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.

Allelic size standards and reference genotypes to unify international cocoa (Theobroma cacao L.) microsatellite data

Standardisation of microsatellite allele profiles between laboratories is of fundamental importance to the transferability of genetic fingerprint data and the identification of clonal individuals held at multiple sites. Here we describe two methods of standardisation applied to the microsatellite fingerprinting of 429 Theobroma cacao L. trees representing 345 accessions held in the worlds largest Cocoa Intermediate Quarantine facility: the use of a partial allelic ladder through the production of 46 cloned and sequenced allelic standards (AJ748464 to AJ48509), and the use of standard genotypes selected to display a diverse allelic range. Until now a lack of accurate and transferable identification information has impeded efforts to genetically improve the cocoa crop. To address this need, a global initiative to fingerprint all international cocoa germplasm collections using a common set of 15 microsatellite markers is in progress. Data reported here have been deposited with the International Cocoa Germplasm Database and form the basis of a searchable resource for clonal identification. To our knowledge, this is the first quarantine facility to be completely genotyped using microsatellite markers for the purpose of quality control and clonal identification. Implications of the results for retrospective tracking of labelling errors are briefly explored.

Evaluation of cacao (Theobroma cacao L.) clones against seven Colombian isolates of Moniliophthora roreri (Cif.) Evans et al., representing four major genetic groupings of the pathogen in cacao

Artificial pod inoculation was used to compare the relative aggressiveness of seven Colombian isolates of Moniliophthora roreri (the causal agent of moniliasis or frosty pod disease), representing four major genetic groupings of the pathogen in cacao (cocoa), when applied to five diverse cacao genotypes (ICS-1, ICS-95, TSH-565, SCC-61 and CAP-34) at La Suiza Experimental Farm, Santander Department, Colombia. The following variables were evaluated 9 weeks after inoculation of 2- to 3-month-old pods with spore suspensions (1.2 x 10(5) spores mL(-1)): (i) disease incidence (DI); (ii) external severity (ES); and (iii) internal severity (IS). IS was found to be of greatest value in classifying the reaction of the host genotype against M. roreri. Genetic variation reported between isolates and cacao genotypes was not matched by similar diversity in their aggressiveness. All isolates were generally highly aggressive against most cacao genotypes, with only two isolates showing reduced IS and ES reactions. There was considerable variation between clones in the IS and ES scores, but one cultivated clone (ICS-95) displayed a significant level of resistance against all seven isolates. This clone may be useful in cacao breeding initiatives for resistance to moniliasis of cacao.

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.

Cryopreservation of cocoa (Theobroma cacao L.) somatic embryos for long-term germplasm storage

Cryopreservation using encapsulation-dehydration was developed for the long-term conservation of cocoa (Theobroma cacao L.) germplasm. Survival of individually encapsulated somatic embryos after desiccation and cryopreservation was achieved through optimization of cryoprotectants (abscisic acid (ABA) and sugar), duration of osmotic and evaporative dehydration, and embryo development stage. Up to 63% of the genotype SPA4 early-cotyledonary somatic embryos survived cryopreservation following 7 days preculture with 1 M sucrose and 4 h silica exposure (16% moisture content in bead). This optimized protocol was successfully applied to three other genotypes, e.g. EET272, IMC14 and AMAZ12, with recovery frequencies of 25, 40 and 72%, respectively (but the latter two genotypes using 0.75 M sucrose). Recovered SPA4 somatic embryos converted to plants at a rate of 33% and the regenerated plants were phenotypically comparable to non-cryopreserved somatic embryo-derived plants.

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.

Investigation on Cacao swollen shoot virus (CSSV) pollen transmission through cross-pollination

DNA- and RNA-based polymerase chain reaction (PCR) systems were used with Cacao swollen shoot virus (CSSV) primers designed from conserved regions of the six published genomic sequences of CSSV to investigate whether the virus is transmissible from infected trees through cross-pollination to seeds and seedlings. Pollen was harvested from CSSV infected cocoa trees and used to cross-pollinate flowers of healthy cocoa trees (recipient parents) to generate enough cocoa seeds for the PCR screening. Adequate precautions were taken to avoid cross-contamination during duplicated DNA extractions and only PCR results accompanied by effective positive and negative controls were scored. Results from the PCR analyses showed that samples of cocoa pod husk, mesocarp and seed tissues (testa, cotyledon and embryo) from the cross-pollinations were PCR negative for CSSV DNA. Sequential DNA samples from new leaves of seedlings resulting from the cross-pollinated trees were consistently PCR negative for presence of portions of CSSV DNA for over 36 months after germination. A reverse transcription-PCR analysis performed on the seedlings showed negative results, indicating absence of functional CSSV RNA transcripts in the seedlings. None of the seedlings exhibited symptoms characteristic of the CSSV disease, and all infectivity tests on the seedlings were also negative. Following these results, the study concluded that although CSSV DNA was detected in pollen from CSSV infected trees, there was no evidence of pollen transmission of the virus through cross-pollination from infected cocoa parents to healthy cocoa trees. Keywords:badnavirus;CSSV;PCR;pollen;seed transmission;Theobroma cacao

Genetic variability in partitioning to the yield component of cacao (Theobroma cacao L.)

Biomass partitioning of cacao (Theobroma cacao L.) was studied in seven clones and five hybrids in a replicated experiment in Bahia, Brazil. Over an eighteen month period, a seven- fold difference in dry bean yield was demonstrated between genotypes, ranging from the equivalent of 200 to 1389 kg.ha-1. During the same interval, the increase in trunk cross-sectional area ranged from 11.1 cm2 for clone EEG-29 to 27.6 cm2 for hybrid PA-150 * MA-15. Yield efficiency increment (the ratio of cumulative yield to the increase in trunk circumference), which indicated partitioning between the vegetative and reproductive components, ranged from 0.008 kg.cm-2 for clone CP-82 to 0.08 kg.cm-2 for clone EEG-29. An examination of biomass partitioning within the pod of the seven clones revealed that the beans accounted for between 32.0% (CP-82) and 44.5% (ICS-9) of the pod biomass. The study demonstrated the potential for yield improvement in cacao by selectively breeding for more efficient partitioning to the yield component.

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.

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.

Investigation on Cacao swollen shoot virus (CSSV) pollen transmission through cross-pollination

DNA- and RNA-based polymerase chain reaction (PCR) systems were used with Cacao swollen shoot virus (CSSV) primers designed from conserved regions of the six published genomic sequences of CSSV to investigate whether the virus is transmissible from infected trees through cross-pollination to seeds and seedlings. Pollen was harvested from CSSV infected cocoa trees and used to cross-pollinate flowers of healthy cocoa trees (recipient parents) to generate enough cocoa seeds for the PCR screening. Adequate precautions were taken to avoid cross-contamination during duplicated DNA extractions and only PCR results accompanied by effective positive and negative controls were scored. Results from the PCR analyses showed that samples of cocoa pod husk, mesocarp and seed tissues (testa, cotyledon and embryo) from the cross-pollinations were PCR negative for CSSV DNA. Sequential DNA samples from new leaves of seedlings resulting from the cross-pollinated trees were consistently PCR negative for presence of portions of CSSV DNA for over 36 months after germination. A reverse transcription-PCR analysis performed on the seedlings showed negative results, indicating absence of functional CSSV RNA transcripts in the seedlings. None of the seedlings exhibited symptoms characteristic of the CSSV disease, and all infectivity tests on the seedlings were also negative. Following these results, the study concluded that although CSSV DNA was detected in pollen from CSSV infected trees, there was no evidence of pollen transmission of the virus through cross-pollination from infected cocoa parents to healthy cocoa trees.

Photosynthetic activity and early growth of four Cacao genotypes as influenced by different shade regimes under West African dry and wet season conditions

The physiological performance of four cacao clones was examined under three artificial shade regimes over the course of a year in Ghana. Plants under light shade had significantly higher photosynthetic rates in the rainy seasons whereas in the dry season there was a trend of higher photosynthetic rates under heavy shade. The results imply that during the wet seasons light was the main limiting factor to photosynthesis whereas in the dry season vapour pressure deficit was the major factor limiting photosynthesis through stomatal regulation. Leaf area was generally lower under heavier shade but the difference between shade treatments varied between clones. Such differences in leaf area allocation appeared to underlie genotypic differences in final biomass production in response to shade. The results suggest that shade for young cacao should be provided based on the current ambient environment and genotype.

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.