypothesis that cherelle wilt is analogous to a fruit-thinning mechanism.     

Fine-root growth dynamics in cacao (Theobroma cacao)

Summary Mulches in five combinations of clear plastic, black plastic, and undecomposed leaves covered by or on top of black or clear plastic film to modify soil temperature, were used to study the enzyme activity of the soil and numbers of bacteria able to produce degradative enzymes. Yield of pepper and broccoli planted through the mulches was measured.The numbers of bacteria in the soil able to excrete protease, phosphatase, cellulase, and lipase were correlated with one another suggesting that the same group of organisms excreted the different enzymes. The activity of enzymes in the soil was similarly correlated. Bacterial urease-producers and the urease activity in the soil were not correlated with the number of other enzyme producers or activity of other enzymes in soil suggesting an independent population of urease producers degraded the urea fertilizer used. Temperature differences of 11–14°C provided by the various mulches appeared insufficient to create important differences in populations and biochemical activities of soil bacteria.Yield of broccoli was greatest early in the season in soil warmed under black polyethylene plastic and later in the season in the soil cooled with leaf mulch over black plastic. Yield of pepper was greatest in mid-to-late summer on soil warmed with black plastic, but the highest yield was obtained in early fall in soil with clear and black plastic over leaf mulch.     

The Use of Enzyme-Linked Immunosorbent Assay (ELISA) for Detection of Cacao Swollen Shoot Virus (CSSV) in Theobroma cacao

Cacao swollen shoot virus (CSSV) was readily detected in different parts of Theobroma cacao using the ELISA technique. Different plant tissues contained varying amounts of CSSV; highest concentrations were found in leaf lamina. Methods to preserve the serological activity of CSSV were evaluated, and best results obtained with samples stored in a buffer or freeze-dried.     

Dispersion models and sampling of cacao mirid bug Sahlbergella singularis (Hemiptera: Miridae) on Theobroma Cacao in southern Cameroon

The spatio-temporal distribution of Sahlbergella singularis Haglung, a major pest of cacao trees (Theobroma cacao) (Malvaceae), was studied for 2 yr in traditional cacao forest gardens in the humid forest area of southern Cameroon. The first objective was to analyze the dispersion of this insect on cacao trees. The second objective was to develop sampling plans based on fixed levels of precision for estimating S. singularis populations. The following models were used to analyze the data: Taylor's power law, Iwao's patchiness regression, the Nachman model, and the negative binomial distribution. Our results document that Taylor's power law was a better fit for the data than the Iwao and Nachman models. Taylor's b and Iwao's β were both significantly >1, indicating that S. singularis aggregated on specific trees. This result was further supported by the calculated common k of 1.75444. Iwao's α was significantly <0, indicating that the basic distribution component of S. singularis was the individual insect. Comparison of negative binomial (NBD) and Nachman models indicated that the NBD model was appropriate for studying S. singularis distribution. Optimal sample sizes for fixed precision levels of 0.10, 0.15, and 0.25 were estimated with Taylor's regression coefficients. Required sample sizes increased dramatically with increasing levels of precision. This is the first study on S. singularis dispersion in cacao plantations. Sampling plans, presented here, should be a tool for research on population dynamics and pest management decisions of mirid bugs on cacao.     

The status of cacao (Theobroma cacao,sterculiaceae) in the western hemisphere

There are, at the present time, effectively no long-range, ongoing programs in any tropical country of the western hemisphere dedicated to the improvement of cacao (Theobroma cacao, Sterculiaceae). While some effort is currently made to obtain new acquisitions of cacao cultivars exhibiting desirable characteristics and to maintain genepools of these trees, there are few data from field trials to prove and substantiate these qualities. In addition, there is a growing concern regarding the disparities between predicted yields of cacao trees through the use of “hybrid” seed and from actual production under field conditions. This has stimulated an awareness of the current inadequate understanding of the genetics of cacao and the lack of comprehension as to which cultivars, under distinct ecological conditions, are precocious, resistant to disease, or heavy bearing, or indeed demonstrate those traits vital to the success of farming programs adapted to today’s market conditions. This paper examines the events that have led to the current status of selection, development, and breeding of cacao. Alternative approaches are suggested.     

Cacao domestication I: the origin of the cacao cultivated by the Mayas

Criollo cacao (Theobroma cacao ssp. cacao) was cultivated by the Mayas over 1500 years ago. It has been suggested that Criollo cacao originated in Central America and that it evolved independently from the cacao populations in the Amazon basin. Cacao populations from the Amazon basin are included in the second morphogeographic group: Forastero, and assigned to T. cacao ssp. sphaerocarpum. To gain further insight into the origin and genetic basis of Criollo cacao from Central America, RFLP and microsatellite analyses were performed on a sample that avoided mixing pure Criollo individuals with individuals classified as Criollo but which might have been introgressed with Forastero genes. We distinguished these two types of individuals as Ancient and Modern Criollo. In contrast to previous studies, Ancient Criollo individuals formerly classified as 'wild', were found to form a closely related group together with Ancient Criollo individuals from South America. The Ancient Criollo trees were also closer to Colombian-Ecuadorian Forastero individuals than these Colombian-Ecuadorian trees were to other South American Forastero individuals. RFLP and microsatellite analyses revealed a high level of homozygosity and significantly low genetic diversity within the Ancient Criollo group. The results suggest that the Ancient Criollo individuals represent the original Criollo group. The results also implies that this group does not represent a separate subspecies and that it probably originated from a few individuals in South America that may have been spread by man within Central America.     

The regulation of triacylglycerol biosynthesis in cocoa (Theobroma cacao) L.

Developing cocoa cotyledons accumulate initially an unsaturated oil which is particularly rich in oleate and linoleate. However, as maturation proceeds, the characteristic high stearate levels appear in the storage triacylglycerols. In the early stages of maturation, tissue slices of developing cotyledons (105 days post anthesis, dpa) readily accumulate radioactivity from [¹⁴C]acetate into the diacylglycerols and label predominantly palmitate and oleate. In older tissues (130 dpa), by contrast, the triacylglycerols are extensively labelled and, at the same time, there is an increase in the percentage labelling of stearate. Thus, the synthesis of triacylglycerol and the production of stearate are co-ordinated during development. The relative labelling of the phospholipids (particularly phosphatidylcholine) was rather low at both stages of development which contrasts with oil seeds that accumulate a polyunsaturated oil (e.g. safflower). Microsomal membrane preparations from the developing cotyledons readily utilised an equimolar [¹⁴C]acyl-CoA substrate (consisting of palmitate, stearate and oleate) and glycerol 3-phosphate to form phosphatidate, diacylglycerol and triacylglycerol. Analysis of the [¹⁴C]acyl constituents at the sn-1 and sn-2 positions of phosphatidate and diacylglycerol revealed that the first acylase enzyme (glycerol 3-phosphate acyltransferase) selectively utilised palmitate over stearate and excluded oleate, whereas the second acylase (lysophosphatidate acyltransferase) was highly selective for the unsaturated acyl-CoA. On the other hand, the third acylase (diacylglycerol acyltransferase) exhibited an almost equal selectivity for palmitate and stearate. Thus, stearate is preferentially enriched at position sn-3 of triacylglycerol at 120–130 dpa because of the relatively higher selectivity of the diacylglycerol acyltransferase for this fatty acid compared with those of the other two acylation enzymes.Abbreviation dpa days post anthesis We are grateful to Drs. G. Pettipher (Cadbury-Schweppes, Reading, UK), M. End and P. Hadley (Department of Horticulture, University of Reading) for the supply of cocoa pods and to the Agricultural and Food Research Council for financial support. We also wish to thank Dr. S. Stymne (Swedish University of Agricultural Sciences, Uppsala, Sweden) for a generous gift of acyl-CoA substrates.     

Genetic variation in resistance to vascular-streak dieback in cocoa (Theobroma cacao)

Summary Genetic variation in disease resistance to vascular-streak dieback (Vsd) was studied in 54 cocoa hybrid progenies from Trinitario x Amazonian crosses. The disease was assessed by the number of Vsd infected branches per tree, total length of infected branches, average depth of disease penetration within branches and percentages of infected and dead plants. Highly significant differences due to general combining ability for both Trinitario female parents and Amazonian male parents were obtained for all characters. Percent of dead plants was the only character where specific combining ability was also important. Gene effects were predominantly additive for most characters, indicating that selection for progenies resistant to Vsd is effective in the present breeding population. There is strong evidence that resistance to Vsd is in the form of horizontal resistance. It is polygenic and largely inherited as additive genes. Therefore, breeding for resistance is an effective means of controlling Vsd in Papua New Guinea.     

The globalization of a ceremonial tree: The case of cacao (Theobroma cacao) among the Mopan Maya

The uses, perceptions, and economic significance of cacao have radically changed in the past 25 years among the Mopan Maya in southern Belize. Cacao was once perceived as a ceremonial crop with little cash value. Over the past 25 years though, cacao has become the most important cash crop grown by the Mopan Maya. The Mopan Maya grow organic cacao that has allowed them to tap into a specialized, high-end chocolate market. However, the emergence of cacao as an important cash crop has altered traditional uses and created conflicts in villages where increasing acreage of reservation lands are planted with cacao, thereby assigning a commercial value to previously communal lands.     

Effects of temperature on growth and water relations of cacao (Theobroma cacao var.Comum) seedlings

Growth of 55-day-oldTheobroma cacao var.Comum seedlings varied with temperature regimes, various plant parts, growth parameters, and time of harvesting. Over a 60-day period the optimal day-temperature regimes were near 33.3°C for dry weight increase and relative growth rates of seedlings and leaves; 30.5°C for increase in leaf area, height growth, and leaf abscission; 22.2°C for dry weight increase of stems or roots, stem diameter growth, and root-shoot ratio. The rates of increase in dry weights of stems or roots as well as root-shoot ratios declined progressively at temperatures above 22.2°C Partitioning of dry matter was affected by temperature regime, with proportionally more photosynthate retained by shoots and less translocated to roots at high temperatures. The progressive decrease in the root-shoot ratio at temperatures above 22.2°C may decrease drought tolerance of seedlings because roots will be less capable of absorbing endugh water to replace transpirational losses. This was shown by more negative shoot water potentials at high temperatures. Research supported by the College of Agricultural and Life Science. University of Wisconsin, Madison, Wi, USA and the Centro de Pesquisas do Cacau (CEPLAC/CEPEC), 45600 Itabuna, Ba, Brazil.     

Genetic Diversity and Natural Population Structure of Cacao (Theobroma cacao L.) from the Brazilian Amazon Evaluated by Microsatellite Markers

A sample of 94 accessions of Theobroma cacao L. (cacao), representing four populations from the Brazilian Amazon (Acre, Rondônia, lower Amazon and upper Amazon) were analyzed using microsatellite markers to assess the genetic diversity and the natural population structure. From the 19 microsatellite loci tested, 11 amplified scorable products, revealing a total of 49 alleles, including two monomorphic loci. The Brazilian upper Amazon population contained the largest genetic diversity, with the most polymorphic loci, the highest observed heterozygosity; and the majority of rare alleles, thereby this region might be considered part of the center of diversity of the species. The observed heterozygosity for all the Brazilian populations (H _o = 0.347) was comparable with values reported for other similar upper Amazon Forastero cacao populations, with the Acre and Rondônia displaying the lowest values. The lower Amazon population, traditionally defined as highly homozygous, presented an unexpectedly high observed heterozygosity (H _o = 0.372), disclosing rare and distinct alleles, with large identity with the upper Amazon population. It was hypothesized that part of the lower Amazon population might derive from successive natural or intentional introduction of planting material from other provenances, mainly upper Amazon. Most of the loci exhibited a lower observed heterozygosity than expected, suggesting that self-pollination might be more common than usually assumed in cacao, but excess of homozygotes might also derive from sub-grouping (Wahlund effect) or from sampling related individuals. Most of the gene diversity was found to occur within groups, with small differentiation between the four Brazilian Amazon populations, typical of species with high gene flow.     

Genetic diversity and relationships of cacao (Theobroma cacao L.) in southern Mexico

 Neotropical tree crops are affected by a combination of biological and human factors that complicate the study of genetic diversity and crop evolution. Genetic diversity and relationships among southern Mexican populations and horticultural collections of Theobroma cacao (chocolate, cocoa, cacao) are examined in light of the agricultural practices of the Maya. Collections of cacao were obtained from the extremes of its geographic range including archeological sites in southern Mexico where cacao was first domesticated. Genetic diversity was assayed by 57 informative random amplified polymorphic DNA (RAPD) marker loci. A unique sample of the total diversity found in this study exists in the southern Mexican populations. These populations are significantly different from all other cacao with regards to their profile of RAPD bands, including the ‘criollo’ variety, their morphological and geographical group. A population of cacao found in a sinkhole (cenote) in northern Yucatan with genetic affinities to populations in Chiapas suggests the Maya maintained plants far away from their native habitat. This finding concurs with known agroforestry practices of the Maya. Modern efforts to increase germplasm of tropical tree crops such as cacao should carefully examine archeological sites where genetic diversity, either deliberately or by chance, was collected and maintained by ancient cultures. Received: 21 May 1997 / Accepted: 9 October 1997     

Over-expression of a cacao class I chitinase gene in Theobroma cacao L. enhances resistance against the pathogen, Colletotrichum gloeosporioides

Theobroma cacao L. plants over-expressing a cacao class I chitinase gene (TcChi1) under the control of a modified CaMV-35S promoter were obtained by Agrobacterium-mediated transformation of somatic embryo cotyledons. Southern blot analysis confirmed insertion of the transgene in eight independent lines. High levels of TcChi1 transgene expression in the transgenic lines were confirmed by northern blot analysis. Chitinase activity levels were measured using an in vitro fluorometric assay. The transgene was expressed at varying levels in the different transgenic lines with up to a sixfold increase of endochitinase activity compared to non-transgenic and transgenic control plants. The in vivo antifungal activity of the transgene against the foliar pathogen Colletotrichum gloeosporioides was evaluated using a cacao leaf disk bioassay. The assay demonstrated that the TcChi1 transgenic cacao leaves significantly inhibited the growth of the fungus and the development of leaf necrosis compared to controls when leaves were wound inoculated with 5,000 spores. These results demonstrate for the first time the utility of the cacao transformation system as a tool for gene functional analysis and the potential utility of the cacao chitinase gene for increasing fungal pathogen resistance in cacao.