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.

Cacao swollen shoot virus in Nigeria: analysis of a pathogen and its vectors

Cacao swollen shoot virus (CSSV) causes the Cacao swollen shoot virus disease (CSSVD) and significantly reduces production in West African cacao. This study characterised the current status of the disease in the major cacao growing States in Nigeria and attempted a clarification on the manner of CSSV transmission. Two separate field surveys and sample collections were conducted in Nigeria in summer 2012 and spring 2013. PCR-based screening of cacao leaf samples and subsequent DNA sequencing showed that the disease continues to persist in Ondo and Oyo States and in new cacao sites in Abia, Akwa Ibom, Cross River and Edo States. Mealybug samples collected were identified using a robust approach involving environmental scanning electron microscopy, histology and DNA barcoding, which highlighted the importance of integrative taxonomy in the study. The results show that the genus Planococcus (Planococcus citri (Risso) and/or Planococcus minor (Maskell)) was the most abundant vector (73.5%) at the sites examined followed by Formicococcus njalensis (Laing) (19.0 %). In a laboratory study, the feeding behaviour of Pl. citri, Pseudococcus longispinus (Targioni-Tozzetti) and Pseudococcus viburni (Signoret) on cacao were investigated using electrical penetration graph (EPG) analysis. EPG waveforms reflecting intercellular stylet penetration (C), extracellular salivation (E1e), salivation in sieve elements (E1), phloem ingestion (E2), derailed stylet mechanics (F), xylem ingestion (G) and non-probing phase (Np) were analysed. Individual mealybugs exhibited marked variation within species and significantly differed (p ≤ .05) between species for E1e and E1. PCR-based assessments of the retention time for CSSV in viruliferous Pl. citri, Ps. longispinus and Ps. viburni fed on a non-cacao diet showed that CSSV was still detectable after 144 hours. These unusually long durations for a pathogen currently classified as a semi-persistent virus have implications for the design of non-malvaceous barrier crops currently being considered for the protection of new cacao plantings.

ICGD 2003: International Cocoa Germplasm Database Version 5.2. CD-ROM

The current version of this database on CD-ROM contains information on 14 127 cocoa (Theobroma cacao) clones and their 14 112 synonyms, the origin and history of the clones and the clone names, and accession lists for 48 of the major cocoa gene banks including quarantine stations. Also included are morphological data for leaves, fruits and seeds, disease reactions, quality and agronomic characters, and reference information on common abbreviations and acronyms, cocoa gene bank addresses and a full bibliography (with hyperlinked reference to data). New additions are 748 photographs and drawings of 428 individual clones in 11 different locations. Also included are 376 profiles for 15 simple sequence repeat primer pairs on 331 clones held in the University of Reading Intermediate Cocoa Quarantine Facility. Minimum system requirements are Windows 95 or later, a Pentium 166 with 32 MB RAM, CD-ROM drive and a minimum 20 MB hard disk space. A user guide is included in the package.

Optimal planning of cocoa clonal selection programmes

Data from three cocoa (Theobroma cacao) clonal selection trials are used to investigate the genetic and environmental components of variation in yield and the percentage of total pods affected by black pod disease (Phytophtora pod rot). Simulations based on these estimated components of variation are then used to discuss the best choice in future of numbers of clones, replicates and years of harvest to maximise selection advances in the traits measured. The three main conclusions are the need to increase the number of clones at the expense of the number of replicates of each clone, the diminishing returns from additional years of harvesting and the importance of widening the genetic base of the clones chosen to be tested.

Pre-Columbian ring ditch construction and land use on a “chocolate forest island” in the Bolivian Amazon

We present a palaeoecological investigation of pre-Columbian land use in the savannah “forest island” landscape of north-east Bolivian Amazonia. A 5700 year sediment core from La Luna Lake, located adjacent to the La Luna forest island site, was analysed for fossil pollen and charcoal. We aimed to determine the palaeoenvironmental context of pre-Columbian occupation on the site and assess the environmental impact of land use in the forest island region. Evidence for anthropogenic burning and Zea mays L. cultivation began ~2000 cal a BP, at a time when the island was covered by savannah, under drier-than-present climatic conditions. After ~1240 cal a BP burning declined and afforestation occurred. We show that construction of the ring ditch, which encircles the island, did not involve substantial deforestation. Previous estimates of pre-Columbian population size in this region, based upon labour required for forest clearance, should therefore be reconsidered. Despite the high density of economically useful plants, such as Theobroma cacao, in the modern forest, no direct pollen evidence for agroforestry was found. However, human occupation is shown to pre-date and span forest expansion on this site, suggesting that here, and in the wider forest island region, there is no truly pre-anthropogenic ‘pristine’ forest.