Cacao Germplasm Research Articles

First Report of Phytophthora palmivora, Causal Agent of Black Pod, on Cacao in Puerto Rico.

Black pod or Phytophthora pod rot is the most economically important and widespread disease of cacao, Theobroma cacao L. Total losses due to Phytophthora exceed $400 million worldwide (1), and several species are known to attack cacao with P. palmivora (E.J. Butler) E. J. Butler as the most common. All plant parts are infected, but pod infections are particularly damaging. Symptoms resembling those of black pod disease were observed at the National Plant Germplasm Collection System of cacao at the USDA-ARS Tropical Agriculture Research Station (TARS) in Mayaguez, Puerto Rico for a number of years. During May of 2005, to determine the etiology of the disease, small, surface disinfested sections of pod lesions were placed on water agar and incubated for 4 days. The formation of papillate, deciduous, ellipsoidal to ovoid sporangia produced on sympodial sporangiophores on fruits, fit the description of P. palmivora and the identification was confirmed on cultures on water agar (2). Chlamydospores were readily observed in diseased pods and observed in pure cultures on V8 agar (2). Eight, single hyphal tips were transferred to potato dextrose agar (PDA) (Sigma-Aldrich, St. Louis, MO) and maintained as stock cultures. For pathogenicity tests, healthy mature pods were surface disinfested and placed in a humidity chamber lined with moist paper towels. Eight isolates were tested on four fruits per isolate and the pathogenicity test was repeated once. Inoculum was prepared by growing each isolate on PDA for 5 days with irradiation at 24°C, adding approximately 3.0 ml of water to each plate, dislodging the sporangia with a glass rod, mixing the suspension, estimating spore numbers with a hemacytometer, and adjusting to 104 sporangia per ml. A small, sterile scalpel was used to make an approximately 20.0 mm cut on the fruit epidermis, and approximately 0.2 ml of inoculum was placed on the wound. Pods were evaluated daily for 2 weeks. For molecular analysis, each of the eight cultures were grown in 50% potato dextrose broth to produce mycelia for DNA extraction using the FastDNA kit (Q-Biogen1, Irvine, CA). The internal transcribed spacer (ITS) region of the ribosomal RNA gene cluster was amplified, purified, and sequenced for all eight isolates. The ITS sequences of GenBank Accession Nos. DQ987915 to DQ987922 were identical and exhibited strong similarity (>99% identity) to that of three previously described isolates of P. palmivora from cacao (GenBank Accession Nos. AF 228097, AF467093, and AF467089). P. palmivora has been reported on citrus, coconut, black pepper, and Arracacia xanthorrhiza in Puerto Rico (2,3) and inoculum may have originated from these host or imported on cacao planted into the cacao collection before 2000. USDA-ARS-TARS is the official site for the cacao germplasm collection, thus, a detailed integrated pest management plan that includes the evaluation for resistance, sanitation measures, and use of fungicides to reduce disease levels has been implemented. Decreasing incidence and severity of this disease is a top priority. To our knowledge, this is the first report of P. palmivora on cacao in Puerto Rico.

Field Guide efficacy in the identification of reallocated clonally propagated accessions of cacao (Theobroma cacao L.)

Misidentification is a major constraint to the conservation and utilization of cacao genetic resources. One solution for rapid and accurate identification is to produce a Field Guide for each recognized cacao early generation original population. Each Field Guide in this series of compendia would be comprised of pod digital images with complementary morphological descriptors, molecular fingerprint data with kinship analyses and agronomic data useful to cacao cultivar development. This Field Guide Concept culminated in the production of the first compendium entitled, Field Guide to the ICS Clones of Trinidad. In the current study, this Field Guide was used to verify the identity of 69 trees representing a possible 15 ICS clone genotypes introduced nearly 50 years ago to Costa Rica. Phase one of this study involved identity verification of the trees in Costa Rica using the pod digital image and morphological descriptor data in the Field Guide to the ICS Clones of Trinidad. The error rate was 3.5% for misidentification in the field in Costa Rica. In the second and final phase, SSR fingerprint data were generated for each of the 69 trees and analyzed for verification to the original ICS genotype in the Field Guide. Misidentification was reduced to 0% with the addition of SSR fingerprint analyses. The 69 trees in this study clustered into two groups or sub-populations clearly differentiated by Discriminant analyses with six SSR primers. A 46% cost reduction in SSR fingerprinting of the ICS clones was realized by combining use of the pod image and morphological descriptor data in the Field Guide with genetic diversity estimates derived from these six primers. A Field Guide approach to the identification of reference genotypes for cacao germplasm is discussed.

Accuracy and Reliability of High‐Throughput Microsatellite Genotyping for Cacao Clone Identification

Microsatellite‐based DNA fingerprinting has been increasingly applied to cacao (Theobroma cacao L.) genotype identification. However, the accuracy and reliability of using high throughput microsatellite analysis for cacao clone identification have not yet been rigorously assessed. Despite the use of highly robust fingerprinting protocols, cacao genotype identification has been affected by genotyping errors, which potentially mislead the result of clone identification. In this paper, we calculated the probability of identity for 15 selected microsatellite loci. We then quantified the genotyping error rate through repeated genotyping and simulated the impact of the genotyping error on cacao clone identification. Allelic dropout (ADO), or failure to amplify one allele for a heterozygous locus, and false allele (FA), or an amplicon size error by the polymerase, accounted for 48 and 52% of the genotyping inconsistencies, respectively. The result of simulation showed that 99% of the consensus genotype can be generated for the ambiguous loci through a minimum of three polymerase chain reaction (PCR) repetitions. On the basis of the error rate and probability of identity (PID), we designed a genotyping scheme and applied it to the cacao germplasm held in the USDA cacao collection at Mayaguez, Puerto Rico. Out of the 141 samples, we unambiguously identified nine duplicated groups consisting of 34 cacao accessions. This genotyping scheme is being implemented in large scale fingerprinting of cacao germplasm.

Assessing the Genetic Diversity in the International Cocoa Genebank, Trinidad (ICG,T) using Isozyme Electrophoresis and RAPD

Random amplified polymorphic DNA (RAPD) and isozyme electrophoresis (IE) techniques were used to estimate the level of genetic diversity in a sample of cacao germplasm existing at the International Cocoa Genebank, Trinidad. Twenty-six cocoa populations represented by 459 cocoa genotypes were analysed using IE and 22 populations represented by 353 cocoa genotypes were analysed using RAPD. Despite few differences in the classification of the populations, both techniques revealed three major groups: the indigenous trees, the cultivated Trinitario and the cultivated trees from Ecuador. Two-thirds of the partitioned diversity were found within populations and one-third between the populations, with both techniques.

Assessment of Resistance to Witches'-Broom Disease in Clonal and Segregating Populations of Theobroma cacao.

Fourteen clonal genotypes of cacao (Theobroma cacao) and their open-pollinated progenies were tested for their resistance to witches'-broom disease (Crinipellis perniciosa) using an agar-droplet inoculation method in separate, replicated greenhouse experiments. Nine measures of resistance were evaluated for precision, ability to discriminate between levels for resistance, and their correlation to field resistance. Correlations among the various measures of resistance were also ascertained. Data were re-examined to determine the accuracy and precision associated with individual plant measurement as well as the optimum sample size to assess segregating populations for resistance. The agar-droplet method was able to effectively discriminate various levels of resistance. Incubation period provided a precise, sensitive, valid, and heritable measure of resistance to C. perniciosa. Furthermore, incubation period was highly correlated with broom size and moderately correlated with broom frequency, two epidemiologically important components of resistance. The study also shows that 25 to 30 open-pollinated progeny are sufficient to provide a good estimate of a parent's resistance. The implications of the findings for identifying resistance among cacao germplasm collections as well as within segregating populations for resistance breeding are discussed.

Verification of identities in global cacao germplasm collections

The ex situ conservation of cacao genetic resources is in the form of field genebanks. Misidentified trees though, represent a serious problem to (i) curators, who need to capture genetic diversity, and (ii) users of germplasm material, who must be certain of the identities of the accessions for proper evaluation of their results. The presence of mislabelled trees was assessed from data compiled in the International Cocoa Germplasm Database (ICGD) 2000 v4.1CD-ROM, which contains the published records of cacao accessions in global holdings. Circumstantial evidence revealed that many germplasm collections, especially those in Brazil (CEPEC), Colombia (ICA), Costa Rica (CATIE), Cote d'Ivoire, Mexico, Malaysia, Togo, Trinidad (ICG,T) and Venezuela (EEC) probably possessed misidentified material. The severity of the problem ranged from 15–44 % and is suggested as an area for immediate resolution. Based upon the morphological data provided by the ICGD CD-ROM, several accessions were highly suspect, including 'EET 19 [ECU]', 'EET 48 [ECU]', 'EET 162 [ECU]', 'ICS 1', 'ICS 6', 'ICS 43', 'ICS 89', 'ICS 100', 'IMC 67', 'PA 121 [PER]', 'POUND 7 [POU]', 'SCA 6', 'SPA 9 [COL]', 'UF 29', 'UF 613' and 'UF 667'. However, the identification of mislabelled trees should depend ultimately on molecular analysis. Hence, the use of voucher specimens for verification is forwarded and the concept of a public domain database for microsatellite analyses of germplasm holdings is raised.

Genetic similarity of Theobroma cacao L. accessions maintained in duplicate at the cacao research center germplasm collection based on RAPD markers

The objective of this work was to use RAPD markers to analyze the genetic similarity of 18 accessions maintained in 37 rows in the germplasm collection of Cacao Research Center (CEPEC/CEPLAC), Itabuna, Brazil. Within each row a plant was chosen at random, from which genomic DNA was extracted. DNA sample of each row was amplified by the RAPD. Fifty one RAPD markers were generated, and then transformed to a matrix of binary data. These binary data were used to estimate genetic similarities and to perform cluster analyses. Genetic similarities of 0.70, 0.94 and 0.96 were observed between plants of the accessions RB 29, Ca 1 and C. Sul 4, respectively. The mistakes in the identification of accessions in different germplasm collections seems to be more common than expected. Also, considering the great importance of the germplasm collections, an international effort would be necessary for the characterization of all important cacao germplasm collections around the world.

Genetic Variability in Cultivated Cacao Populations in Bahia, Brazil, Detected by Isozymes and RAPD Markers

The objective of this work was to study genetic variability in accessions from the CEPLAC cacao germplasm collection, which constituted the parents of hybrids recommended by CEPLAC, to infer about the diversity of cacao plantations in Bahia. The 21 clones or accessions utilized were SIC, SIAL (local selections) groups and ICS, IMC, PA, SCA, TSA and UF groups (foreign selections).

Analysis of Genetic Diversity in Theobroma cacao with Emphasis on Witches' Broom Disease Resistance

To facilitate the identification of Theobroma cacao L. that possess desirable traits to meet changing production and market conditions, there is a need to understand the genetic relationships among T. cacao germplasm. In addition, new cultivars are needed to provide more broadly based resistance to devastating diseases such as witches' broom disease [Crinipellis perniciosa (Stahel) Singer]. A subset of 270 T. cacao accessions based on (i) witches' broom disease resistance data, (ii) genetic characterization experiments, and (iii) a random sampling of recently acquired accessions was selected from the extensive germplasm collection at the Centro de Pesquisa do Cacau (CEPEC; Itabuna, Bahia, Brazil) in collaboration with Fazenda Almirante, a division of M&M Mars Incorporated in Itajuipe, Bahia, Brazil. Estimates of genetic distance based on random amplified polymorphic DNA (RAPD) markers were used to evaluate the genetic diversity among the selected accessions. Incorporation of recently acquired accessions (Accessions 181–270) did not increase the breadth of the distribution of genetic diversity already present within the “original” group sampled (Accessions 1–180) suggesting new accessions collected from already sampled geographic regions do not increase the existing genetic diversity in the germplasm collection. In addition, differences in RAPD marker frequencies were associated with accessions that had a high threshold of tolerance to witches' broom disease. Most accessions exhibiting tolerance to witches' broom disease were from the Upper Amazon region, with the exception of SGU 26, a hybrid from Guatemala. This suggests that the Upper Amazon is not the only region to have genes for resistance to witches' broom disease and stresses the need for further collection and examination of germplasm from other regions.

Cacao resistance to Phytophthora: Effect of pathogen species, inoculation depths and pod maturity

Two species of Phytophthora (P. palmivora and P. capsici) and inoculations at two depths (3 mm and 9 mm) were tested each on 10 clones of Theobroma cacao to determine their effects on pod resistance. Ripe and unripe pods were also assessed to determine the influence of physiological status of the pod on the expression of resistance. The two pathogens tested (P. palmivora and P. capsici) differed significantly in their reactions on pods, with P. palmivora being more aggressive than P. capsici. However, the lack of interaction between clones and pathogen species and the similarity in the ranking of clones based on lesion size suggested that selection for resistant clones can be based on one of the two pathogens, preferably the more aggressive one. Pod reactions differed between inoculation depths (3 mm and 9 mm), and between pod maturity stages (ripe and unripe pods) with relatively larger lesions being recorded at 9 mm depth and on unripe pods as compared to those observed at 3 mm depth and on unripe pods, respectively. The magnitude of increase in lesion sizes, however, varied with genotypes, indicating that inoculation depth and pod maturity stage should be standardized in screening cacao germplasm for resistance to Phytophthora.

Allozyme Diversity in a Germplasm Collection of Theobroma cacao L.

Allozyme data for eight polymorphic loci encoding six enzymes were used to describe 86 clones from the USDA/ACRI Theobroma cacao germplasm collection. To further characterize gene diversity, the total population was subdivided two ways: by geographical origin and by morphological type. Contingency ξ2 analysis showed the Caribbean and Central American groups to be distinct from South American clones Among types, the Forasteros differed from the Tnnitarios at four of six loci Some differences were due to the presence or absence of certain alleles. Phenetic trees were constructed using gene frequency data averaged over groups Most gene diversity was found to exist within, rather than between, groups; this differentiation was somewhat higher among morphological types than among geographical origins. A core collection of clones selected across all morphological types, using both morphological and allozyme descriptors, might provide an optimal method of maintaining a germplasm collection of this species.

Numerical taxonomic studies on cacao (Theobroma cacao L.) in Trinidad

Morphological and biochemical characterization of cacao germplasm has been undertaken in the International Cocoa Genebank, Trinidad (ICG, T) to provide useful information for breeders and other users of the crop and in order to facilitate assessment of the genetic diversity of the collection. Use of the full list of standardized morphological descriptors recommended by the International Board for Plant Genetic Resources (IBPGR) prolongs the process of data collation. The work reported in this paper describes an attempt to develop a much shorter list, which contains the most discriminative or taxonomically useful descriptors. Twelve taxonomically useful quantitative descriptors have been identified, using numerical taxonomic techniques.

Characterisation of cacao germplasm using isoenzyme markers. 1. A preliminary survey of diversity using starch gel electrophoresis and standardisation of the procedure

A preliminary screening of isoenzymes in cacao has been carried out with a view to the use of the data in characterising cacao germplasm. Staining was carried out for 24 enzyme systems in leaf tissue. Seventeen of these showed activity, and in seven enzyme systems, reproducible variation in banding patterns was obtained. A single enzyme, peroxidase, was extracted from woody tissue and also exhibited repeatable variation. It was considered that by running extracts from leaves and wood on starch gels, and staining for these 8 enzyme systems, valuable information would be obtained. This could be used to identify clonal material for breeding. It could also be used to supplement the usual morphological descriptors for characterising material in genebanks.