The profile distribution of total and extractable boron in cacao-growing soils in southwestern Nigeria

The profile distribution of total and extractable B was determined in 16 Nigerian cacao-growing soil profiles formed from different parent materials. Total B for all soils ranged from 8 to 54μgg⁻¹ with a mean of 24μgg⁻¹. The soils formed from sandstones in the rainforest zone contained higher amounts of total B than soils derived from basement complex. Boron extractable in hot water, in 0.1% CaCl₂, and in 1N NH₄OAc varied from 0.13 to 1.38, 0.44 to 1.20 0.03 to 0.56μgg⁻¹ respectively. The corresponding means were 0.66, 0.75 and 0.27μgg⁻¹ B. Soils on metamorphic rocks gave the highest values. All extractable B values were related to organic matter while only CaCl₂-extractable B correlated with total B. Generally total and extractable B values were higher in the top soils than in the subsoils.     

Theobroma cacao L.: a genetic linkage map and quantitative trait loci analysis

A genetic linkage map of Theobroma cacao (cocoa) has been constructed from 131 backcross trees derived from a cross between a single tree of the variety Catongo and an F1 tree from the cross of Catongo by Pound 12. The map comprises 138 markers: 104 RAPD loci, 32 RFLP loci and two morphologic loci. Ten linkage groups were found which cover 1068 centimorgans (cM). Only six (4%) molecular-marker loci show a significant deviation from the expected 11 segregation ratio.The average distance between two adjacent markers is 8.3 cM. The final genome-size estimates based on two-point linkage data ranged from 1078 to 1112 cM for the cocoa genome. This backcross progeny segregates for two apparently single gene loci controlling (1) anthocyanidin synthesis (Anth) in seeds, leaves and flowers and (2) self-compatibility (Autoc). The Anth locus was found to be 25 cM from Autoc and two molecular markers co-segregate with Anth. The genetic linkage map was used to localize QTLs for early flowering, trunk diameter, jorquette height and ovule number in the BC₁ generation using both single-point ANOVA and interval mapping. A minimum number of 2–4 QTLs (P<0.01) involved in the genetic expression of the traits studied was detected. Coincident map locations of a QTL for jorquette height and trunk diameter suggests the possibility of pleiotropic effects in cocoa for these traits. The combined estimated effects of the different mapped QTLs explained between 11.2% and 25.8% of the phenotypic variance observed in the BC₁ population.     

Comparison of two cacao (Theobroma cacao L.) clones for the effect of pollination intensity on fruit set and seed content

We compared the influence of pollination intensity (PI) on fruit set and seed number per pod in two cacao (Theobroma cacao L.) clones, IFC5 (Forastero Lower-Amazon Amelonado), which is self-compatible and known to produce a high number of seeds per pod under open pollination, and SCA6 (Forastero Upper-Amazon), which is self-incompatible and known to produce a low number of seeds per pod under open pollination. With both clones, PI had a positive effect on fruit set, with the maximum rate requiring more than 150 pollen grains per pod. One-half of the maximum rate of fruit set was reached with 39 pollen grains per pod for SCA6, and 78 for IFC5. With SCA6, a significant positive effect of PI on seed number per pod was also observed, with maximum seed number requiring more than 200 pollen grains per pod. In contrast, seed numbers with IFC5 were approximately equal over the PI range 100–800 pollen grains per pod. Patterns of seed number per pod were compared after: (1) open insect pollination, (2) low-intensity hand pollination and (3) high-intensity hand pollination. The patterns obtained with IFC5 under open pollination showed a peak of 40–50 seeds per pod, whereas the distribution patterns were more even with SCA6. The pattern of seed number under open pollination was similar to that obtained with high-intensity hand pollination for IFC5, and with low-intensity hand pollination for SCA6. We concluded that the high number of seeds per pod observed with IFC5 under natural insect pollination may be explained by a high number of compatible self-pollen grains on the stigma and by a severe drop of low-pollinated flowers eliminating potential lowfilled fruits. With SCA6, however, the number of compatible pollen grains deposited on the stigma was probably low under open pollination, and the flowers required lower pollen quantities to set fruit, which resulted in a high frequency of low seed numbers per fruit. This difference in the capacity to set low-seeded fruits might be considered as an adaptive trait related to the mode of reproduction.     

Microsatellite markers developed from Theobroma cacao L. expressed sequence tags

Theobroma cacao L. expressed sequence tags (ESTs) were converted into useful genetic markers for fingerprinting individuals and genetic linkage mapping. Primers were designed to microsatellite‐containing ESTs. Twenty‐two T. cacao accessions, parents of various mapping populations segregating for disease resistance and crop yield characteristics, were tested. Twenty‐seven informative loci were discovered with 26 primer pairs. The number of detected alleles ranged from two to 11 and averaged 4.4 per locus. All 27 markers could be mapped into at least one of the existing F₁ or F₂ populations segregating for agronomically important traits.     

Comparative analysis of expressed genes from cacao meristems infected by Moniliophthora perniciosa

BACKGROUND AND AIMS: Witches' broom disease is caused by the hemibiotrophic basidiomycete Moniliophthora perniciosa, and is one of the most important diseases of cacao in the western hemisphere. Because very little is known about the global process of such disease development, expressed sequence tags (ESTs) were used to identify genes expressed during the Theobroma cacao-Moniliophthora perniciosa interaction. METHODS: Two cDNA libraries corresponding to the resistant (RT) and susceptible (SP) cacao-M. perniciosa interactions were constructed from total RNA, using the DB SMART Creator cDNA library kit (Clontech). Clones were randomly selected, sequenced from the 5' end and analysed using bioinformatics tools including in silico analysis of the differential gene expression. KEY RESULTS: A total of 6884 ESTs were generated from the RT and SP cDNA libraries. These ESTs were composed of 2585 singlets and 341 contigs for a total of 2926 non-redundant sequences. The redundancy of the libraries was low and their specificity high when compared with the few other cacao libraries already published. Sequence analysis allowed the assignment of a putative functional category for 54 % of sequences, whereas approx. 22 % of sequences corresponded to unknown function and approx. 24 % of sequences did not show any significant similarity with other proteins present in the database. Despite the similar overall distribution of the sequences in functional categories between the two libraries, qualitative differences were observed. Genes involved during the defence response to pathogen infection or in programmed cell death were identified, such as pathogenesis related-proteins, trypsin inhibitor or oxalate oxidase, and some of them showed an in silico differential expression between the resistant and the susceptible interactions. CONCLUSIONS: As far as is known this is the first EST resource from the cacao-M. perniciosa interaction and it is believed that it will provide a significant contribution to the understanding of the molecular mechanisms of the resistance and susceptibility of cacao to M. perniciosa, to develop strategies to control witches' broom, and as a source of polymorphism for molecular marker development and marker-assisted selection.     

Isolation of endophytic endospore-forming bacteria from Theobroma cacao as potential biological control agents of cacao diseases

Sixty-nine endospore-forming bacterial endophytes consisting of 15 different species from five genera were isolated from leaves, pods, branches, and flower cushions of Theobroma cacao as potential biological control agents. Sixteen isolates had in vitro chitinase production. In antagonism studies against cacao pathogens, 42% inhibited Moniliophthora roreri, 33% inhibited Moniliophthora perniciosa, and 49% inhibited Phytophthora capsici. Twenty-five percent of isolates inhibited the growth of both Moniliophthora spp., while 22% of isolates inhibited the growth of all three pathogens. Isolates that were chitinolytic and tested negative on Bacillus cereus agar were tested with in planta studies. All 14 isolates colonized the phyllosphere and internal leaf tissue when introduced with Silwet L-77, regardless of the tissue of origin of the isolate. Eight isolates significantly inhibited P. capsici lesion formation (p = 0.05) in detached leaf assays when compared to untreated control leaves. ARISA with bacilli specific primers amplified 21 OTUs in field grown cacao leaves, while eubacteria specific primers amplified 58 OTUs. ARISA analysis of treated leaves demonstrated that inundative application of a single bacterial species did not cause a long-term shift of native bacterial communities. This research illustrates the presence of endospore-forming bacterial endophytes in cacao trees, their potential as antagonists of cacao pathogens, and that cacao harbors a range of bacterial endophytes.     

Application of chemical and biological agents for the management of frosty pod rot (Moniliophthora roreri) in Costa Rican cocoa (Theobroma cacao)

This article describes two field trials carried out at La Lola, Costa Rica, to assess control measures against frosty pod rot of cocoa (Theobroma cacao) caused by Moniliophthora (Crinipellis) roreri. In the first, factorial, trial the control agents were applied using motorised mistblowers (MMs) and hydraulic sprayers fitted with a narrow angle cone nozzle. There was an interaction between agents and application methods; together with previous application data for the most active fungicide (copper hydroxide), these trials indicate that best yields are achieved with sprays that maximise deposits on pods. We describe the droplet size spectra produced by a Stihl SR400 MM under a range of conditions because this has become the standard method of fungicide application in this series of trials at La Lola. The factor that had the largest effect on droplet size spectrum was the presence or the absence of a detachable baffle plate in front of the air‐shear nozzle. In both trials described here, MMs were fitted with baffle plates, a formulation pump and restrictor transmitting 550 mL min^?1 to deliver an estimated equivalent of 190 L ha^?1. Copper hydroxide as prophylactic applications at 1500 g a.i. ha^?1 have, to date, shown the most consistent (but incomplete) improvement in healthy pod yield. Use of copper fungicides may be cost effective when farm‐gate cocoa prices exceed approximately $1.25 kg^?1. In these trials, isolates of the hyperparasitic fungi Clonostachys byssicola and Trichoderma asperellum and two off‐patent triazole fungicides (bitertanol and triadimenol) made no significant improvement to healthy yields. The systemic oxathiin fungicide flutolanil, at a dosage of 300 g a.i. ha^?1, appears to protect pods substantially at early stages but gives proportionately less control of M. roreri than copper at later stages of pod development.     

Extraction and fractionation of glucosyltransferase inhibitors from cacao bean husk

The re-utilization of cacao bean husk, a waste generated from the chocolate industry, would bring benefits both environmentaly and economicaly. This study relates to a process for effectively separating and fractionating a cacao bean husk fraction having high inhibitory activity against glucosyltransferase (GTF) for the prevention of tooth decay (anticaries activity). Since the GTF inhibitory activity is known to be rendered by polyphenols, the separation process was also able to aim at high recovery of polyphenols which benefits human health. In this study, cacao bean husk extract, obtained under optimal conditions, extraction with 50% (v/v) aq. (aqueous) acetone solution at 60 °C for 4 h followed by 50% (v/v) aq. ethanol using a styrene-based resin, showed significantly higher inhibitory activity (2 and 12 folds, respectively) against GTF and a similar polyphenol content, compared to two other commercial anti-GTF polyphenol products.