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.     

The influence of emergent trees on rainfall distribution in a cacao agroforest (Sulawesi,Indonesia)

Emergent trees may have an influence on the volume and the spatial distribution of water input into agroforestry stands and may thus affect water availability for the main crops. Our goal was to analyze the influence of such trees on rainfall distribution in a cacao agroforest area in the rainforest margin zone of Central Sulawesi, Indonesia. The emergent trees studied belong to the species Bischofia javanica (Phyllanthaceae) and were 15 m high remnants from the natural forest. A set of 96 throughfall gauges was systematically distributed underneath canopies of cacao only, and underneath canopies of cacao plus emergent trees (cacao plus trees). From an earlier study we knew that stemflow can safely be estimated with less than 1% of gross precipitation (Pg).     

Slow death of Atlantic forest trees in cocoa agroforestry in southeastern Brazil

Cocoa (Theobroma cacao) is cultivated in the states of Bahia and Espírito Santo in eastern Brazil under the so-called cabruca system, where the understorey of native Atlantic forest is cleared and the canopy is thinned out to provide adequate shading for the cocoa trees. Apart from its economic and social role, the cabruca system is said to be important for the conservation of Atlantic forest biodiversity. In this paper we studied tree species richness and forest structure of cabrucas to examine the demographic health of these forests and discuss their long-term survival. Data were collected in 20 farms located alongside a 30km track of the northern margin of the Rio Doce, in northern Espírito Santo. All trees 5cm DBH were identified and their diameter was measured in 80 plots (600m²), totalling 4.8ha of sampled area. Recorded trees were also allocated to four different regeneration phases (pioneers, early secondary, late secondary and climax). The inventory resulted in 507 trees belonging to 105 species in 39 families. This species richness is much lower than in less disturbed forests located in the region. Pioneers and early secondary species dominate the cabruca forest in terms of number of species (56.2%), density (71.0%) and basal area (72.3%). The distribution of diameter frequency showed an imbalance in tree regeneration. Most trees in the range of 5–30cm DBH were pioneers (40.7%), or early secondary species (32.6%), while late secondary and climax trees were less frequent (10.2 and 16.5% of the sampled trees, respectively). The dominance of species of early regeneration phases was also observed for trees >30cm DBH (69.0% of pioneers or early secondary and 31.0% of late secondary or climax species). The results indicated that the cabruca forests are not only less diverse and less dense than secondary or primary forests of the region, but also, and more importantly, their natural succession and gap dynamics are being severely impaired. As a consequence, cabrucas present a structure where tree species of late successional phases are becoming increasingly rare while pioneers and early secondary species are becoming dominant. If current management practices of thinning and clearing of native trees are not improved, the long-term survival of these forests is questionable and their role in maintaining biodiversity in the long run is limited.     

Vascular Streak Dieback of cacao in Southeast Asia and Melanesia: in planta detection of the pathogen and a new taxonomy

Vascular Streak Dieback (VSD) disease of cacao (Theobroma cacao) in Southeast Asia and Melanesia is caused by a basidiomycete (Ceratobasidiales) fungus Oncobasidium theobromae (syn. =Thanatephorus theobromae). The most characteristic symptoms of the disease are green-spotted leaf chlorosis or, commonly since about 2004, necrotic blotches, followed by senescence of leaves beginning on the second or third flush behind the shoot apex, and blackening of infected xylem in the vascular traces at the leaf scars resulting from the abscission of infected leaves. Eventually the shoot apex is killed and infected branches die. In susceptible cacao the fungus may grow through the xylem down into the main stem and kill a mature cacao tree. Infections in the stem of young plants prior to the formation of the first 3-4 lateral branches usually kill the plant. Basidiospores released from corticioid basidiomata developed on leaf scars or along cracks in the main vein of infected leaves infect young leaves. The pathogen commonly infects cacao but there are rare reports from avocado. As both crops are introduced to the region, the pathogen is suspected to occur asymptomatically in native vegetation. The pathogen is readily isolated but cultures cannot be maintained. In this study, DNA was extracted from pure cultures of O. theobromae obtained from infected cacao plants sampled from Indonesia. The internal transcribed spacer region (ITS), consisting of ITS1, 5.8S ribosomal RNA and ITS2, and a portion of nuclear large subunit (LSU) were sequenced. Phylogenetic analysis of ITS sequences placed O. theobromae sister to Ceratobasidium anastomosis groups AG-A, AG-Bo, and AG-K with high posterior probability. Therefore the new combination Ceratobasidium theobromae is proposed. A PCR-based protocol was developed to detect and identify C. theobromae in plant tissue of cacao enabling early detection of the pathogen in plants. A second species of Ceratobasidium, Ceratobasidium ramicola, identified through ITS sequence analysis, was isolated from VSD-affected cacao plants in Java, and is widespread in diseased cacao collected from Indonesia.     

System level analysis of cacao seed ripening reveals a sequential interplay of primary and secondary metabolism leading to polyphenol accumulation and preparation of stress resistance

Theobroma cacao and its popular product, chocolate, are attracting attention due to potential health benefits including antioxidative effects by polyphenols, anti‐depressant effects by high serotonin levels, inhibition of platelet aggregation and prevention of obesity‐dependent insulin resistance. The development of cacao seeds during fruit ripening is the most crucial process for the accumulation of these compounds. In this study, we analyzed the primary and the secondary metabolome as well as the proteome during Theobroma cacao cv. Forastero seed development by applying an integrative extraction protocol. The combination of multivariate statistics and mathematical modelling revealed a complex consecutive coordination of primary and secondary metabolism and corresponding pathways. Tricarboxylic acid (TCA) cycle and aromatic amino acid metabolism dominated during the early developmental stages (stages 1 and 2; cell division and expansion phase). This was accompanied with a significant shift of proteins from phenylpropanoid metabolism to flavonoid biosynthesis. At stage 3 (reserve accumulation phase), metabolism of sucrose switched from hydrolysis into raffinose synthesis. Lipids as well as proteins involved in lipid metabolism increased whereas amino acids and N‐phenylpropenoyl amino acids decreased. Purine alkaloids, polyphenols, and raffinose as well as proteins involved in abiotic and biotic stress accumulated at stage 4 (maturation phase) endowing cacao seeds the characteristic astringent taste and resistance to stress. In summary, metabolic key points of cacao seed development comprise the sequential coordination of primary metabolites, phenylpropanoid, N‐phenylpropenoyl amino acid, serotonin, lipid and polyphenol metabolism thereby covering the major compound classes involved in cacao aroma and health benefits.     

Effects of VA mycorrhiza on the growth of cacao seedlings under nursery conditions in Venezuela

The effects of vesicular-arbuscular mycorrhizae inoculation on the growth of cacao seedlings (Theobroma cacao var. Ocumare 60) grown for 5 months in a nursery were studied. The effects of introduced VAM fungi (Glomus occultum, Acaulospora appendicula, Glomus manihotis, Acaulospora morrowae and Scutellospora pellucida) in soils treated with copper oxychloride or methyl bromide, were compared with the indigenous VAM and with their respective non-inoculated controls.Cacao seedlings responded well to indigenous VAM fungi, which included Scutellospora calospora as the dominant species, inducing increases significant in height, dry weight and foliar uptake of P, Cu and Zn in relation to the sterile control. G. occultum and A. appendicula increased the height of cacao seedlings but to an extent not yet statistically significant through the duration of the experiment. S. pellucida and A. appendicula doubled the phosphorus uptake of cacao seedlings. The methyl bromide sterilization induced the lowest Cu and Zn uptake in the cacao seedlings but this effect was ameliorated with VAM inoculation. Manifest deficiency symptoms were not observed in the treatments.Copper oxychloride treatment depressed growth to the same level as the sterile control although its residual effects did not kill VAM. It could change the competitive relations among the VAM species and in this case seemed to affect adversely the more efficient native fungi.The influence of the presence and long permanence of large cotyledons in cacao on the results obtained is discussed.     

Spatial ecology and conservation of two sloth species in a cacao landscape in limón,Costa Rica

The spatial ecology of sloths was studied in an agricultural landscape in Limón Province, Costa Rica. Two sloth species, the three-toed sloth (Bradypus variegatus) and the two-toed sloth (Choloepus hoffmanni), actively used and traveled through a cacao agroforest and its contiguous living fence rows and riparian forests. This agroecosystem was embedded in an agricultural landscape dominated by banana and pineapple plantations and pastures with dispersed trees. The two-toed sloth (C. hoffmanni) was found in 101 tree species and used 34 for food; the three-toed sloth (B. variegatus) was found in 71 tree species and used 15 for food. Choice of preferred species differed between the two sloth species. Trees commonly used by sloths for food and/or refuge in the cacao agroforest included Erythrina poeppigiana, Cecropia obtusifolia, Leucaena leucocephala; in the living fence rows, Cordia alliodora, Erythrina poeppigiana, Ocotea sinuata and Trophis racemosa; in the riparian forests, Coussapoa villosa, Cecropia obtusifolia, Hura crepitans, Pterocarpus officinalis and Spondias mombin; and in the pastures with dispersed trees, Cordia alliodora, Coussapoa villosa, Erythrina poeppigiana, Ocotea sinuata and Hura crepitans. This study demonstrates the importance of the cacao agroforest as well as arboreal elements in other land uses in providing resources for sloth conservation in a larger agricultural landscape.     

Combining-ability for cacao (Theobroma cacao L.) yield components under southern Bahia conditions

The objective of this study was to assess five cacao cultivars (selfs) and 20 hybrids with regard to their general-and specific-combining ability for yield components using method 1, model I, of the diallel analysis system. The selfings and the hybrids were obtained through controlled crossings, tested in the field in a random block design with four replications and plots containing 16 plants. The experiment was set up in the Centro de Pesquisas do Cacau, in Itabuna, Bahia, Brasil, in 1975. The characteristics studied were: the number of healthy and collected fruits per plant (NHFP and NCFP), the weight of humid seeds per plant and per fruit (WHSP and WHSF), and the percentage of diseased fruits per plant (PDFP), for 5 years (1986–1990). The F-test values, highly significant for general combining ability (GCA) and specific combining ability (SCA), demonstrated the existence of variability for both effects. However, the effects of SCA were greater than those of GCA, when compared in terms of the average squared effects. This condition held for the characteristics NHFP, NCFP and WHSP, which shows the relative importance of the non-additive genetic effects over the additive effects. The reciprocal effects did not show significance. Breeding methods which explore the additive portion of genetic variance should be employed for obtaining higher-yielding cacao and high seed weight. For this, the segregant populations should involve cultivars CEPEC 1, SIAL 169 and ICS 1. Combinations involving the cultivar ICS 1 presented the most favorable results for the characteristics WHSP and WHSF, where the hybrid SIAL 169 x ICS 1 and its reciprocal were outstanding.     

Ferns,frogs, lizards,birds and bats in forest fragments and shade cacao plantations in two contrasting landscapes in the Atlantic forest,Brazil

The traditional shade cacao plantations (cabrucas) of southern Bahia, Brazil, are biologically rich habitats, encompassing many forest-dwelling species. However, a critical question for the conservation management of this specific region, and the highly fragmented Atlantic forest in general, is to what extent the conservation value of cabrucas relies on the presence of primary forest habitat in the landscape. We investigated the relative importance of cabrucas and forests for the conservation of five diverse biological groups (ferns, frogs, lizards, birds and bats) in two contrasting landscapes in southern Bahia, one dominated by forest with some interspersed cabrucas, and one dominated by cabrucas with interspersed forest fragments. The community structure (richness, abundance and diversity) of all biological groups differed between cabrucas and forests, although these differences varied among groups. A high number of forest species was found in the cabrucas. However, there were pronounced differences between the two landscapes with regard to the ability of cabrucas to maintain species richness. Irrespective of the biological group considered, cabrucas located in the landscape with few and small forest fragments supported impoverished assemblages compared to cabrucas located in the landscape with high forest cover. This suggests that a greater extent of native forest in the landscape positively influences the species richness of cabrucas. In the landscape with few small forest fragments interspersed into extensive areas of shade cacao plantations, the beta diversity of birds was higher than in the more forested landscape, suggesting that forest specialist species that rarely ventured into cabrucas were randomly lost from the fragments. These results stress both the importance and the vulnerability of the small forest patches remaining in landscapes dominated by shade plantations. They also point to the need to preserve sufficient areas of primary habitat even in landscapes where land use practices are generally favorable to the conservation of biodiversity.     

Shade cacao plantations (<Emphasis Type="Italic">Theobroma cacao</Emphasis>) and bat conservation in southern Bahia,Brazil

Nearly 40% of the remaining Atlantic Forest in southern Bahia state, Brazil, is a rustic agroforest of cacao (Theobroma cacao). These traditional shade plantations, locally known as ‘cabrucas’, are habitats for forest dwelling species. Here we investigated the potential role of this traditional crop as a support for bat assemblages in southern Bahia, establishing the extent to which the bat community structure found in this agricultural system is influenced by the proximity of forest tracts. We compared the bat community attributes (richness, diversity and dominance) and species-level response (capture frequency) between native forest tracts and shade cacao plantations located in two distinct landscapes, which varied in total amount of forest (patch sizes and total forested area) and in their proximity to forest tracts. The cabrucas contiguous to forest tracts showed a rich and abundant bat community, with samples showing capture rates, species richness, diversity and evenness significantly higher than those reported for forest tracts. This situation changes, however, when shade plantations are located at some distance from forest tracts ( > 1000 m). Bat communities in these isolated cabrucas are less diverse than those found in forests and nearby cabrucas, but in both cases, species usually associated with pristine habitats were found. These findings suggest that cabrucas per se are not forest surrogates, and their potential to harbor forest dwelling bat species is closely linked with the existence of nearby forest tracts that may act as a source for species populations. Therefore, the entire landscape should be considered for management, taking into account that maintenance of cabrucas together with the preservation and restoration of forest patches is probably direly needed if one wishes to conserve the bat diversity in southern Bahia for the long term.