Drought mitigation in cocoa (Theobroma cacao L.) through developing tolerant hybrids.

BACKGROUND: Cocoa, being a shade loving crop cannot withstand long periods of water stress. Breeding for drought tolerance is the need of the hour due to change in climatic condition and extension of crop to non-traditional areas. Hybrids were produced by crossing four tolerant genotypes in all possible combination. The cross GV1 55 x M 13.12 didn't yield any fruit due to cross incompatibility between these genotypes. Various biochemical parameters act as the true indicators to select tolerant and susceptible types. The major biochemical parameters considered after imposing stress included proline, nitrate reductase activity, superoxide dismutase content and glycine betaine. RESULTS: The drought tolerant hybrids were having high amount of proline, superoxide dismutase enzyme and glycine betaine content. Normally, plants having drought stress show low amount of nitrate reductase activity. However, in case of hybrids, the drought tolerant hybrids were having higher NR activity than the susceptible hybrids. The highest amount of NR was found in the control plants kept at fully irrigated conditions. CONCLUSIONS: This experiment showed the role of different biochemical enzymes and osmolytes in giving tolerance to plants during drought stress. Logistic regression analysis selected proline and nitrate reductase as the two biochemical markers for identifying efficient drought tolerant genotypes in the future breeding programmes.

Genome-wide analysis of ZAT gene family revealed GhZAT6 regulates salt stress tolerance in G. hirsutum.

High salt environments can induce stress in different plants. The genes containing the ZAT domain constitute a family that belongs to a branch of the C2H2 family, which plays a vital role in responding to abiotic stresses. In this study, we identified 169 ZAT genes from seven plant species, including 44 ZAT genes from G. hirsutum. Phylogenetic tree analysis divided ZAT genes in six groups with conserved gene structure, protein motifs. Two C2H2 domains and an EAR domain and even chromosomal distribution on At and Dt sub-genome chromosomes of G. hirsutum was observed. GhZAT6 was primarily expressed in the root tissue and responded to NaCl and ABA treatments. Subcellular localization found that GhZAT6 was located in the nucleus and demonstrated transactivation activity during a transactivation activity assay. Arabidopsis transgenic lines overexpressing the GhZAT6 gene showed salt tolerance and grew more vigorously than WT on MS medium supplemented with 100 mmol NaCl. Additionally, the silencing of the GhZAT6 gene in cotton plants showed more obvious leaf wilting than the control plants, which were subjected to 400 mmol NaCl treatment. Next, the expressions of GhAPX1, GhFSD1, GhFSD2, and GhSOS3 were significantly lower in the GhZAT6-silenced plants treated with NaCl than the control. Based on these findings, GhZAT6 may be involved in the ABA pathway and mediate salt stress tolerance by regulating ROS-related gene expression.

Genetic and environmental links between traits of cocoa beans and pods clarify the phenotyping processes to be implemented.

The average weight of cocoa beans is not generally taken into account during breeding processes, although it is a trait of interest. Several studies indicate that the weight of the beans has a high heritability in Theobroma cacao. However, the values obtained from different countries for the same clone often vary. In this study, we analyzed the effect of different factors on the weight of the beans. Apart from the clone effect, three main factors had an impact: i) the number of beans per pod: a good filling of the pod with beans tended to limit the weight of the beans, ii) the position of the beans in the pod: beans in the apical part of the pod were significantly lighter than the others and iii) the longer the duration of the fructification cycle the heavier the beans were (positive genetics correlation). These results lead us to propose protocols aimed at normalizing the phenotypic values of the genetic material. To obtain a reliable estimate of the bean weight, the following is proposed: either to use beans obtained from manual pollination to saturate the pods with beans, or to systematically use the number beans in the pods as a covariable.

Mitigation of Cd toxicity by Mn in young plants of cacao, evaluated by the proteomic profiles of leaves and roots.

Cd is a non-essential metal and highly toxic to plants, animals and humans, even at very low concentrations. Cd has been found in cocoa beans and in their products, as in the case of chocolate. Mn plays an important role in photosynthetic and can interact with Cd and attenuate its toxic effects on plants. The objective of this work was to evaluate the mechanisms of Mn response in the mitigation of Cd toxicity in young plants of the CCN 51 cacao genotype submitted to 0.8 mmol Cd kg-1, 1.6 mmol Mn kg-1 or the combination of 0.4 mmol Cd kg-1 + 0.8 mmol Mn kg-1 soil, together with the control treatment (without addition of Cd and Mn in soil), by means of analysis of changes in the profile of exclusive proteins (EP) and differentially accumulated proteins (DAP). Leaf and root proteins were extracted and quantified from the different treatments, followed by proteomic analysis. About eight DAP and 38 EP were identified in leaves, whereas in roots 43 DAP and 21 EP were identified. Some important proteins induced in the presence of Cd and repressed in the presence of Cd + Mn or vice versa, were ATPases, isoflavone reductase, proteasome and chaperonin. It was concluded that proteins involved in oxidoreduction and defense and stress response processes, in addition to other processes, were induced in the presence of Cd and repressed in the presence of Cd + Mn. This demonstrated that Mn was able to mitigate the toxic effects of Cd on young plants of the CCN 51 cocoa genotype.

An overview of the physical and biochemical transformation of cocoa seeds to beans and to chocolate: Flavor formation.

Chocolate is a widely consumed product worldwide due to its exquisite flavor, which comes from the unique and fascinating cocoa flavor. This flavor depends on little controllable variables such as the genotype and the agroecological niche, and on the other side, on postharvest operations: (1) cocoa transformation from seeds to beans that comprises cocoa seeds preconditioning, fermentation, and drying, and (2) the production of chocolate from the bean in which roasting is highlighted. Postharvest transformation operations are critically important because during these, cocoa flavor is formed, allowing the differentiation of two categories: bulk and specialty cocoa. In this sense, this article presents an overview of cocoa postharvest operations, the variables and phenomena that influence and control the physical and biochemical transformation from seeds to cocoa beans, and their relation to the formation of chocolate flavor. Moreover, research perspectives in terms of control and management of postharvest practices in order to obtain cocoa with differentiated and specialty characteristics "from bean to bar" are discussed.

Phenotypic plasticity to photon flux density of physiological, anatomical and growth traits in a modern Criollo cocoa clone.

Cocoa grows under shade, but some cultivars develop successfully in full sunlight. In order to characterize the response to photosynthetic photon flux density (PPFD) of a Modern Criollo cocoa clone, gas exchange, photochemical activity and leaf traits, and their relation to growth were measured in seedlings growing in a greenhouse at three different values of PPFD, as well as in adults in full sunlight and shade in the field. Plants showed changes in physiological, biochemical, and morpho-anatomical traits in response to the different light conditions, and in the phenotypic plasticity of these variables. Seedlings subjected to high PPFD in the greenhouse showed decreases in photosynthetic rate, apparent quantum yield of CO2 fixation and photochemical quenching, and increases in non-photochemical quenching, suggesting down-regulation of PSII. In contrast, trees under full sunlight in the field showed a marked reduction in maximum quantum yield of PSII, indicating photoinhibition and supporting that cocoa is a shade tolerant crop. Cocoa showed higher plasticity of physiological and biochemical variables than morpho-anatomical variables in response to PPFD. Effects of time under treatment in the greenhouse and plant age (greenhouse vs field) on plasticity were observed. The acclimation observed in some of the variables studied after 6 months in high light did not represent a particular advantage to seedlings, since relative growth rate was lower than in low- and medium-light seedlings.

Witches' broom resistant genotype CCN51 shows greater diversity of symbiont bacteria in its phylloplane than susceptible genotype catongo.

BACKGROUND: Theobroma cacao L. (cacao) is a perennial tropical tree, endemic to rainforests of the Amazon Basin. Large populations of bacteria live on leaf surfaces and these phylloplane microorganisms can have important effects on plant health. In recent years, the advent of high-throughput sequencing techniques has greatly facilitated studies of the phylloplane microbiome. In this study, we characterized the bacterial microbiome of the phylloplane of the catongo genotype (susceptible to witch's broom) and CCN51 (resistant). Bacterial microbiome was determined by sequencing the V3-V4 region of the bacterial 16S rRNA gene. RESULTS: After the pre-processing, a total of 1.7 million reads were considered. In total, 106 genera of bacteria were characterized. Proteobacteria was the predominant phylum in both genotypes. The exclusive genera of Catongo showed activity in the protection against UV radiation and in the transport of substrates. CCN51 presented genus that act in the biological control and inhibition in several taxonomic groups. Genotype CCN51 presented greater diversity of microorganisms in comparison to the Catongo genotype and the total community was different between both. Scanning electron microscopy analysis of leaves revealed that on the phylloplane, many bacterial occur in large aggregates in several regions of the surface and isolated nearby to the stomata. CONCLUSIONS: We describe for the first time the phylloplane bacterial communities of T. cacao. The Genotype CCN51, resistant to the witch's broom, has a greater diversity of bacterial microbioma in comparison to Catongo and a greater amount of exclusive microorganisms in the phylloplane with antagonistic action against phytopathogens.

Physiological, ultrastructural, biochemical and molecular responses of young cocoa plants to the toxicity of Cr (III) in soil.

The objective of this study was to evaluate Cr toxicity in young plants of the CCN 51 Theobroma cacao genotype at different concentrations of Cr3+ in the soil (0, 100, 200, 400 and 600 mg kg-1) through physiological, ultrastructural, antioxidant and molecular changes. Doses of 400 and 600 mg Cr3+ kg-1 soil severely affected foliar gas exchange, promoted by damages in photosynthetic machinery evidenced by the decrease in CO2 fixation. Decreased expression of psbA and psbO genes, changes in enzymatic activity and lipid peroxidation also affected leaf gas exchange. A hormesis effect was observed at 100 mg Cr3+ kg-1 soil for the photosynthetic activity. As a metal exclusion response, the roots of the cocoa plants immobilized, on average, 75% of the total Cr absorbed. Ultrastructural changes in leaf mesophyll and roots, with destruction of mitochondria, plasmolysis and formation of vesicles, were related to the oxidative stress promoted by excess ROS. The activity of the antioxidant enzymes SOD, APX, GPX and CAT and the amino acid proline coincided with the greater expression of the sod cyt gene demonstrating synchronicity in the elimination of ROS. It was concluded, therefore, that the tolerance of the cocoa plants to the toxicity of Cr3+ depends on the concentration and time of exposure to the metal. Higher doses of Cr3+ in the soil promoted irreversible damage to the photosynthetic machinery and the cellular ultrastructure, interfering in the enzymatic and non-enzymatic systems related to oxidative stress and gene expression. However, the low mobility of the metal to the leaf is presented as a strategy of tolerance to Cr3+.

Path analysis of phenotypic traits in young cacao plants under drought conditions.

Drought is worldwide considered one of the most limiting factors of Theobroma cacao production, which can be intensified by global climate changes. In this study, we aimed to investigate the phenotypic correlation among morphological characteristics of cacao progenies submitted to irrigation and drought conditions and their partitions into direct and indirect effects. Path analysis with phenotypic plasticity index was used as criteria for estimation of basic and explanatory variables. The experiment was conducted in a greenhouse at the Cacao Research Center (CEPEC), Ilhéus, Bahia, Brazil, in a randomized block 21 x 2 factorial arrangement [21 cacao progenies obtained from complete diallel crosses and two water regimes (control and drought)] and six replications. In general, drought conditions influenced biomass production in most progenies, causing significant reductions in total leaf area, leaf number, leaf biomass, fine-roots length (diameter <1 mm), root volume and root area for considered drought intolerant. All progenies showed alterations in growth due to drought. Phenotypic plasticity was most strongly pronounced in root volume. Stem and root diameters, as well as stem dry biomass were the growth variables with the greatest direct effects on root volume under drought conditions, these characters being indicated in screening of cacao progenies drought tolerant.

Cocoa agroforestry is less resilient to sub-optimal and extreme climate than cocoa in full sun.

Cocoa agroforestry is perceived as potential adaptation strategy to sub-optimal or adverse environmental conditions such as drought. We tested this strategy over wet, dry and extremely dry periods comparing cocoa in full sun with agroforestry systems: shaded by (i) a leguminous tree species, Albizia ferruginea and (ii) Antiaris toxicaria, the most common shade tree species in the region. We monitored micro-climate, sap flux density, throughfall, and soil water content from November 2014 to March 2016 at the forest-savannah transition zone of Ghana with climate and drought events during the study period serving as proxy for projected future climatic conditions in marginal cocoa cultivation areas of West Africa. Combined transpiration of cocoa and shade trees was significantly higher than cocoa in full sun during wet and dry periods. During wet period, transpiration rate of cocoa plants shaded by A. ferruginea was significantly lower than cocoa under A. toxicaria and full sun. During the extreme drought of 2015/16, all cocoa plants under A. ferruginea died. Cocoa plants under A. toxicaria suffered 77% mortality and massive stress with significantly reduced sap flux density of 115 g cm-2  day-1 , whereas cocoa in full sun maintained higher sap flux density of 170 g cm-2  day-1 . Moreover, cocoa sap flux recovery after the extreme drought was significantly higher in full sun (163 g cm-2  day-1 ) than under A. toxicaria (37 g cm-2  day-1 ). Soil water content in full sun was higher than in shaded systems suggesting that cocoa mortality in the shaded systems was linked to strong competition for soil water. The present results have major implications for cocoa cultivation under climate change. Promoting shade cocoa agroforestry as drought resilient system especially under climate change needs to be carefully reconsidered as shade tree species such as the recommended leguminous A. ferruginea constitute major risk to cocoa functioning under extended severe drought.

Can flavonoid-rich chocolate modulate arterial elasticity and pathological uterine artery Doppler blood flow in pregnant women? A pilot study.

INTRODUCTION: Dark chocolate has shown beneficial effects on cardiovascular health and might also modulate hypertensive complications in pregnancy and uteroplacental blood flow. Increased uteroplacental resistance is associated with systemic arterial stiffness. We aimed to investigate the short-term effect of flavonoid-rich chocolate on arterial stiffness and Doppler blood flow velocimetry indexes in pregnant women with compromised uteroplacental blood flow. METHODS: Doppler blood flow velocimetry and digital pulse wave analysis (DPA) were performed in 25 women pregnant in the second and third trimesters with uterine artery (UtA) score (UAS) 3-4, before and after 3 days of ingestion of chocolate with high flavonoid and antioxidant contents. UtA pulsatility index (PI), UtA diastolic notching, UAS (semiquantitative measure of PI and notching combined), and umbilical artery PI were calculated, and DPA variables representing central and peripheral maternal arteries were recorded. RESULTS: Mean UtA PI (p = .049) and UAS (p = .025) significantly decreased after chocolate consumption. There were no significant changes in UtA diastolic notching or any DPA indexes of arterial stiffness/vascular tone. CONCLUSION: Chocolate may have beneficial effects on the uteroplacental circulation, but in this pilot study, we could not demonstrate effects on arterial vascular tone as assessed by DPA.

Deciphering the Theobroma cacao self-incompatibility system: from genomics to diagnostic markers for self-compatibility.

Cocoa self-compatibility is an important yield factor and has been described as being controlled by a late gameto-sporophytic system expressed only at the level of the embryo sac. It results in gametic non-fusion and involves several loci. In this work, we identified two loci, located on chromosomes 1 and 4 (CH1 and CH4), involved in cocoa self-incompatibility by two different processes. Both loci are responsible for gametic selection, but only one (the CH4 locus) is involved in the main fruit drop. The CH1 locus acts prior to the gamete fusion step and independently of the CH4 locus. Using fine-mapping and genome-wide association studies, we focused analyses on restricted regions and identified candidate genes. Some of them showed a differential expression between incompatible and compatible reactions. Immunolocalization experiments provided evidence of CH1 candidate genes expressed in ovule and style tissues. Highly polymorphic simple sequence repeat (SSR) diagnostic markers were designed in the CH4 region that had been identified by fine-mapping. They are characterized by a strong linkage disequilibrium with incompatibility alleles, thus allowing the development of efficient diagnostic markers predicting self-compatibility and fruit setting according to the presence of specific alleles or genotypes. SSR alleles specific to self-compatible Amelonado and Criollo varieties were also identified, thus allowing screening for self-compatible plants in cocoa populations.

Combining ability, heritability and genotypic relations of different physiological traits in cacao hybrids.

Selecting parents and evaluating progenies is a very important step in breeding programs and involves approaches such as understanding the initial stages of growth and characterizing the variability among genotypes for different parameters, such as physiological, growth, biomass partitioning and nutrient translocation to the aerial part. In these cases, facilitating tools can be used to understand the involved gene dynamics, such as diallel crosses and genetic and phenotypic correlations. Our main hypothesis is that the contrasting phenotypes of these parental genotypes of cocoa used are due to genetic factors, and progenies derived from crosses of these parental genotypes are useful for breeding programs related to plant architecture, physiological parameters and translocation of mineral nutrients. We aimed to evaluate the combining abilities in progenies of cacao (Theobroma cacao L) originating from contrasting parents for canopy vigor. Emphasis was given to the evaluation of morphological and physiological parameters and the phenotypic and genotypic correlations to understand the dynamics of the action of the genes involved, as well as in expression profile from genes of gibberellins biosynthesis pathway in the parents. Fifteen F1 progenies were obtained from crosses of six clones (IMC 67, P4B, PUCALA, SCA 6, SCA 24 and SJ 02) that were evaluated in a randomized complete block design with four replicates of 12 plants per progeny, in a balanced half table diallel scheme. It is possible to identify and select plants and progenies of low, medium and high height, as there is expressive genetic variability for the evaluated parameters, some of these on higher additive effects, others on larger nonadditive effects and others under a balance of these effects. Most physiological parameters evaluated show that for selection of plants with the desired performance, no complex breeding methods would be necessary due to the high and medium heritability observed. Strong genetic components were observed from many of the correlations, which indicate the possibility to formulate selection indices for multi-traits, such as dwarfism or semidwarfism, tolerance to increase of leaf sodium concentrations and maintenance of the photosynthetic apparatus integrity under these conditions. Additionally, plants with higher carbon fixation, better water use, higher carboxylation efficiency and greater magnesium accumulation in leaves can be selected.

Cacao Phylloplane: The First Battlefield against Moniliophthora perniciosa, Which Causes Witches' Broom Disease.

The phylloplane is the first contact surface between Theobroma cacao and the fungus Moniliophthora perniciosa, which causes witches' broom disease (WBD). We evaluated the index of short glandular trichomes (SGT) in the cacao phylloplane and the effect of irrigation on the disease index of cacao genotypes with or without resistance to WBD, and identified proteins present in the phylloplane. The resistant genotype CCN51 and susceptible Catongo presented a mean index of 1,600 and 700 SGT cm-2, respectively. The disease index in plants under drip irrigation was reduced by approximately 30% compared with plants under sprinkler irrigation prior to inoculation. Leaf water wash (LWW) of the cacao inhibited the germination of spores by up to 98%. Proteins from the LWW of CCN51 were analyzed by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by tandem mass spectrometry. The gel showed 71 spots and identified a total of 42 proteins (28 from the plant and 14 from bacteria). Proteins related to defense and synthesis of defense metabolites and involved in nucleic acid metabolism were identified. The results support the hypothesis that the proteins and water-soluble compounds secreted to the cacao phylloplane participate in the defense against pathogens. They also suggest that SGT can contribute to the resistance of cacao.

Diallel Analysis and Growth Parameters as Selection Tools for Drought Tolerance in Young Theobroma cacao Plants.

This study aimed to estimate the combining ability, of T. cacao genotypes preselected for drought tolerance through diallel crosses. The experiment was conducted under greenhouse conditions at the Cacao Research Center (CEPEC), Ilhéus, Bahia, Brazil, in a completely randomized block design, in an experimental arrangement 21 x 2 [21 complete diallel crosses and two water regimes (control and stressed)]. In the control, soil moisture was kept close to field capacity, with predawn leaf water potential (ΨWL) ranging from -0.1 to -0.5 MPa. In the drought regime, the soil moisture was reduced gradually by decreasing the amount of water application until ΨWL reached -2.0 to -2.5 MPa. Significant differences (p < 0.05) were observed for most morphological attributes analyzed regarding progenies, water regime and their interactions. The results of the joint diallel analysis revealed significant effects between general combining ability (GCA) x water regimes and between specific combining ability (SCA) x water regimes. The SCA 6 genetic material showed high general combining ability for growth variables regardless of the water regime. In general, the water deficit influenced the production of biomass in most of the evaluated T. cacao crosses, except for SCA-6 x IMC-67, Catongo x SCA, MOC-01 x Catongo, Catongo x IMC-67 and RB-40 x Catongo. Multivariate analysis showed that stem diameter (CD), total leaf area (TLA), leaf dry biomass (LDB), stem dry biomass (SDB), root dry biomass (RDB), total dry biomass (TDB), root length (RL), root volume (RV), root diameter (RD) <1 mm and 1 <(RD) <2 mm were the most important growth parameters in the separation of T. cacao genotypes in to tolerant and intolerant to soil water deficit.

Association mapping of seed and disease resistance traits in Theobroma cacao L.

MAIN CONCLUSION: Microsatellite and single nucleotide polymorphism markers that could be used in marker assisted breeding of cacao were identified for number of filled seeds, black pod resistance and witches' broom disease resistance. An association mapping approach was employed to identify markers for seed number and resistance to black pod and witches' broom disease (WBD) in cacao (Theobroma cacao L.). Ninety-five microsatellites (SSRs) and 775 single nucleotide polymorphisms (SNPs) were assessed on 483 unique trees in the International Cocoa Genebank Trinidad (ICGT). Linkage disequilibrium (LD) and association mapping studies were conducted to identify markers to tag the phenotypic traits. Decay of LD occurred over an average 9.3 cM for chromosomes 1-9 and 2.5 cM for chromosome 10. Marker/trait associations were generally identified based on general linear models (GLMs) that incorporated principal components from molecular information on relatedness factor. Seven markers (mTcCIR 8, 66, 126, 212; TcSNP368, 697, 1370) on chromosomes 1 and 9 were identified for number of filled seeds (NSEED). A single marker was found for black pod resistance (mTcCIR280) on chromosome 3, whereas six markers on chromosomes 4, 5, 6, 8, and 10 were detected for WBD (mTcCIR91, 183; TcSNP375, 720, 1230 and 1374). It is expected that this association mapping study in cacao would contribute to the knowledge of the genetic determinism of cocoa traits and that the markers identified herein would prove useful in marker assisted breeding of cacao.

Vulnerability to climate change of cocoa in West Africa: Patterns, opportunities and limits to adaptation.

The West African cocoa belt, reaching from Sierra Leone to southern Cameroon, is the origin of about 70% of the world's cocoa (Theobroma cacao), which in turn is the basis of the livelihoods of about two million farmers. We analyze cocoa's vulnerability to climate change in the West African cocoa belt, based on climate projections for the 2050s of 19 Global Circulation Models under the Intergovernmental Panel on Climate Change intermediate emissions scenario RCP 6.0. We use a combination of a statistical model of climatic suitability (Maxent) and the analysis of individual, potentially limiting climate variables. We find that: 1) contrary to expectation, maximum dry season temperatures are projected to become as or more limiting for cocoa as dry season water availability; 2) to reduce the vulnerability of cocoa to excessive dry season temperatures, the systematic use of adaptation strategies like shade trees in cocoa farms will be necessary, in reversal of the current trend of shade reduction; 3) there is a strong differentiation of climate vulnerability within the cocoa belt, with the most vulnerable areas near the forest-savanna transition in Nigeria and eastern Côte d'Ivoire, and the least vulnerable areas in the southern parts of Cameroon, Ghana, Côte d'Ivoire and Liberia; 4) this spatial differentiation of climate vulnerability may lead to future shifts in cocoa production within the region, with the opportunity of partially compensating losses and gains, but also the risk of local production expansion leading to new deforestation. We conclude that adaptation strategies for cocoa in West Africa need to focus at several levels, from the consideration of tolerance to high temperatures in cocoa breeding programs, the promotion of shade trees in cocoa farms, to policies incentivizing the intensification of cocoa production on existing farms where future climate conditions permit and the establishment of new farms in already deforested areas.

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 the design of a high-resolution melt (HRM) assay capable of rapid identification of the commonly encountered mealybug pests of cacao. CONCLUSIONS: HRM analysis readily differentiated between mealybug pests of cacao that cannot 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.

Making biodiversity-friendly cocoa pay: combining yield, certification, and REDD for shade management.

The twin United Nations' Millennium Development Goals of biodiversity preservation and poverty reduction both strongly depend on actions in the tropics. In particular, traditional agroforestry could be critical to both biological conservation and human livelihoods in human-altered rainforest areas. However, traditional agroforestry is rapidly disappearing, because the system itself is economically precarious, and because the forest trees that shade traditional crops are now perceived to be overly detrimental to agricultural yield. Here, we show a case where the commonly used agroforestry shade metric, canopy cover, would indeed suggest complete removal of shade trees to maximize yield, with strongly negative biodiversity and climate implications. However, a yield over 50% higher was achievable if approximately 100 shade trees per hectare were planted in a spatially organized fashion, a win-win for biodiversity and the smallholder. The higher yield option was detected by optimizing simultaneously for canopy cover, and a second shade metric, neighboring tree density, which was designed to better capture the yield value of ecological services flowing from forest trees. Nevertheless, even a 50% yield increase may prove insufficient to stop farmers converting away from traditional agroforestry. To further increase agroforestry rents, we apply our results to the design of a sustainable certification (eco-labelling) scheme for cocoa-based products in a biodiversity hotspot, and consider their implications for the use of the United Nations REDD (reducing emissions from deforestation and forest degradation) program in agroforestry systems. Combining yield boost, certification, and REDD has the potential to incentivize eco-friendly agroforestry and lift smallholders out of poverty, simultaneously.

Molecular, physiological and biochemical responses of Theobroma cacao L. genotypes to soil water deficit.

Six months-old seminal plants of 36 cacao genotypes grown under greenhouse conditions were subjected to two soil water regimes (control and drought) to assess, the effects of water deficit on growth, chemical composition and oxidative stress. In the control, soil moisture was maintained near field capacity with leaf water potentials (ΨWL) ranging from -0.1 to -0.5 MPa. In the drought treatment, the soil moisture was reduced gradually by withholding additional water until ΨWL reached values of between -2.0 to -2.5 MPa. The tolerant genotypes PS-1319, MO-20 and MA-15 recorded significant increases in guaiacol peroxidase activity reflecting a more efficient antioxidant metabolism. In relation to drought tolerance, the most important variables in the distinguishing contrasting groups were: total leaf area per plant; leaf, stem and total dry biomass; relative growth rate; plant shoot biomass and leaf content of N, Ca, and Mg. From the results of these analyses, six genotypes were selected with contrasting characteristics for tolerance to soil water deficit [CC-40, C. SUL-4 and SIC-2 (non-tolerant) and MA-15, MO-20, and PA-13 (tolerant)] for further assessment of the expression of genes NCED5, PP2C, psbA and psbO to water deficit. Increased expression of NCED5, PP2C, psbA and psbO genes were found for non-tolerant genotypes, while in the majority of tolerant genotypes there was repression of these genes, with the exception of PA-13 that showed an increased expression of psbA. Mutivariate analysis showed that growth variables, leaf and total dry biomass, relative growth rate as well as Mg content of the leaves were the most important factor in the classification of the genotypes as tolerant, moderately tolerant and sensitive to water deficit. Therefore these variables are reliable plant traits in the selection of plants tolerant to drought.