Tracing the native ancestors of the modern Theobroma cacao L. population in Ecuador

The native Theobroma cacao L. population from Ecuador, known as Nacional, is famous for its fine cocoa flavour. From the beginning of the twentieth century, however, it has been subjected to genetic erosion due principally to successive introductions of foreign germplasm whose hybrid descendants gradually replaced the native plantations, implying a decrease in cocoa quality. We attempted to trace this native cacao within a wide pool of modern Ecuadorian cacao population. Three hundred and twenty-two cacao accessions collected from different geographical areas along the pacific coast of Ecuador and maintained in two living collections were analysed using 40 simple-sequence repeat markers. Most of Ecuadorian cacao accessions displayed a high diversity and heterozygosity level. A factorial analysis of correspondence (FAC) showed a continuous variation among them, with a few ones, grouped at an extreme side of the FAC cloud, showing higher levels of homozygosity and lower introgression level by foreign cacaos. A paternity analysis revealed that these highly homozygous individuals are the most probable ancestors of the modern Nacional hybrid pool. These particular accessions studied could represent the native Nacional cacao present in Ecuador before the foreign introductions. Their identification will help to conserve valuable genetic material and to improve cocoa quality in new cacao varieties.     

Host preferences of the cocoa pod borer,Conopomorpha cramerella,the main threat to cocoa production in Southeast Asia

Insects ensure the survival of their offspring by depositing their eggs in suitable environments. Even generalist egg-laying insects often show preferences for specific host plants. The cocoa pod borer (CPB), Conopomorpha cramerella (Snellen) (Lepidoptera: Gracillariidae), successfully infests and reproduces on relatively few host plants, but has a major economic impact only on cocoa, Theobroma cacao L. (Malvaceae). Choice tests were performed in the laboratory to compare the frequency of insect visits, the duration of the visits, and the number of eggs laid on the fruits for each combination of host plants tested – that is, cocoa clones or fruits of rambutan (Nephelium lappaceum L.), Fijian longan (Pometia pinnata JR Forst & G Forst, both Sapindaceae), and langsat (Lansium domesticum Corrêa, Malvaceae). Our laboratory study showed that, when given the choice, CPB significantly favored cocoa pods over other host fruits (rambutan, langsat, or Fijian longan). Females also deposited more eggs on unripe fruits than on ripe fruits of similar size. The preference to lay eggs on specific cocoa clones in the bioassays did not reflect the level of the clone resistance to CPB damage reported from the field. Consequently, oviposition preference of female CPB does not seem to be the main factor explaining field resistance of some cocoa clones to CPB infestation.     

Large Mammals in an Agroforestry Mosaic in the Brazilian Atlantic Forest

The forest‐like characteristics of agroforestry systems create a unique opportunity to combine agricultural production with biodiversity conservation in human‐modified tropical landscapes. The cacao‐growing region in southern Bahia, Brazil, encompasses Atlantic forest remnants and large extensions of agroforests, locally known as cabrucas, and harbors several endemic large mammals. Based on the differences between cabrucas and forests, we hypothesized that: (1) non‐native and non‐arboreal mammals are more frequent, whereas exclusively arboreal and hunted mammals are less frequent in cabrucas than forests; (2) the two systems differ in mammal assemblage structure, but not in species richness; and (3) mammal assemblage structure is more variable among cabrucas than forests. We used camera‐traps to sample mammals in nine pairs of cabruca‐forest sites. The high conservation value of agroforests was supported by the presence of species of conservation concern in cabrucas, and similar species richness and composition between forests and cabrucas. Arboreal species were less frequently recorded, however, and a non‐native and a terrestrial species adapted to open environments (Cerdocyon thous) were more frequently recorded in cabrucas. Factors that may overestimate the conservation value of cabrucas are: the high proportion of total forest cover in the study landscape, the impoverishment of large mammal fauna in forest, and uncertainty about the long‐term maintenance of agroforestry systems. Our results highlight the importance of agroforests and forest remnants for providing connectivity in human‐modified tropical forest landscapes, and the importance of controlling hunting and dogs to increase the value of agroforestry mosaics.     

Effect of Xenorhabdus and Photorhabdus bacteria and their exudates on Moniliophthora roreri

Frosty pod rot or moniliasis is a very destructive disease of cocoa plantations in Latin America. The conventional methods to control this disease such as the use of chemical pesticides have failed or are too expensive for smallholders. Nowadays, biological alternatives are being investigated to improve yields and to keep moniliasis controlled. Xenorhabdus and Photorhabdus bacteria and their exudates were evaluated against Moniliophthora roreri. All bacteria demonstrated antifungal action reaching up to 97% after 13 days of exposure, whereas some exudates demonstrated more than 70%. Moniliophthora roreri did not sporulate in bacteria presence and sporulation was reduced by their exudates in 70%. The bacteria and exudates were compared with a commercial fungicide used by Venezuelan farmers; better performance of the bacteria and exudates was found and the fungicide was unable to control M. roreri. These experiments showed promising results of Xenorhabdus and Photorhabdus as biocontrol agents for being included in the integrated disease management programmes in Venezuela.     

Growth response ofTheobroma cacao L. seeldings to inoculation with vesicular-arbuscular mycorrhizal fungi

Summary The effects of vesicular-arbuscular mycorrhiza (VAM) on the growth and phosphorus uptake of cocoa seedlings (Theobroma cacao L.) grown for 100 days in polythene bags, were studied at five levels of phosphorus fertilization in both steamed and unsterile Bungor Series soil (a fine clayey, kaolinitic isohyperthermic Typic Paleudult). The cocoa seedlings responded well to phosphorus fertilization and mycorrhizal treatments. Plants inoculated with VAM fungi (Gigaspora spp.) gave the most vigorous growth and higher phosphorus in the leaf tissues in unsterile soil compared to plants grown in steamed soil. However, the mycorrhizal effect was significantly more pronounced (P<0.01) in plants grown in steamed than in unsterile soil. High levels of phosphorus application depressed mycorrhizal development. Phosphorus fertilizer applied at the rates of 250 and 500 ug g⁻¹ soil gave maximum root colonization and spore counts in both soil types used.     

Early growth and nutritional response to resource competition in cocoa-shade intercropped systems

Intercropping is often promoted for effective mutualism between species, thus compensating for external inputs. However, for optimal farm design resulting in superior production and nutrition, an accurate assessment of plant inter- and intra-specific competition is required. In predominant shade tree-cocoa (Theobroma cacao) systems, inconclusive evidence remains on species interactions, limitations to resource availability and subsequent growth and nutritional response, particularly in early growth. We examined cocoa biomass and foliar nutrition as well as nutrient supply through rates of decomposition and N mineralization after 1-year growth. Our approach employed fertilization and mixed planting treatments in an additive design of cocoa in monoculture (control), under artificial shade, and intercropped under two separate shade species (Terminalia superba and Newbouldia laevis). Intercropping had no effect on cocoa biomass production in comparison to monoculture cocoa. However, artificial shading stimulated foliage and root production both with and without fertilization, suggesting strong effects of light regulation on growth in the absence of belowground competition. Nutritionally, intercropping suppressed K uptake in cocoa foliage as K concentration was reduced by 20–25%, signifying dilution of this nutrient, presumably due to interspecific competition for mobile elements. Foliar N content under N. laevis was raised, where N concentration kept up with growth under this intercropped species. Intercropping also delayed decomposition rates, suggesting slower but sustained release of available nutrients into the topsoil. Cocoa under artificial shade, both with and without fertilization, exhibited the greatest nutrient responses as compared to unfertilized monoculture cocoa, where P uptake was stimulated most (175 and 112%), followed by K (69 and 71%), and then N (54 and 42%). Intercropping with shade trees failed to increase cocoa biomass, however, nutrient uptake was sustained for N and P, suggesting low interspecific competition. When fertilizers are undesirable or unavailable, intercropping of appropriately selected shade trees will not competitively suppress early growth of cocoa but will improve light regulation and nutritional status of cocoa saplings.     

The relationship between lignin and morphological characteristics of the tracheary elements from cacao(Theobroma cacao L.) Hulls

Tracheary elements (TEs) were physically separated from the hulls of cacao pods(Theobroma cacao L). Their morphological features were extensively investigated with scanning electron microscopy and chemical characterization. Spiral TEs were covered with a thin layer of primary wall that had a web-like structure on its outer surface. These TEs had a spiral circularity diameter of 8.2 ± 0.6 μm and an estimated secondary wall thickness of about 2.1 ± 0.2 μm. Polarized microscopy analysis revealed that the cellulose microfibrils were aligned parallel to that thickening. Lignin content was 36.1%, with a 0.13:1.00 molar ratio of syringyl to guaiacyl units and a 1.09:1.00 molar ratio of erythronic acid and threonic acid. Total yields of the alkaline nitrobenzene oxidation and ozonation products were 324.5 and 148.8 μmol g^-1 of extract-free TEs, respectively. Based on these morphological and lignin characteristics, we conclude that fully ripened cacao hulls exhibit the same features of secondary wall thickening as those seen at an earlier stage.     

Genetic transformation of cocoa leaf cells using Agrobacterium tumefaciens

Leaf strips from cocoa tree (Theobroma cacao L.) clones ICS-16 and SIC-5 were cocultivated with the supervirulent Agrobacterium tumefaciens strain A281-Kan. A281-Kan contains a wild-type Ti plasmid and an additional plasmid, pGPTV-Kan, which confers kanamycin resistance to transformed plant cells after integration and expression of the neomycin phosphotransferase II (nptII) gene. Transformed cells were selected on callusing medium containing 100 g ml^-1 kanamycin. NptII assays confirmed that kanamycin-resistant cultures of ICS-16 and SIC-5 expressed the nptII gene, whereas control cultures did not. Genomic Southern blot analyses demonstrated single T-DNA insertions into ICS-16 and SIC-5. T-DNA/cocoa DNA border regions from transformed cultures were cloned and sequenced, revealing that in both transformed cell lines, the right T-DNA border was at the 5 end of the 25 bp right border repeat. Cocoa DNA probes from the T-DNA/cocoa DNA insertion sites were used in Southern blot analyses and showed that T-DNA from pGPTV-Kan had inserted into a unique region in ICS-16 and into a repetitive region in SIC-5. This study establishes that foreign genes can be inserted and expressed in cocoa using A. tumefaciens-mediated gene transfer.     

Geometric isomers of sex pheromone components do not affect attractancy of Conopomorpha cramerella in cocoa plantations

The cocoa pod borer (CPB), Conopomorpha cramerella (Snellen), sex pheromone was previously identified as a blend of (E,Z,Z)‐ and (E,E,Z)‐4,6,10‐hexadecatrienyl acetates and corresponding alcohols. These pheromone components were synthesized by modification of an existing method and the relative attractiveness of synthetic blends that included different levels of non‐target pheromone components and chemical purities was tested in a cocoa field using Delta traps. Male captures were not significantly different among traps baited with pheromone blends containing 5% to 47% (based on four identified pheromone components) of other geometric acetates [(E,Z,E)‐, (Z,Z,Z)‐, (Z,E,Z)‐ and (Z,E,E)‐4,6,10‐hexadecatrienyl acetates], indicating that C. cramerella males did not discriminate among the pheromone components and other geometric isomers in the blends. Therefore, neither antagonistic nor synergistic effects from other pheromone geometric isomers were observed. The modified synthetic pathway offers the prospect of more economical production of CPB sex pheromone. During 17 weeks when C. cramerella monitoring coincided with the main cocoa pod harvest period in 2013–2014, CPB trap catch data from some blends showed a good correlation with the number of pods with C. cramerella infestation symptoms.     

Conservation of Vascular Epiphyte Diversity in Shade Cacao Plantations in the Chocó Region of Ecuador

To assess the contributions of rustic shade cacao plantations to vascular epiphyte conservation, we compared epiphyte species richness, abundance, composition, and vertical distributions on shade trees and in the understories of six plantations and adjacent natural forests. On three phorophytes and three 10 × 10 m understory plots in each of the agroforestry plantations and natural forests, 54 and 77 species were observed, respectively. Individual-based rarefaction curves revealed that epiphyte species richness was significantly higher on forest phorophytes than on cacao farm shade trees; detailed analyses showed that the differences were confined to the inner and outer crown zones of the phorophytes. No differences in epiphyte species richness were found in understories. Araceae, Piperaceae, and Pteridophyta were less species-rich in plantations than in forests, while there were no differences in Orchidaceae and Bromeliaceae. Regression analysis revealed that epiphyte species richness on trunks varied with canopy cover, while abundance was more closely related to soil pH, canopy cover, and phorophyte height. For crown epiphytes, phorophyte diameter at breast height (dbh) explained much of the variation in species richness and abundance. There were also pronounced downward shifts in the vertical distributions of epiphyte species in agroforests relative to natural forests. The results confirm that epiphyte diversity, composition, and vertical distributions are useful indicators of human disturbance and showed that while the studied plantations serve to preserve portions of epiphyte diversity in the landscape, their presence does not fully compensate for the loss of forests.