Cryopreservation of cocoa (Theobroma cacao L.) somatic embryos by vitrification

Losses of cultivated cocoa (Theobroma cacao L.) due to diseases and continued depletion of forests that harbour the wild progenitors of the crop make ex situ conservation of cocoa germplasm of paramount importance. In order to enhance security of in situ germplasm collections, 2-3 mm floral-derived secondary somatic embryos were cryopreserved by vitrification. This work demonstrates the most uncomplicated clonal cocoa cryopreservation. Optimal post-cryostorage survival (74.5%) was achieved by 5 d preculture of SSEs on 0.5 M sucrose medium followed by 60 min dehydration in cold PVS2. To minimise free radical related cryo-injury, cation sources were removed from the embryo development solution and/or the recovery medium, the former treatment resulting in a significant benefit. After optimisation with cocoa genotype AMAZ 15, the same protocol was effective across all five additional cocoa genotypes tested. For the multiplication of clones, embryos regenerated following cryopreservation were used as explant sources, and vitrification was found to maintain their embryogenic potential.

Evaluación in vitro de hongos nativos antagonistas de Moniliophthora rereri (Cif. & Par., Evans et al.,) en el cultivo de cacao (Theobroma cacao L.)

El cacao es afectado por una diversidad de microorganismos patógenos, entre ellos el hongo Moniliophthora roreri (Cif. & Par., Evans et al.,) causante de la moniliasis, que es una de las enfermedades de mayor importancia de este cultivo. El uso irracional de plaguicidas sintéticos para el manejo de esta enfermedad ha generado problemas al agroecosistema y la salud humana, por lo que se está demandando el uso de alternativas agroecol��gicas. En este contexto, el objetivo principal de esta investigación fue generar información acerca de la capacidad antagónica in vitro mostrada por hongos nativos contra la moniliasis del cacao. Se recolectaron muestras de suelo, hojas, corteza, frutos sanos y enfermos en tres zonas cacaoteras de Nicaragua para la caracterización morfol��gica del patógeno y de los potenciales microorganismos antagonistas. Las pruebas de antagonismo “in vitro” se realizaron a través de la técnica del cultivo dual. Se evaluó el crecimiento radial del patógeno y de los antagonistas, el porcentaje de inhibición del crecimiento radial (PICR) del patógeno y la capacidad de control biol��gico de los hongos antagonistas. Se probaron cuatro técnicas para la conservación de los hongos antagonistas. Se obtuvieron cuatro aislados del patógeno M. roreri y 17 aislados de hongos antagonistas. El PICR del patógeno ejercida por aislados del antagonista Trichoderma varió de 40.13% a 46.77%, en los aislados del antagonista Paecilomyces el PICR oscil�� entre 59.38% y 67.43%, en el único aislado del antagonista Clonostachys el PICR fue de 62.33%-67.22%. Los 17 aislados se ubicaron en las clases 1, 2 y 3 de la escala de valoración de antagonismo. La mejor técnica de conservación de hongos antagonistas y de M. roreri fue la de conservación con glicerol. Los resultados de este estudio indican que en el agroecosistema de cacao existen microorganismos nativos que tienen potencial para ser usados como agentes de control biol��gico del patógeno M. roreri.

Influence of freezable/non-freezable water and sucrose on the viability of Theobroma cacao somatic embryos following desiccation and freezing

Encapsulated cocoa (Theobroma cacao L.) somatic embryos subjected to 0.08-1.25 M sucrose treatments were analyzed for embryo soluble sugar content, non-freezable water content, moisture level after desiccation and viability after desiccation and freezing. Results indicated that the higher the sucrose concentration in the treatment medium, the greater was the extent of sucrose accumulation in the embryos. Sucrose treatment greatly assisted embryo post-desiccation recovery since only 40% of the control embryos survived desiccation, whereas a survival rate of 60-95% was recorded for embryos exposed to 0.5-1.25 M sucrose. The non-freezable water content of the embryos was estimated at between 0.26 and 0.61 g H2O g(-1)dw depending on the sucrose treatment, and no obvious relationship could be found between the endogenous sucrose level and the amount of non-freezable water in the embryos. Cocoa somatic embryos could withstand the loss of a fraction of their non-freezable water without losing viability following desiccation. Nevertheless, the complete removal of potentially freezable water was not sufficient for most embryos to survive freezing.

Agroforestry systems of timber species and cacao: survival and growth during the early stages

In recent times, increased emphasis has been placed on diversifying the types of trees to shade cacao (Theobroma cacao L.) and to achieve additional services. Agroforestry systems that include profitable and native timber trees are a viable alternative but it is necessary to understand the growth characteristics of these species under different environmental conditions. Thus, timber tree species selection should be based on plant responses to biotic and abiotic factors. The aims of this study were (1) to evaluate growth rates and leaf area indices of the four commercial timber species: Cordia thaisiana, Cedrela odorata, Swietenia macrophylla and Tabebuia rosea in conjunction with incidence of insect attacks and (2) to compare growth rates of four Venezuelan Criollo cacao cultivars planted under the shade of these four timber species during the first 36 months after establishment. Parameters monitored in timber trees were: survival rates, growth rates expressed as height and diameter at breast height and leaf area index. In the four Cacao cultivars: height and basal diameter. C. thaisiana and C. odorata had the fastest growth and the highest survival rates. Growth rates of timber trees will depend on their susceptibility to insect attacks as well as to total leaf area. All cacao cultivars showed higher growth rates under the shade of C. odorata. Growth rates of timber trees and cacao cultivars suggest that combinations of cacao and timber trees are a feasible agroforestry strategy in Venezuela.

Differential effects of temperature on fruit development and bean quality of contrasting genotypes of cacao (Theobroma cacao)

The effects of temperature and light integral on fruit growth and development of five cacao genotypes (Amelonado, AMAZ 15/15, SCA 6, SPEC 54/1 and UF 676) were studied in semi-controlled environment glasshouses in which the thermal regimes of cacao-growing regions of Brazil, Ghana and Malaysia were simulated. Fruit losses because of physiological will (cherelle will) were greater at higher temperatures and also differed significantly between genotypes, reflecting genetic differences in competition for assimilates between vegetative and reproductive components. Short-term measurements of fruit growth indicated faster growth rates at higher temperatures. In addition, a significant negative linear relationship between temperature and development time was observed. There was an effect of genotype on this relationship, such that time to fruit maturation at a given temperature was greatest for the clone UF 676 and least for AMAZ 15/15. Analysis of base temperatures, derived from these relationships indicated genetic variability in sensitivity of cacao fruit growth to temperature (base temperatures ranged from 7.5 degrees C for Amelonado and AMAZ 15/15 to 12.9 for SPEC 54/1). Final fruit size was a positive function of beam number for all genotypes and a positive function of light integral for Amelonado in the Malaysia simulated environment (where the temperature was almost constant). In simulated environments where temperature was the main variable (Brazil and Ghana) increases in temperature resulted in a significant decrease in final pod size for one genotype (Amelonado) in Brazil and for two genotypes (SPEC 54/1 and UF 676) in Ghana. It was hypothesised that pod growth duration (mediated by temperature), assimilation and beam number are all determinants of final pod size but that under specific conditions one of these factors may override the others. There was variability between genotypes in the response of beam size and beam lipid content to temperature. Negative relationships between temperature and bean size were found for Amelonado and UF 676. Lipid concentration was a curvilinear function of temperature for Amelonado and UF 676, with optimal temperatures of 23 degrees C and 24 degrees C, respectively. The variability observed here of different cacao genotypes to temperature highlights the need and opportunities for appropriate matching of planting material with local environments.

A qualitative host-pathogen interaction in the Theobroma cacao-Moniliophthora perniciosa pathosystem

The aim of this study was to test whether resistance of clones of Theobroma cacao ( cocoa) varied between isolates of Moniliophthora (formerly Crinipellis) perniciosa, the cause of witches' broom disease. Developing buds of vegetatively propagated T. cacao grown in greenhouses in the UK were inoculated with 16 000 spores of M. perniciosa per meristem in water, under conditions where water condensed on the inoculated shoot for at least 12 h after inoculation. The proportion of successful inoculations varied between clones and was inversely correlated with time to symptom production or broom formation. A specific interaction was demonstrated among three single-spore isolates of M. perniciosa and the clone Scavina 6 (SCA 6) and a variety of susceptible clones. Isolates Castenhal-I and APC3 were equally likely to infect SCA 6 and the other clones, but isolate Gran Couva A9 never infected SCA 6, although it was as virulent on the other clones. The interaction was maintained when the wetness period was extended to 70 h. Offspring of SCA 6 x Amelonado matings were all susceptible to both Castenhal-I and GC-A5, with no evidence of greater variability in susceptibility to GC-A5 than Castanhal-I. This suggests recessive inheritance of a single homozygous factor conferring resistance to GC-A5, from SCA 6. The progenies were slightly more susceptible to Castanhal-I than GC-A5. The implications for managing the disease are discussed.

The effects of temperature and light integral on early vegetative growth and chlorophyll fluorescence of four contrasting genotypes of cacao (Theobroma cacao)

The effect of temperature on early vegetative growth, leaf chlorophyll fluorescence and chlorophyll content was examined on four genotypes of cacao (Amelonado, AMAZ 15–15, SCA 6 and SPEC 54/1). A controlled environment glasshouse was used to simulate the temperature conditions of three cacao-growing regions (Bahia, Brazil; Tafo, Ghana and Lower Perak, Malaysia) over the course of a year. Base temperatures calculated from increments in main stem growth varied from 18.6°C for AMAZ 15/15 to 20.8°C for SPEC 54/1. Temporal variation in Fv/Fm observed for two of the clones (SCA 6 and SPEC 54/1) in two of the compartments were correlated with temperature differences over time. Significant differences were also recorded between genotypes in leaf chlorophyll content. It was shown that variation over time in leaf chlorophyll content could be quantified accurately as a function of temperature and light integral. The results imply that genetic variability exists in cacao in response to temperature stress.

Progressive erosion of genetic and epigenetic variation in callus-derived cocoa (Theobroma cacao) plants

Relatively little is known about the timing of genetic and epigenetic forms of somaclonal variation arising from callus growth. We surveyed for both types of change in cocoa (Theobroma cacao) plants regenerated from calli of various ages, and also between tissues from the source trees. For genetic change, we used 15 single sequence repeat (SSR) markers from four source trees and from 233 regenerated plants. For epigenetic change, we used 386 methylation-sensitive amplified polymorphism (MSAP) markers on leaf and explant (staminode) DNA from two source trees and on leaf DNA from 114 regenerants. Genetic variation within source trees was limited to one slippage mutation in one leaf. Regenerants were far more variable, with 35% exhibiting at least one mutation. Genetic variation initially accumulated with culture age but subsequently declined. MSAP (epigenetic) profiles diverged between leaf and staminode samples from source trees. Multivariate analysis revealed that leaves from regenerants occupied intermediate eigenspace between leaves and staminodes of source plants but became progressively more similar to source tree leaves with culture age. Statistical analysis confirmed this rather counterintuitive finding that leaves of ‘late regenerants’ exhibited significantly less genetic and epigenetic divergence from source leaves than those exposed to short periods of callus growth.

Canopy characteristics of contrasting clones of cacao (Theobroma cacao)

Canopy characteristics (leaf area index, fractional light interception, extinction coefficient) of mature trees of ten clonally propagated cacao cultivars were measured over a period of 14 months at an experimental site in Bahia, Brazil. Differences in leaf area index between clones became more pronounced over time. When an approximately constant leaf area index was reached (after about nine months), LAI varied between clones from 2.8 to 4.5. Clonal differences in the relationship between leaf area index and fractional light interception implied differences in canopy architecture, as reflected by the range of extinction coefficients (mean values ranged from 0.63 for the clone TSH-565 to 0.82 for CC-10). The results demonstrate the potential for breeding more photosynthetically efficient cacao canopies.

High-resolution melt and morphological analyses of mealybugs (Hemiptera: Pseudococcidae) from cacao: tools for the control of Cacao Swollen Shoot Virus spread

BACKGROUND: Mealybugs (Hemiptera: Coccoidea: Pseudococcidae) are key vectors of badnaviruses, including Cacao Swollen Shoot Virus (CSSV) the most damaging virus affecting cacao (Theobroma cacao L.). The effectiveness of mealybugs as virus vectors is species dependent and it is therefore vital that CSSV resistance breeding programmes in cacao incorporate accurate mealybug identification. In this work the efficacy of a CO1-based DNA barcoding approach to species identification was evaluated by screening a range of mealybugs collected from cacao in seven countries. RESULTS: Morphologically similar adult females were characterised by scanning electron microscopy and then, following DNA extraction, were screened with CO1 barcoding markers. A high degree of CO1 sequence homology was observed for all 11 individual haplotypes including those accessions from distinct geographical regions. This has allowed for the design of a High Resolution Melt (HRM) assay capable of rapid identification of the commonly encountered mealybug pests of cacao. CONCLUSIONS: HRM Analysis (HRMA) readily differentiated between mealybug pests of cacao that can not necessarily be identified by conventional morphological analysis. This new approach, therefore, has potential to facilitate breeding for resistance to CSSV and other mealybug transmitted diseases.

The influence of shade and organic fertilizer treatments on the physiology and establishment of Theobroma cacao clones

Aims: This experiment aimed to determine whether the soil application of organic fertilizers can help the establishment of cacao and whether shade alters its response to fertilizers. Study Design: The 1.6 ha experiment was conducted over a period of one crop year (between April 2007 and March 2008) at the Cocoa Research Institute of Ghana. It involved four cacao genotypes (T 79/501, PA 150, P 30 [POS] and SCA 6), three shade levels (‘light’, ‘medium’ and ‘heavy’) and two fertilizer treatments (‘no fertilizer’, and ‘140 kg/ha of cacao pod husk ash (CPHA) plus poultry manure at 1,800 kg/ha). The experiment was designed as a split-plot with the cacao genotypes as the main plot factor and shade x fertilizer combinations as the sub-plots. Methodology: Gliricidia sepium and plantains (Musa sapientum) were planted in different arrangements to create the three temporary shade regimes for the cacao. Data were collected on temperature and relative humidity of the shade environments, initial soil nutrients, soil moisture, leaf N, P and K+ contents, survival, photo synthesis and growth of test plants. Results: The genotypes P 30 [POS] and SCA 6 showed lower stomatal conductance under non-limiting conditions. In the rainy seasons, plants under light shade had the highest CO2 assimilation rates. However, in the dry season, plants under increased shade recorded greater photosynthetic rates (P = .03). A significant shade x fertilizer interaction (P = .001) on photosynthesis in the dry season showed that heavier shade increases the benefits that young cacao gets from fertilizer application in that season. Conversely, shade should be reduced during the wet seasons to minimize light limitation to assimilation. Conclusion: Under ideal weather conditions young cacao exhibits genetic variability on stomatal conductance. Also, to optimize plant response to fertilizer application shade must be adjusted taking the prevailing weather condition into account.

Atividade antioxidante do extrato seco de cacau orgânico (Theobroma cacao): estudo de estabilidade e teste de aceitação de cremes acrescidos deste extrato

Conselho Nacional de Desevolvimento Científico e Tecnol��gico (CNPq)

Effect of Cocoa ( Theobroma cacao ) Plantation Types on the Distribution of Anuran Communities in Ore, Ondo State, Nigeria

Anuran species diversity and abundance were evaluated in different farming status of cocoa plantation in Ore, Ondo State, Nigeria. Applying the combination of visual encounter survey (VES) and acoustical survey (AES), the different farms surveyed were categorized as; 1) pure cocoa farms with pesticides applied (PCWP); 2) pure cocoa farms without pesticides application; 3) intercropped cocoa farms with pesticides applied (ICWP); and 4) intercropped cocoa farms without pesticides application (ICNP). The surrounding primary/secondary forest (PSFV) was sampled applying the transect method. A mean total of 690±2.6 anurans belonging to 28 species, 14 genera and 9 families were recorded during the study. Out of these, 10, 19, 17, 22 and 26 species were recorded respectively from PCWP, PCNP, ICWP, ICNP and PSFV. The anuran species richness was significantly different between the different cocoa plantation status and the forest sites (F4, 10 = 20.55, P< 0.01). The highest mean number of individuals (190±9.5) was observed at ICNP followed by PSFV (183±5.7), while the least was at PCWP (77±8.0). There was also significant difference (F4, 10 = 150.48, P< 0.01) between the abundance of anuran species at the various sites. Diversity indices (Shannon and Margalef) showed that the forest sites had the greatest (3.204 and 4.799) respectively while the pesticide using pure cocoa farms had the least (1.853 and 2.072). Generally, the result of the study clearly indicated that pesticide use cocoa farms were significantly lower compared to other sites. Farmers are encouraged to engage in intercropping (polyculture) especially with food and economic crops in a biodiversity friendly manner which could have similar characteristics of a natural ecosystem, thereby enhancing the biological diversity of agro-ecosystems.

Viabilidade econômica do cultivo de cacaueiro e bananeira irrigados no Maranhão

Este trabalho teve por objetivo estudar a viabilidade econômica da produção consorciada de cacau e banana na região do Perímetro Irrigado Tabuleiro de São Bernardo nos municípios de Magalhães de Almeida e Araioses, estado do Maranhão (MA), tendo em vista a ampla disponibilidade de recursos naturais para o cultivo e a potencialidade de ser uma região de escape das principais doenças que afetam as culturas de cacau e banana. O trabalho verifica a oportunidade de negócio de produção de cacau e banana na região do rio Parnaíba. O estudo utiliza projeções de fluxo de caixa e os desconta para o Valor Presente L��quido (VPL) para aferir a viabilidade econômica do negócio de produção de cacaueiro e bananeira nesta região do Brasil. Foram realizadas pesquisas bibliográficas para analisar as características edafoclimáticas da microrregião, as características fisiol��gicas do cacaueiro e seu consórcio com a bananeira para o sombreamento temporário necessário durante a fase de crescimento do cacaueiro. Foi sugerido o pacote de alta tecnologia para a produção de cacaueiro, dada a necessidade de irrigação para o seu cultivo. Diferentes produtividades e preços de cacau e banana foram utilizados para analisar a sensibilidade económica deste negócio. Ao projetar um fluxo de caixa, foi possível fazer uma análise sobre o negócio, viabilidade e riscos. Deve-se notar que não há grandes áreas produtoras de cacau na região do rio Parnaíba. Porém, há pequenas áreas de cacau em regiões semiáridas (ou de cerrado). Assim, a produção vegetal carece de comprovação de produção em larga escala para atestar de modo prático algumas das premissas econômico-financeiras adotadas para analisar a viabilidade da produção de cacau e banana no Perímetro Irrigado Tabuleiro de São Bernardo.

Estudos biol��gicos de cochonilhas do gênero Planococcus (Hemiptera: Pseudococcidae) em diferentes hospedeiros

This work was carried out to study the development of populations of the genus Planococcus in different plant species, aiming at the verification of host specificity. The mealybugs Planococcus minor were obtained from cocoa plants (Theobroma cacao L.) and coffee crop (Coffea canephora L.), and Planococcus citri from citrus seedlings (Citrus sinensis L. Osbeck). Single eggs were transferred to Petri dishes containing leaf discs of cocoa (T. cacao cultivar Catongo), coffee (Coffea arabica L. cultivar Mundo Novo) and citrus (C. sinensis cultivar Pera Rio). They were maintained on a water-agar slide at 1%, with the abaxial surface turned upwards. The dishes were sealed with a PVC plastic film and kept in climatized chambers regulated at 25 [plus or minus] 1[degrees] C, 70 [plus or minus] 10% RH and 12 hours of photophase. For the mealybugs coming from cocoa plants, the citrus substrate prolonged the nymphal development of the males (26.0 days). In the nymphal period of females and males coming from coffee plants, the coffee substrate allowed a shorter duration of that period (19.2 and 21.3 days, respectively) and, in addition to having providing a longer longevity to the females (59.1 days). The highest rates of mortality were obtained when they were reared on the cocoa substrate, regardless of the host plant from which they were originally collected, and when kept on citrus, for insects collected on coffee plants. P. citri and P. minor have higher preference for coffee substrate regardless of the original host, thus showing partial host specificity.